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WO2025067472A1 - Procédés pour la culture de lymphocytes t gamma delta - Google Patents

Procédés pour la culture de lymphocytes t gamma delta Download PDF

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Publication number
WO2025067472A1
WO2025067472A1 PCT/CN2024/121884 CN2024121884W WO2025067472A1 WO 2025067472 A1 WO2025067472 A1 WO 2025067472A1 CN 2024121884 W CN2024121884 W CN 2024121884W WO 2025067472 A1 WO2025067472 A1 WO 2025067472A1
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Prior art keywords
cells
culture medium
less
days
γδt
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Inventor
Chenyu SHU
Fei Xu
Yi Xu
Ting GENG
Yafeng Zhang
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Nanjing Legend Biotechnology Co Ltd
Legend Biotech Ireland Ltd
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Nanjing Legend Biotechnology Co Ltd
Legend Biotech Ireland Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2307—Interleukin-7 (IL-7)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2315—Interleukin-15 (IL-15)
    • 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
    • C12N2510/00—Genetically modified cells

Definitions

  • This disclosure generally relates to methods of preparing ⁇ T cells and uses thereof.
  • ⁇ T cells play fundamental roles in cancer immunotherapy.
  • MHC major histocompatibility complex
  • ⁇ T cells exhibit potent cancer antigen recognition independent of classical peptide MHC complexes, making it an attractive candidate for allogeneic cancer adoptive immunotherapy.
  • ⁇ T cells can recognize a wide range of antigens, such as lipids, phospho-antigens, and peptides, in MHC-dependent and –independent manner; suggesting that these cells can also exert anti-tumor effects against tumors with low mutational burdens and downregulated MHC.
  • antigens such as lipids, phospho-antigens, and peptides
  • the disclosure is related to a method for culturing ⁇ T cells comprising: (1) culturing cells from a sample in a first culture medium comprising interleukin-15 (IL-15) , interferon- ⁇ (IFN- ⁇ ) , and interleukin-2 (IL-2) ; and (2) culturing the cells obtained in step (1) in a second culture medium comprising IL-15, IFN- ⁇ , and IL-2.
  • IL-15 interleukin-15
  • IFN- ⁇ interferon- ⁇
  • IL-2 interleukin-2
  • the ⁇ T cells obtained after step (2) comprising V ⁇ 1 T cells, V ⁇ 2 T cells and V ⁇ 1-V ⁇ 2-T cells.
  • At least about 5%of the ⁇ T cells obtained after step (2) are V ⁇ 1 T cells.
  • At least about 5%of the ⁇ T cells obtained after step (2) are V ⁇ 2 T cells.
  • At least about 1%of ⁇ T cells obtained after step (2) are V ⁇ 1-V ⁇ 2-T cells.
  • the first culture medium and/or the second culture medium further comprises an AKT inhibitor, GSK-3 inhibitor, AMPK inhibitor, PI3K inhibitor, mTOR inhibitor, RSK inhibitor, PDK-1 inhibitor, IKK inhibitor, NF- ⁇ B inhibitor, BCL-2 inhibitor, ERK inhibitor, MEK inhibitor, Raf-1 inhibitor, EGFR inhibitor, DAC inhibitor, HDAC inhibitor or CDK46 inhibitor.
  • the AKT inhibitor is selected from the group consisting of ipatasertib, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, Perifosine, Oridonin, Herbacetin, Tehranolide, Isoliquiritigenin, Scutellarin, and Honokiol.
  • the AKT inhibitor is MK-2206 and is present in an amount of 0.1-10 ⁇ g/ml.
  • the IL-15 in the first culture medium and/or the second culture medium is present in a concentration of about 1-500 ng/ml.
  • the IL-15 in the first culture medium is present in a concentration of about 1-30 ng/ml.
  • the IL-15 in the second culture medium is present in a concentration of about 1-200 ng/ml.
  • the IFN- ⁇ in the first culture medium and/or the second culture medium is present in a concentration of about 1-500 ng/ml.
  • the IFN- ⁇ in the first culture medium is present in a concentration of about 50-150 ng/ml.
  • the IFN- ⁇ in the second culture medium is present in a concentration of about 1-100 ng/ml.
  • the IL-2 in the first culture medium and/or the second culture medium is present in a concentration of about 1-500 IU/ml.
  • the IL-2 in the first culture medium is present in a concentration of about 50-150 IU/ml.
  • the IL-2 in the second culture medium is present in a concentration of about 50-150 IU/ml.
  • the concentration of IL-15 in the second culture medium is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times higher than the concentration of IL-15 in the first culture medium.
  • the concentration of IFN- ⁇ in the first culture medium is at least 1 or 2 times higher than the concentration of IFN- ⁇ in the second culture medium.
  • the first culture medium and/or the second culture medium further comprises IL-4 and IL-1 ⁇ .
  • the IL-4 in the first culture medium is present in a concentration of about 1-500 ng/ml.
  • the IL-4 in the first culture medium is present in a concentration of about 50-150 ng/ml.
  • the IL-1 ⁇ in the first culture medium is present in a concentration of about 1-200 ng/ml.
  • the IL-1 ⁇ in the first culture medium is present in a concentration of about 1-30 ng/ml.
  • the first culture medium and/or the second culture medium further comprises IL-21.
  • the IL-21 in the first culture medium is present in a concentration of about 1-200 ng/ml.
  • the IL-21 in the first culture medium is present in a concentration of about 1-30 ng/ml.
  • the first culture medium and/or the second culture medium does not comprise IL-21.
  • the first culture medium and/or the second culture medium does not comprise IL-7.
  • step (1) further comprises stimulating the ⁇ T cells by an anti- ⁇ TCR antibody.
  • the anti- ⁇ TCR antibody specifically binds to the constant chain of TCR gamma or delta chain.
  • the anti- ⁇ TCR antibody is a V H H comprising a CDR1, a CDR2, and a CDR3, respectively comprising the amino acid sequences of: (1) SEQ ID NOs: 10, 11, and 12; or (2) SEQ ID NOs: 13, 14, and 15.
  • the V H H comprises the amino acid sequence of SEQ ID NO: 1, 3, or an amino acid sequence having at least 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity thereto.
  • the anti- ⁇ TCR antibody comprises: (1) HCDR1, HCDR2 and HCDR3 comprising the amino acid sequences of SEQ ID NOs: 16, 17, and 18, respectively; and/or, LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOs: 19, 20, and 21, respectively; or (2) HCDR1, HCDR2 and HCDR3 comprising the amino acid sequences of SEQ ID NOs: 22, 23, and 24, respectively; and/or, LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOs: 25, 26, and 27, respectively.
  • the anti- ⁇ TCR antibody comprises: (1) a VH comprising the amino acid sequence of SEQ ID NO: 5 or an amino acid sequence having at least 80%sequence identity thereto; and/or, a VL comprising the sequence of SEQ ID NO: 6 or an amino acid sequence having at least 80%sequence identity thereto; or (2) a VH comprising the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence having at least 80%sequence identity thereto; and/or, a VL comprising the sequence of SEQ ID NO: 9 or an amino acid sequence having at least 80%sequence identity thereto.
  • the anti- ⁇ TCR antibody comprises an amino sequence that is at least 80%, 90%or 100%identical to SEQ ID NO: 4 and 7.
  • the anti- ⁇ TCR antibody is immobilized on a cell culture plate.
  • the anti- ⁇ TCR antibody is immobilized on the cell culture plate at 0.1-5 ⁇ g/ml per well.
  • the first culture medium comprises the anti- ⁇ TCR-specific antibody.
  • the first culture medium and/or the second culture medium does not comprise anti-CD28 antibody.
  • the sample prior to step (1) , is enriched for ⁇ T cells.
  • ⁇ T cells are depleted prior to step (1) , between step (1) and step (2) , or after step (2) .
  • NK cells are depleted prior to step (1) , between step (1) and step (2) , or after step (2) .
  • the sample is selected from blood, peripheral blood, umbilical cord blood, lymphoid tissue, bone marrow, spleen, induced pluripotent stem cells or skin tissues.
  • the sample comprises peripheral blood mononuclear cells (PBMCs) .
  • PBMCs peripheral blood mononuclear cells
  • the cells are cultured for 3-9 days during step (1) .
  • the heterologous nucleic acid encodes a CAR that comprises an amino acid sequence that is at least 80%, 90%, or 100%identical to SEQ ID NO: 2.
  • the disclosure is related to a pharmaceutical composition
  • a pharmaceutical composition comprising the cell preparation described herein, and a pharmaceutically acceptable carrier.
  • IL-21 refers to a polypeptide derived from a wild-type IL-21 or a functional variant thereof.
  • the IL-21 may be a wildtype IL-21 (e.g., human IL-21) .
  • the IL-21 may have one or more mutations (e.g., insertions, deletions, or substitutions) .
  • the IL-21 may be a human IL-21.
  • the IL-21 may be a recombinant IL-21.
  • IL-2 refers to a polypeptide derived from a wild-type IL-2 or a functional variant thereof.
  • the IL-2 may be a wildtype IL-2 (e.g., human IL-2) .
  • the IL-2 may have one or more mutations (e.g., insertions, deletions, or substitutions) .
  • the IL-2 may be a human IL-2.
  • the IL-2 may be a recombinant IL-2.
  • the term “AKT inhibitor” or “AKT pathway inhibitor” refers to any compound that has the effect of preferentially reducing and/or blocking the activity of AKT.
  • the inhibitor may act directly on AKT, for example by preventing phosphorylation of AKT or de-phosphorylating AKT, for example at Ser473 and/or Thr308, or alternatively, the inhibitor may act via the inhibition of an upstream activator (or multiple activators) of AKT in the PI3K/AKT/mTOR signaling pathway or other pathway involved in apoptosis, or via the activation of an upstream inhibitor of AKT. It is preferred that the AKT inhibitor acts to reduce and/or block the activity of AKT via multiple pathways such that effective inhibition is achieved.
  • cancer refers to cells having the capacity for uncontrolled autonomous growth. Examples of such cells include cells having an abnormal state or condition characterized by rapidly proliferating cell growth. The term is meant to include cancerous growths, e.g., tumors; oncogenic processes, metastatic tissues, and malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness.
  • malignancies of the various organ systems such as respiratory, cardiovascular, renal, reproductive, hematological, neurological, hepatic, gastrointestinal, and endocrine systems; as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and/or testicular tumors, non-small cell carcinoma of the lung, and cancer of the small intestine.
  • Cancer that is “naturally arising” includes any cancer that is not experimentally induced by implantation of cancer cells into a subject, and includes, for example, spontaneously arising cancer, cancer caused by exposure of a patient to a carcinogen (s) , cancer resulting from insertion of a transgenic oncogene or knockout of a tumor suppressor gene, and cancer caused by infections, e.g., viral infections.
  • a carcinogen s
  • cancer resulting from insertion of a transgenic oncogene or knockout of a tumor suppressor gene and cancer caused by infections, e.g., viral infections.
  • the term “carcinoma” is art recognized and refers to malignancies of epithelial or endocrine tissues. The term also includes carcinosarcomas, which include malignant tumors composed of carcinomatous and sarcomatous tissues.
  • an “adenocarcinoma” refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures.
  • the term “sarcoma” is art recognized and refers to malignant tumors of mesenchymal derivation.
  • hematopoietic neoplastic disorders includes diseases involving hyperplastic/neoplastic cells of hematopoietic origin.
  • a hematopoietic neoplastic disorder can arise from myeloid, lymphoid or erythroid lineages, or precursor cells thereof.
  • a hematologic cancer is a cancer that begins in blood-forming tissue, such as the bone marrow, or in the cells of the immune system. Examples of hematologic cancer include e.g., leukemia, lymphoma, and multiple myeloma etc.
  • the terms “subject” and “patient” are used interchangeably throughout the specification and describe an animal, human or non-human, to whom treatment according to the methods of the present disclosure is provided.
  • Veterinary and non-veterinary applications are contemplated in the present disclosure.
  • Human patients can be adult humans or juvenile humans (e.g., humans below the age of 18 years old) .
  • patients include but are not limited to mice, rats, hamsters, guinea-pigs, rabbits, ferrets, cats, dogs, and primates.
  • non-human primates e.g., monkey, chimpanzee, gorilla, and the like
  • rodents e.g., rats, mice, gerbils, hamsters, ferrets, rabbits
  • lagomorphs e.g., swine (e.g., pig, miniature pig)
  • equine canine, feline, bovine, and other domestic, farm, and zoo animals.
  • polypeptide, ” “peptide, ” and “protein” are used interchangeably to refer to polymers of amino acids of any length of at least two amino acids.
  • chimeric antigen receptor refers to genetically engineered receptors, which can be used to graft one or more antigen specificity onto immune effector cells, such as T cells.
  • Some CARs are also known as “artificial T-cell receptors, ” “chimeric T cell receptors, ” or “chimeric immune receptors. ”
  • the CAR may comprise an extracellular antigen binding domain specific for one or more antigens (such as tumor antigens) , a transmembrane domain, and an intracellular signaling domain of a T cell and/or other receptors.
  • CAR-T cell refers to a T cell that expresses a CAR.
  • T-cell receptor refers to an endogenous or modified T-cell receptor comprising an extracellular antigen binding domain that binds to a specific antigenic peptide bound in an MHC molecule.
  • the TCR may comprise a TCR ⁇ polypeptide chain and a TCR ⁇ polypeptide chain.
  • the TCR may comprise a TCR ⁇ polypeptide chain and a TCR ⁇ polypeptide chain.
  • the TCR may specifically bind a tumor antigen.
  • TCR-T refers to a T cell that expresses a recombinant TCR.
  • heterologous antigen receptor such as a heterologous TCR or CAR
  • TCR or CAR can alter the immunogenic specificity of the T cells so that they recognize or display improved recognition for one or more tumor antigens that are present on the surface of the cancer cells of an individual with cancer.
  • CDR complementarity determining region
  • the precise boundaries of these amino acid residues can be defined according to various numbering systems known in the art, for example, according to the definitions in the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991) , Chothia numbering system (Chothia &Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al.
  • the CDRs of the antibodies of the disclosure may be defined according to Kabat, AbM, IMGT, or Chothia numbering system, or any combination thereof. Unless otherwise indicated or clear from the context, the CDRs of the antibodies of the disclosure are preferably defined according to AbM numbering system.
  • FIGS. 1A-1K show the testing results on cell count (FIG. 1A) , viability (FIG. 1B) , CAR positive rate (FIG. 1C) , cell subtype percentages (FIG. 1D) , cytotoxicity (FIG. 1E) , expansion (FIG. 1F) and cytokine secretion (FIGS. 1G-1K) of the ⁇ T cells prepared under different conditions (with or without IL-2 and/or IL-7) .
  • CAR-expressing V ⁇ 1 T cells “CAR-V ⁇ 1”
  • CAR-expressing V ⁇ 2 T cells “CAR-V ⁇ 2”
  • CAR-V ⁇ 2 are used as control cells.
  • FIGS. 2A-2K show the testing results on cell count (FIG. 2A) , viability (FIG. 2B) , CAR positive rate (FIG. 2C) , cell subtype percentages (FIG. 2D) , cytotoxicity (FIG. 2E) , expansion (FIG. 2F) and cytokine secretion (FIGS. 2G-2K) of the ⁇ T cells prepared under different conditions (with or without IL-4 and/or IL-1 ⁇ ) .
  • FIGS. 3A-3I show the testing results on cell count (FIG. 3A) , viability (FIG. 3B) , CAR positive rate (FIG. 3C) , cell subtype percentages (FIG. 3D) , cytotoxicity (FIG. 3E) , expansion (FIG. 3F) and cytokine secretion (FIGS. 3G-3I) of the ⁇ T cells prepared under different conditions (with or without MK-2206) .
  • FIGS. 4A-4I show the testing results on cell count (FIG. 4A) , viability (FIG. 4B) , CAR positive rate (FIG. 4C) , cell subtype percentages (FIG. 4D) , cytotoxicity (FIG. 4E) , expansion (FIG. 4F) and cytokine secretion (FIGS. 4G-4I) of the ⁇ T cells prepared under different conditions (with or without IL-21) .
  • FIGS. 5A-5I show the testing results on cell count (FIG. 5A) , viability (FIG. 5B) , CAR positive rate (FIG. 5C) , cell subtype percentages (FIG. 5D) , cytotoxicity (FIG. 5E) , expansion (FIG. 5F) and cytokine secretion (FIGS. 5G-5I) of the ⁇ T cells prepared under different conditions (with AS287963, OKT3, or CD3/CD28 beads) .
  • FIGS. 6A-6F show intrinsic anti-tumor responses (without CAR expression) of the ⁇ T cells prepared without transduction, against RPMI-8226 (FIG. 6A) , K562 (FIG. 6B) , NCI-H929 (FIG. 6C) , U937 (FIG. 6D) , Huh7 (FIG. 6E) and SK-Hep-1 (FIG. 6F) cells.
  • FIGS. 7A-7B show testing results from the three-Way MLR assay testing, where ⁇ T cells (1.5 ⁇ 10 4 cells per well) , NCI-H929 tumor cells (1.5 ⁇ 10 4 cells per well) and allogeneic/autologous PBMCs (1.5 ⁇ 10 6 cells per well, stain with CellTrace Violet Reagent) were co-cultured in a final volume of 4 mL medium (base medium+10%Hi-FBS) per well within 12-well plates for 7 day.
  • FIG. 7A shows the cell numbers of ⁇ T cells.
  • FIG. 7B shows the cell number of CAR ⁇ T cells.
  • FIGS. 8A-8K show the testing results on cell count (FIG. 8A) , viability (FIG. 8B) , CAR positive rate (FIG. 8C) , purity (FIG. 8D) , cell subtype percentages (FIG. 8E) , cell phenotype percentages (FIG. 8F) , cytotoxicity (FIG. 8G) , expansion (FIG. 8H) and cytokine secretion (FIGS. 8I-8K) of the ⁇ T cells.
  • FIGS. 9A-9D show the in vivo anti-tumor effects of the ⁇ T cells transfected with a BCMA CAR vector, as tested in a RPMI-8226 xenograft model (FIGS. 9A-9B) and a z-138 xenograft model (FIGS. 9C-9D) .
  • FIG. 9A and FIG. 9C show the tumor volume data.
  • FIG. 9B and FIG. 9D show the percentage of CAR-T cells in peripheral blood.
  • FIGS. 10A-H show the testing results on cell expansion fold (FIG. 10A) , viability (FIG. 10B) , purity (FIG. 10C) , cell subtype percentages (FIG. 10D) , CAR positive rate (FIG. 10E) , cytotoxicity (FIG. 10F) , total T expansion of cytotoxicity (FIG. 10G) and CAR+ T expansion (FIGS. 10H) of cytotoxicity.
  • CDR complementarity determining region
  • ⁇ T cells are a subset of T cells that provide a link between the innate and adaptive immune responses. These cells undergo V- (D) -J segment rearrangement to generate antigen-specific ⁇ T cell receptors ( ⁇ TCRs) , and ⁇ T cells and can be directly activated via the recognition of an antigen by either the ⁇ TCR or other, non-TCR proteins, acting independently or together to activate ⁇ T cell effector functions.
  • ⁇ T cells represent a small fraction of the overall T cell population in mammals, approximately 1-5%of the T cells in peripheral blood and lymphoid organs, and they appear to reside primarily in epithelial cell-rich compartments like skin, liver, digestive, respiratory, and reproductive tracks.
  • ⁇ TCRs which recognize antigens bound to major histocompatibility complex molecules (MHC)
  • MHC major histocompatibility complex molecules
  • ⁇ TCRs can directly recognize bacterial antigens, viral antigens, stress antigens expressed by diseased cells, and tumor antigens in the form of intact proteins or non-peptide compounds.
  • ⁇ T cells are highly cytotoxic against tumor cells. They function through TCR, NCR (natural cytotoxicity receptor) and other mechanisms to recognize and kill tumors. Unlike T cell receptors in ⁇ T cells, the ⁇ TCR recognizes antigens in a MHC-independent manner, thus paving the way for the use of ⁇ T cells as allogeneic, “off-the-shelf” products to treat cancer, since it does not cause GvHD.
  • Human ⁇ T cells consist of three major populations, according to their V ⁇ chains. There are V ⁇ 1, V ⁇ 2 and V ⁇ 3 cells. Typically, V ⁇ 2 T cells are paired with V ⁇ 9 chains within ⁇ TCR complex and are primarily distributed in peripheral blood, while V ⁇ 1 T cells are paired with V ⁇ 2/3/4/5/8/9 within ⁇ TCR complex and can be found in peripheral blood, skin, gut, spleen and liver owing to its diversity.
  • a detailed discussion of ⁇ T cells and their application in cancer immunotherapy can be found in, e.g., Deng, Jiechu, and Hongna Yin. "Gamma delta ( ⁇ ) T cells in cancer immunotherapy; where it comes from, where it will go? .
  • ⁇ T cells can be selectively expanded in vitro by culturing these cells in two phases.
  • these cells can be cultured in a first culture medium comprising interleukin-15 (IL-15) , interferon- ⁇ (IFN- ⁇ ) , and interleukin-2 (IL-2) .
  • IL-15 interleukin-15
  • IFN- ⁇ interferon- ⁇
  • IL-2 interleukin-2
  • these cells can be expanded in a second culture medium containing IL-15, IFN- ⁇ , and IL-2.
  • IL-15 interleukin-15
  • IFN- ⁇ interferon- ⁇
  • IL-2 interleukin-2
  • the cell culture method described herein is very robust, highly reproducible and fully compatible with large-scale clinical applications. It generates sufficient numbers of differentiated ⁇ T cells for use in adoptive immunotherapy of cancer, and in a variety of other therapeutic applications.
  • the ⁇ T cells obtained by the method described herein may be used in cell therapies.
  • ⁇ T cells are consider as a first line of defense against infectious pathogens.
  • ⁇ T cells possess intrinsic cytolytic activity against transformed cells of various origins including B-cell lymphomas, sarcomas and carcinomas.
  • the ⁇ T cells obtained and cultured ex vivo according to the methods of the disclosure may be transfused into a patient for the treatment or prevention of infections, cancer or diseases resulting from immunosuppression.
  • T lymphocytes bearing T cell receptors composed of ⁇ and ⁇ (rather than ⁇ and ⁇ ) chains led to the identification of the distinct ⁇ T cell lineage.
  • ⁇ T cell are infrequent and account for less 10%of human peripheral blood lymphocytes but are substantially enriched in epithelial tissues of healthy individuals.
  • ⁇ T cells develop alongside ⁇ T cells from shared thymic progenitors, although functional activation of ⁇ T cells at peripheral sites occurs more rapidly than that of conventional ⁇ T cells. The reason is ⁇ T cells do not require classic MHC-mediated antigen presentation and like natural killer cells, they instead recognize infected or neoplastic cells via multiple receptor-ligand interactions and promptly react to them in an innate-like fashion. Therefore, ⁇ T cells are usually considered a first-line surveillance mechanism against infection and tumors.
  • V TCR variable
  • TRGV 14 unique V ⁇ alleles
  • TRDV1, TRDV2, and TRDV3 3 unique V ⁇ alleles
  • TRDV8/TRAV38-2 5 V ⁇ alleles that share a common nomenclature with V ⁇ alleles
  • V ⁇ 2 T cells can be activated by phosphoantigens, which is produced at abnormal levels in tumor cells and in individuals exposed to bone-strengthening amino bisphosphonates like zoledronate.
  • V ⁇ 1 T cells are not well study as V ⁇ 2 T cells to date. Different from V ⁇ 2 T cells, V ⁇ 1 T cells account for only up to one-third of circulating ⁇ T cells, but they preferentially reside in peripheral tissues including the gut epithelium, dermis, spleen and liver, where they are the predominant ⁇ T cell subset and contribute to tissue homeostasis. Different ligands have been identified as being recognized by certain V ⁇ 1 TCRs, such as the MHC-like proteins of the CD1 family, including the lipid-presenting proteins CD1c and CD1d.
  • ⁇ CAR-T cells may remain detectable for more than ten years.
  • V ⁇ 3 T cells are rare to virtually absent in the peripheral blood of healthy individuals but are a notable population in the intestines and liver, as well as in the circulation in the context of viral infection or leukaemia. This ⁇ T cell subset seems to share functional similarities with V ⁇ 1 T cells, including the ability to recognize glycolipids presented by CD1d on target cells.
  • the present disclosure provides methods for selectively culturing and expanding ⁇ T cells in culture.
  • the methods as described in the present disclosure are carried out on a sample, which is also referred to herein as a “starting sample” .
  • the methods can use either unfractionated samples or samples that have been enriched for T cells or ⁇ T cells.
  • the samples can be enriched for ⁇ T cells.
  • the sample can be any sample that contains ⁇ T cells or precursors thereof including, but not limited to, blood, bone marrow, lymphoid tissue, thymus, spleen, lymph node tissue, infected tissue, fetal tissue and fractions or enriched portions thereof.
  • the sample is optionally blood including peripheral blood or umbilical cord blood or fractions thereof, including buffy coat cells, leukapheresis products, peripheral blood mononuclear cells (PBMCs) and low-density mononuclear cells (LDMCs) .
  • the sample may be human blood or a fraction thereof.
  • the cells can be obtained from a sample of blood using techniques known in the art such as density gradient centrifugation.
  • whole blood can be layered onto an equal volume of Ficoll-Hypaque TM followed by centrifugation at 400 ⁇ g for 15-30 minutes at room temperature.
  • the interface material will contain low-density mononuclear cells that can be collected and washed in culture medium and centrifuged at 200 ⁇ g for 10 minutes at room temperature.
  • ⁇ T cells in the sample may be depleted.
  • ⁇ T cells in the sample may be depleted using antibodies targeting ⁇ T cells.
  • the antibodies targeting ⁇ T cells may be linked to magnetic beads.
  • ⁇ T cells may be removed along with these magnetic beads.
  • ⁇ T cells in the sample may be depleted using a TCR ⁇ cells depletion kit (Miltenyi, 200-070-407) .
  • Natural killer (NK) cells in the sample may be depleted.
  • NK cells in the sample may be depleted using antibodies targeting NK cells.
  • the antibodies targeting NK cells may be linked to magnetic beads.
  • the antibodies targeting NK cells may specifically bind to CD56.
  • NK cells in the sample may be depleted using a CD56+ cells depletion kit (Miltenyi, 130-050-401) as per the manufacturer’s instructions.
  • ⁇ T cells in the sample may be depleted using a TCR ⁇ cells depletion kit (Miltenyi, 200-070-407) and CD56+ cells depletion kit (Miltenyi, 130-050-401) as per the manufacturer’s instructions, which are incorporated herein by reference in the entirety.
  • ⁇ T cells may be depleted before the first phase, between the first phase and the second phase, or after the second phase.
  • NK cells may be depleted before the first phase, between the first phase and the second phase, or after the second phase.
  • the present disclosure provided methods to produce CAR- ⁇ T cells with high purity, high expansion rate and high transduction rate for clinical use and production. Such cells display high activation, low exhaustion and predominant phenotype. In vitro validation shows superior anti-tumor activity and safer profile than cells obtained from some other existing methods.
  • the methods for expanding human ⁇ T cells have a culturing phase and an expanding phase.
  • these cells can be cultured in a cell culture medium comprising one or more cytokines selected from interleukin-15 (IL-15) , interferon- ⁇ (IFN- ⁇ ) , and interleukin-2 (IL-2) .
  • the cells may be stimulated by a ⁇ TCR-specific antibody.
  • these cells can be expanded in a cell expansion culture medium containing one or more cytokines selected from IL-15, IFN- ⁇ , and IL-2.
  • the cells are cultured and expanded without the need for the use of feeder cells or microbial or viral components.
  • the cells may be cultured and expanded without the need for IL-7.
  • the method for culturing and expanding ⁇ T cells in a sample comprising:
  • culturing cells in the sample in a first culture medium comprising one or more cytokines selected from IL-15, IFN- ⁇ , and IL-2;
  • step (2) (2) culturing the cells obtained in step (1) in a second culture medium comprising one or more cytokines selected from IL-15, IFN- ⁇ , and IL-2.
  • the cells may be stimulated by a ⁇ TCR-specific antibody.
  • the first culture medium may be located in a container (e.g., cell culture plate) that is coated with a ⁇ TCR-specific antibody.
  • the ⁇ TCR-specific antibody may be AS287963, AS281850, AS287435 or AS288180 (an anti- ⁇ TCR antibody, SEQ ID NO: 1, 3, 4 and 7) .
  • the first culture medium may comprise or consist of IL-15, IFN- ⁇ , IL-2, IL-1 ⁇ , IL-4, and IL-21.
  • the first culture medium may lack IL-7, IL-18, and/or IL-12.
  • the first culture medium may lack IL-21.
  • IL-1 ⁇ may be present in an amount that is greater than 1 ng/ml, greater than 2 ng/ml, greater than 3 ng/ml, greater than 4 ng/ml, greater than 5 ng/ml, greater than 6 ng/ml, greater than 7 ng/ml, greater than 8 ng/ml, greater than 9 ng/ml, greater than 10 ng/ml, greater than 11 ng/ml, greater than 12 ng/ml, greater than 13 ng/ml, greater than 14 ng/ml, greater than 15 ng/ml, greater than 16 ng/ml, greater than 17 ng/ml, greater than 18 ng/ml, greater than 19 ng/ml, greater than 20 ng/ml, greater than 25 ng/ml, or greater than 30 ng/ml.
  • IL-1 ⁇ may be present in an amount that is from about 1 to about 1000 ng/ml, from about 5 to 100 ng/ml, from about 10 to 75 ng/ml, from about 10 to 50 ng/ml, from about 10 to 25 ng/ml, from about 10 to 20 ng/ml, from about 12.5 to 17.5 ng/ml, or from about 14 to 16 ng/ml.
  • HPL may be present in an amount that is greater than 1%, greater than 2%, greater than 3%, greater than 4%, greater than 5%, greater than 6%, greater than 7%, greater than 8%, greater than 9%, greater than 10%, greater than 11%, greater than 12%, greater than 15%, greater than 20%, or greater than 25%.
  • HPL may be present in an amount that is less than 1%, less than 2%, less than 3%, less than 4%, less than 5%, less than 6%, less than 7%, less than 8%, less than 9%, less than 10%, less than 11%, less than 12%, less than 15%, less than 20%, or less than 25%.
  • the first cell culture medium and/or the second cell culture may contain a base medium in an amount that is more than 10%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, or more than 95%by volume.
  • the first cell culture medium and/or the second cell culture may contain a base medium in an amount that is less than 10%, less than 20%, less than 30%, less than 40%, less than 50%, less than 60%, less than 70%, less than 80%, less than 90%, or less than 95%by volume.
  • the first cell culture medium and/or the second cell culture can be supplemented with serum or plasma.
  • the amount of plasma in the first and second culture media may be from about 0.5%to about 25%by volume, for example from about 2%to about 20%by volume or from about 2.5%to about 10%by volume, for example is about 10%by volume.
  • the serum or plasma can be obtained from any source including, but not limited to, human peripheral blood, umbilical cord blood, or blood derived from another mammalian species.
  • the plasma may be from a single donor or may be pooled from several donors. If autologous ⁇ T cells are to be used clinically, i.e. reinfused into the same patient from whom the original sample was obtained, then autologous plasma (i.e.
  • the first cell culture medium and/or the second cell culture may be supplemented with human AB serum.
  • the human AB serum may be present in an amount that is greater than 0.5%, greater than 1%, greater than 2%, greater than 3%, greater than 4%, greater than 5%, greater than 6%, greater than 7%, greater than 8%, greater than 9%, greater than 10%, greater than 11%, greater than 12%, greater than 13%, greater than 14%, greater than 15%, greater than 20%, greater than 25%, or greater than 30%by volume.
  • the human AB serum may be present in an amount that is about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, les about 15%, about 20%, about 25%, or about 30%by volume.
  • the human AB serum may be present in an amount that is from about 0.5%to about 30%, from about 1%to about 25%, from about 2%to about 20%, from about 5%to about 20%, from about 5%to about 15%, from about 8%to about 12%, or from about 9%to about 11%by volume.
  • the first cell culture medium and/or the second cell culture may be supplemented with human serum replacement (e.g. human platelet lysate (HPL) ) .
  • human platelet lysate may be present in an amount that is greater than 0.5%, greater than 1%, greater than 2%, greater than 3%, greater than 4%, greater than 5%, greater than 6%, greater than 7%, greater than 8%, greater than 9%, greater than 10%, greater than 11%, greater than 12%, greater than 13%, greater than 14%, greater than 15%, greater than 20%, greater than 25%, or greater than 30%by volume.
  • the human platelet lysate may be present in an amount that is less than 0.5%, less than 1%, less than 2%, greater than 3%, less than 4%, less than 5%, less than 6%, less than 7%, less than 8%, less than 9%, less than 10%, less than 11%, less than 12%, less than 13%, less than 14%, less than 15%, less than 20%, less than 25%, or less than 30%by volume.
  • the human platelet lysate may be present in an amount that is about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 20%, about 25%, or about 30%by volume.
  • the human platelet lysate may be present in an amount that is from about 0.5%to about 30%, from about 1%to about 25%, from about 2%to about 20%, from about 5%to about 20%, from about 5%to about 15%, from about 8%to about 12%, or from about 9%to about 11%by volume.
  • the sample or fraction thereof Prior to culturing the sample or fraction thereof (such as PBMCs) in the first culture medium, the sample or fraction thereof may be enriched for certain cell types and/or depleted for other cell types.
  • the sample or fraction thereof may be enriched for T cells, or enriched for ⁇ T cells, depleted of NK cells or depleted of a ⁇ T cells.
  • the sample prior to culturing the sample in the first culture medium, the sample may be depleted of a ⁇ T cells and/or NK cells.
  • the first culture medium and/or second culture medium may additionally include other ingredients that can assist in the growth and expansion of the ⁇ T cells.
  • other ingredients that can be added include, but are not limited to, plasma or serum, purified proteins such as albumin, a lipid source such as low-density lipoprotein (LDL) , vitamins, amino acids, steroids and any other supplements supporting or promoting cell growth and/or survival.
  • plasma or serum purified proteins such as albumin
  • a lipid source such as low-density lipoprotein (LDL)
  • vitamins, amino acids, steroids and any other supplements supporting or promoting cell growth and/or survival include, but are not limited to, plasma or serum, purified proteins such as albumin, a lipid source such as low-density lipoprotein (LDL) , vitamins, amino acids, steroids and any other supplements supporting or promoting cell growth and/or survival.
  • LDL low-density lipoprotein
  • the method of preparing ⁇ T cells may comprise: (1) culturing cells in the sample in a first culture medium comprising 50-150 ng/ml interleukin-4 (IL-4) , 1-30 ng/ml interleukin-15 (IL-15) , 1-30 ng/ml interleukin-1 ⁇ (IL-1 ⁇ ) , 50-150 ng/ml interferon- ⁇ (IFN- ⁇ ) , 1-30 ng/ml IL-21, and 50-150 IU/ml IL-2; and (2) culturing the cells obtained in step (1) in a second culture medium comprising 1-200 ng/ml IL-15, 1-100 ng/ml IFN- ⁇ , and 50-150 IU/ml IL-2.
  • IL-4 interleukin-4
  • IL-15 1-30 ng/ml interleukin-15
  • IFN- ⁇ interferon- ⁇
  • the method of preparing ⁇ T cells may comprise: (1) culturing cells in the sample in a first culture medium comprising 50-150 ng/ml interleukin-4 (IL-4) , 1-30 ng/ml interleukin-15 (IL-15) , 1-30 ng/ml interleukin-1 ⁇ (IL-1 ⁇ ) , 50-150 ng/ml interferon- ⁇ (IFN- ⁇ ) , and 1-30 ng/ml IL-21; and (2) culturing the cells obtained in step (1) in a second culture medium comprising 1-200 ng/ml IL-15, 1-100 ng/ml IFN- ⁇ , and 50-150 IU/ml IL-2.
  • IL-4 interleukin-4
  • IL-15 1-30 ng/ml interleukin-15
  • IL-1 ⁇ interleukin-1 ⁇
  • IFN- ⁇ interferon- ⁇
  • IL-21 1-30 ng/ml interferon- ⁇
  • the method of preparing ⁇ T cells may comprise: (1) culturing cells in the sample in a first culture medium comprising 50-150 ng/ml interleukin-4 (IL-4) , 1-30 ng/ml interleukin-15 (IL-15) , 1-30 ng/ml interleukin-1 ⁇ (IL-1 ⁇ ) , 50-150 ng/ml interferon- ⁇ (IFN- ⁇ ) , and 50-150 IU/ml IL-2; and (2) culturing the cells obtained in step (1) in a second culture medium comprising 1-200 ng/ml IL-15, 1-100 ng/ml IFN- ⁇ , and 50-150 IU/ml IL-2.
  • IL-4 interleukin-4
  • IL-15 1-30 ng/ml interleukin-15
  • IL-1 ⁇ interleukin-1 ⁇
  • IFN- ⁇ interferon- ⁇
  • IU/ml IL-2 50-150 IU/ml IL-2
  • the method of preparing ⁇ T cells may comprise: (1) culturing cells in the sample in a first culture medium comprising about 50 ng/ml interleukin-4 (IL-4) , about 5 ng/ml interleukin-15 (IL-15) , about 5 ng/ml interleukin-1 ⁇ (IL-1 ⁇ ) , about 50 ng/ml interferon- ⁇ (IFN- ⁇ ) , about 5 ng/ml IL-21, and about 50 IU/ml IL-2; and (2) culturing the cells obtained in step (1) in a second culture medium comprising about 50 ng/ml IL-15, about 25 ng/ml IFN- ⁇ , and about 25 IU/ml IL-2.
  • IL-4 interleukin-4
  • IL-15 interleukin-15
  • IFN- ⁇ interferon- ⁇
  • IFN- ⁇ interferon- ⁇
  • the method of preparing ⁇ T cells may comprise: (1) culturing cells in the sample in a first culture medium comprising about 50 ng/ml interleukin-4 (IL-4) , about 5 ng/ml interleukin-15 (IL-15) , about 5 ng/ml interleukin-1 ⁇ (IL-1 ⁇ ) , about 50 ng/ml interferon- ⁇ (IFN- ⁇ ) , and about 50 IU/ml IL-2; and (2) culturing the cells obtained in step (1) in a second culture medium comprising about 50 ng/ml IL-15, about 25 ng/ml IFN- ⁇ , and about 25 IU/ml IL-2.
  • IL-4 interleukin-4
  • IL-15 interleukin-15
  • IL-1 ⁇ interleukin-1 ⁇
  • IFN- ⁇ interferon- ⁇
  • IU/ml IL-2 interferon- ⁇
  • the method of preparing ⁇ T cells may comprise: (1) culturing cells in the sample in a first culture medium comprising about 120 ng/ml interleukin-4 (IL-4) , about 15 ng/ml interleukin-15 (IL-15) , about 15 ng/ml interleukin-1 ⁇ (IL-1 ⁇ ) , about 100 ng/ml interferon- ⁇ (IFN- ⁇ ) , about 15 ng/ml IL-21, and about 100 IU/ml IL-2; and (2) culturing the cells obtained in step (1) in a second culture medium comprising about 150 ng/ml IL-15, about 75 ng/ml IFN- ⁇ , and about 100 IU/ml IL-2.
  • IL-4 interleukin-4
  • IL-15 interleukin-15
  • IL-1 ⁇ interleukin-1 ⁇
  • IFN- ⁇ interferon- ⁇
  • the method of preparing ⁇ T cells may comprise: (1) culturing cells in the sample in a first culture medium comprising about 120 ng/ml interleukin-4 (IL-4) , about 15 ng/ml interleukin-15 (IL-15) , about 15 ng/ml interleukin-1 ⁇ (IL-1 ⁇ ) , about 100 ng/ml interferon- ⁇ (IFN- ⁇ ) , and about 100 IU/ml IL-2; and (2) culturing the cells obtained in step (1) in a second culture medium comprising about 150 ng/ml IL-15, about 75 ng/ml IFN- ⁇ , and about 100 IU/ml IL-2.
  • IL-4 interleukin-4
  • IL-15 interleukin-15
  • IL-1 ⁇ interleukin-1 ⁇
  • IFN- ⁇ interferon- ⁇
  • the method of preparing ⁇ T cells may comprise: (1) culturing cells in the sample in a first culture medium comprising about 75 ng/ml interleukin-4 (IL-4) , about 10 ng/ml interleukin-15 (IL-15) , about 10 ng/ml interleukin-1 ⁇ (IL-1 ⁇ ) , about 150 ng/ml interferon- ⁇ (IFN- ⁇ ) , about 10 ng/ml IL-21, and about 75 IU/ml IL-2; and (2) culturing the cells obtained in step (1) in a second culture medium comprising about 120 ng/ml IL-15, about 75 ng/ml IFN- ⁇ , and about 100 IU/ml IL-2.
  • IL-4 interleukin-4
  • IL-15 interleukin-15
  • IFN- ⁇ interferon- ⁇
  • the method of preparing ⁇ T cells may comprise: (1) culturing cells in the sample in a first culture medium comprising about 120 ng/ml interleukin-4 (IL-4) , about 10 ng/ml interleukin-15 (IL-15) , about 15 ng/ml interleukin-1 ⁇ (IL-1 ⁇ ) , about 100 ng/ml interferon- ⁇ (IFN- ⁇ ) , about 15 ng/ml IL-21, and about 120 IU/ml IL-2; and (2) culturing the cells obtained in step (1) in a second culture medium comprising about 150 ng/ml IL-15, about 75 ng/ml IFN- ⁇ , and about 100 IU/ml IL-2.
  • IL-4 interleukin-4
  • IL-15 interleukin-15
  • IFN- ⁇ interferon- ⁇
  • the method of preparing ⁇ T cells may comprise: (1) culturing cells in the sample in a first culture medium comprising about 120 ng/ml interleukin-4 (IL-4) , about 10 ng/ml interleukin-15 (IL-15) , about 15 ng/ml interleukin-1 ⁇ (IL-1 ⁇ ) , about 100 ng/ml interferon- ⁇ (IFN- ⁇ ) , and about 120 IU/ml IL-2; and (2) culturing the cells obtained in step (1) in a second culture medium comprising about 150 ng/ml IL-15, about 75 ng/ml IFN- ⁇ , and about 100 IU/ml IL-2.
  • IL-4 interleukin-4
  • IL-15 interleukin-15
  • IL-1 ⁇ interleukin-1 ⁇
  • IFN- ⁇ interferon- ⁇
  • the method of preparing ⁇ T cells may comprise: (1) culturing cells in the sample in a first culture medium comprising about 75 ng/ml interleukin-4 (IL-4) , about 15 ng/ml interleukin-15 (IL-15) , about 15 ng/ml interleukin-1 ⁇ (IL-1 ⁇ ) , about 100 ng/ml interferon- ⁇ (IFN- ⁇ , about 15 ng/ml IL-21, and about 120 IU/ml IL-2; and (2) culturing the cells obtained in step (1) in a second culture medium comprising about 150 ng/ml IL-15, about 75 ng/ml IFN- ⁇ , and about 100 IU/ml IL-2.
  • IL-4 interleukin-4
  • IL-15 interleukin-15
  • IL-1 ⁇ interleukin-1 ⁇
  • IFN- ⁇ interferon- ⁇
  • the method of preparing ⁇ T cells may comprise: (1) culturing cells in the sample in a first culture medium comprising about 110 ng/ml interleukin-4 (IL-4) , about 15 ng/ml interleukin-15 (IL-15) , or about 10 ng/ml interleukin-1 ⁇ (IL-1 ⁇ ) , about 120 ng/ml interferon- ⁇ (IFN- ⁇ ) , about 15 ng/ml IL-21, and about 100 IU/ml IL-2; and (2) culturing the cells obtained in step (1) in a second culture medium comprising about 150 ng/ml IL-15, about 75 ng/ml IFN- ⁇ , and about 100 IU/ml IL-2.
  • IL-4 interleukin-4
  • IL-15 interleukin-15
  • IFN- ⁇ interferon- ⁇
  • the method of preparing ⁇ T cells may comprise: (1) culturing cells in the sample in a first culture medium comprising about 110 ng/ml interleukin-4 (IL-4) , about 15 ng/ml interleukin-15 (IL-15) , or about 10 ng/ml interleukin-1 ⁇ (IL-1 ⁇ ) , about 120 ng/ml interferon- ⁇ (IFN- ⁇ ) , and about 100 IU/ml IL-2; and (2) culturing the cells obtained in step (1) in a second culture medium comprising about 150 ng/ml IL-15, about 75 ng/ml IFN- ⁇ , and about 100 IU/ml IL-2.
  • IL-4 interleukin-4
  • IL-15 interleukin-15
  • IL-1 ⁇ interleukin-1 ⁇
  • IFN- ⁇ interferon- ⁇
  • the method of preparing ⁇ T cells may comprise: (1) culturing cells in the sample in a first culture medium comprising about 75 ng/ml interleukin-4 (IL-4) , about 7 ng/ml interleukin-15 (IL-15) , about 7 ng/ml interleukin-1 ⁇ (IL-1 ⁇ ) , about 100 ng/ml interferon- ⁇ (IFN- ⁇ ) , about 15 ng/ml IL-21, and about 75 IU/ml IL-2; and (2) culturing the cells obtained in step (1) in a second culture medium comprising about 120 ng/ml IL-15, about 30 ng/ml IFN- ⁇ , and about 100 IU/ml IL-2.
  • IL-4 interleukin-4
  • IL-15 interleukin-15
  • IL-1 ⁇ interleukin-1 ⁇
  • IFN- ⁇ interferon- ⁇
  • the method of preparing ⁇ T cells may comprise: (1) culturing cells in the sample in a first culture medium comprising about 110 ng/ml interleukin-4 (IL-4) , about 7 ng/ml interleukin-15 (IL-15) , about 15 ng/ml interleukin-1 ⁇ (IL-1 ⁇ ) , about 75 ng/ml interferon- ⁇ (IFN- ⁇ ) , about 7 ng/ml IL-21, and about 100 IU/ml IL-2; and (2) culturing the cells obtained in step (1) in a second culture medium comprising about 120 ng/ml IL-15, about 75 ng/ml IFN- ⁇ , and about 100 IU/ml IL-2.
  • IL-4 interleukin-4
  • IL-15 interleukin-15
  • IFN- ⁇ interferon- ⁇
  • the method of preparing ⁇ T cells may comprise: (1) culturing cells in the sample in a first culture medium comprising about 110 ng/ml interleukin-4 (IL-4) , about 7 ng/ml interleukin-15 (IL-15) , about 15 ng/ml interleukin-1 ⁇ (IL-1 ⁇ ) , about 75 ng/ml interferon- ⁇ (IFN- ⁇ ) , and about 100 IU/ml IL-2; and (2) culturing the cells obtained in step (1) in a second culture medium comprising about 120 ng/ml IL-15, about 75 ng/ml IFN- ⁇ , and about 100 IU/ml IL-2.
  • IL-4 interleukin-4
  • IL-15 interleukin-15
  • IFN- ⁇ interferon- ⁇
  • the method of preparing ⁇ T cells may comprise: (1) culturing cells in the sample in a first culture medium comprising about 110 ng/ml interleukin-4 (IL-4) , about 7 ng/ml interleukin-15 (IL-15) , about 10 ng/ml interleukin-1 ⁇ (IL-1 ⁇ ) , about 75 ng/ml interferon- ⁇ (IFN- ⁇ ) , about 7 ng/ml IL-21, and about 100 IU/ml IL-2; and (2) culturing the cells obtained in step (1) in a second culture medium comprising about 150 ng/ml IL-15, about 75 ng/ml IFN- ⁇ , and about 75 IU/ml IL-2.
  • IL-4 interleukin-4
  • IL-15 interleukin-15
  • IFN- ⁇ interferon- ⁇
  • the method of preparing ⁇ T cells may comprise: (1) culturing cells in the sample in a first culture medium comprising about 110 ng/ml interleukin-4 (IL-4) , about 15 ng/ml interleukin-15 (IL-15) , about 7 ng/ml interleukin-1 ⁇ (IL-1 ⁇ ) , about 120 ng/ml interferon- ⁇ (IFN- ⁇ ) , about 15 ng/ml IL-21, and about 75 IU/ml IL-2; and (2) culturing the cells obtained in step (1) in a second culture medium comprising about 150 ng/ml IL-15, about 100 ng/ml IFN- ⁇ , and about 100 IU/ml IL-2.
  • IL-4 interleukin-4
  • IL-15 interleukin-15
  • IFN- ⁇ interferon- ⁇
  • the method of preparing ⁇ T cells may comprise: (1) culturing cells in the sample in a first culture medium comprising about 110 ng/ml interleukin-4 (IL-4) , about 15 ng/ml interleukin-15 (IL-15) , about 7 ng/ml interleukin-1 ⁇ (IL-1 ⁇ ) , about 120 ng/ml interferon- ⁇ (IFN- ⁇ ) , and about 75 IU/ml IL-2; and (2) culturing the cells obtained in step (1) in a second culture medium comprising about 150 ng/ml IL-15, about 100 ng/ml IFN- ⁇ , and about 100 IU/ml IL-2.
  • IL-4 interleukin-4
  • IL-15 interleukin-15
  • IFN- ⁇ interferon- ⁇
  • the method of preparing ⁇ T cells may comprise: (1) culturing cells in the sample in a first culture medium comprising about 110 ng/ml interleukin-4 (IL-4) , about 7 ng/ml interleukin-15 (IL-15) , about 15 ng/ml interleukin-1 ⁇ (IL-1 ⁇ ) , about 75 ng/ml interferon- ⁇ (IFN- ⁇ ) , about 15 ng/ml IL-21, and about 75 IU/ml IL-2; and (2) culturing the cells obtained in step (1) in a second culture medium comprising about 150 ng/ml IL-15, about 100 ng/ml IFN- ⁇ , and about 75 IU/ml IL-2.
  • IL-4 interleukin-4
  • IL-15 interleukin-15
  • IFN- ⁇ interferon- ⁇
  • the method of preparing ⁇ T cells may comprise: (1) culturing cells in the sample in a first culture medium comprising about 75 ng/ml interleukin-4 (IL-4) , about 15 ng/ml interleukin-7 (IL-15) , about 7 ng/ml interleukin-1 ⁇ (IL-1 ⁇ ) , about 100 ng/ml interferon- ⁇ (IFN- ⁇ ) , about 7 ng/ml IL-21, and about 100 IU/ml IL-2; and (2) culturing the cells obtained in step (1) in a second culture medium comprising about 120 ng/ml IL-15, or about 75 ng/ml IFN- ⁇ , and about 100 IU/ml IL-2.
  • IL-4 interleukin-4
  • IL-15 interleukin-7
  • IFN- ⁇ interferon- ⁇
  • the method of preparing ⁇ T cells may comprise: (1) culturing cells in the sample in a first culture medium comprising about 75 ng/ml interleukin-4 (IL-4) , about 15 ng/ml interleukin-7 (IL-15) , about 7 ng/ml interleukin-1 ⁇ (IL-1 ⁇ ) , about 100 ng/ml interferon- ⁇ (IFN- ⁇ ) , and about 100 IU/ml IL-2; and (2) culturing the cells obtained in step (1) in a second culture medium comprising about 120 ng/ml IL-15, or about 75 ng/ml IFN- ⁇ , and about 100 IU/ml IL-2.
  • IL-4 interleukin-4
  • IL-15 interleukin-7
  • IFN- ⁇ interferon- ⁇
  • IL-2 interferon- ⁇
  • the method of preparing ⁇ T cells may comprise: (1) culturing cells in the sample in a first culture medium comprising about 110 ng/ml interleukin-4 (IL-4) , about 7 ng/ml interleukin-15 (IL-15) , about 7 ng/ml interleukin-1 ⁇ (IL-1 ⁇ ) , about 100 ng/ml interferon- ⁇ (IFN- ⁇ ) , about 7 ng/ml IL-21, and about 100 IU/ml IL-2; and (2) culturing the cells obtained in step (1) in a second culture medium comprising about 150 ng/ml IL-15, about 50 ng/ml IFN- ⁇ , and about 100 IU/ml IL-2.
  • IL-4 interleukin-4
  • IL-15 interleukin-15
  • IL-1 ⁇ interleukin-1 ⁇
  • IFN- ⁇ interferon- ⁇
  • the method of preparing ⁇ T cells may comprise: (1) culturing cells in the sample in a first culture medium comprising about 75 ng/ml interleukin-4 (IL-4) , about 15 ng/ml interleukin-15 (IL-15) , about 15 ng/ml interleukin-1 ⁇ (IL-1 ⁇ ) , about 100 ng/ml interferon- ⁇ (IFN- ⁇ ) , about 10 ng/ml IL-21, and about 75 IU/ml IL-2; and (2) culturing the cells obtained in step (1) in a second culture medium comprising about 100 ng/ml IL-15, about 75 ng/ml IFN- ⁇ , and about 100 IU/ml IL-2.
  • IL-4 interleukin-4
  • IL-15 interleukin-15
  • IL-1 ⁇ interleukin-1 ⁇
  • IFN- ⁇ interferon- ⁇
  • the method of preparing ⁇ T cells may comprise: (1) culturing cells in the sample in a first culture medium comprising about 75 ng/ml interleukin-4 (IL-4) , about 15 ng/ml interleukin-15 (IL-15) , about 15 ng/ml interleukin-1 ⁇ (IL-1 ⁇ ) , about 100 ng/ml interferon- ⁇ (IFN- ⁇ ) , and about 75 IU/ml IL-2; and (2) culturing the cells obtained in step (1) in a second culture medium comprising about 100 ng/ml IL-15, about 75 ng/ml IFN- ⁇ , and about 100 IU/ml IL-2.
  • IL-4 interleukin-4
  • IL-15 interleukin-15
  • IL-1 ⁇ interleukin-1 ⁇
  • IFN- ⁇ interferon- ⁇
  • IL-2 interferon- ⁇
  • the method of preparing ⁇ T cells may comprise: (1) culturing cells in the sample in a first culture medium comprising about 110 ng/ml interleukin-4 (IL-4) , about 15 ng/ml interleukin-15 (IL-15) , about 15 ng/ml interleukin-1 ⁇ (IL-1 ⁇ ) , about 100 ng/ml interferon- ⁇ (IFN- ⁇ ) , about 15 ng/ml IL-21, and about 75 IU/ml IL-2; and (2) culturing the cells obtained in step (1) in a second culture medium comprising about 150 ng/ml IL-15, about 75 ng/ml IFN- ⁇ , about 100 IU/ml IL-2.
  • IL-4 interleukin-4
  • IL-15 interleukin-15
  • IL-1 ⁇ interleukin-1 ⁇
  • IFN- ⁇ interferon- ⁇
  • the method of preparing ⁇ T cells may comprise: (1) culturing cells in the sample in a first culture medium comprising about 75 ng/ml interleukin-4 (IL-4) , about 7 ng/ml interleukin-about 15 ng/ml interleukin-1 ⁇ (IL-1 ⁇ ) , about 100 ng/ml interferon- ⁇ (IFN- ⁇ ) , about 15 ng/ml IL-21, and about 75 IU/ml IL-2; and (2) culturing the cells obtained in step (1) in a second culture medium comprising about 150 ng/ml IL-15, about 100 ng/ml IFN- ⁇ , and about 100 IU/ml IL-2.
  • IL-4 interleukin-4
  • IFN- ⁇ interferon- ⁇
  • the method of preparing ⁇ T cells may comprise: (1) culturing cells in the sample in a first culture medium comprising about 75 ng/ml interleukin-4 (IL-4) , about 7 ng/ml interleukin-about 15 ng/ml interleukin-1 ⁇ (IL-1 ⁇ ) , about 100 ng/ml interferon- ⁇ (IFN- ⁇ ) , and about 75 IU/ml IL-2; and (2) culturing the cells obtained in step (1) in a second culture medium comprising about 150 ng/ml IL-15, about 100 ng/ml IFN- ⁇ , and about 100 IU/ml IL-2.
  • IL-4 interleukin-4
  • IFN- ⁇ interferon- ⁇
  • the method of preparing ⁇ T cells may comprise: (1) culturing cells in the sample in a first culture medium comprising about 110 ng/ml interleukin-4 (IL-4) , about 15 ng/ml interleukin-15 (IL-15) , about 15 ng/ml interleukin-1 ⁇ (IL-1 ⁇ ) , about 100 ng/ml interferon- ⁇ (IFN- ⁇ ) , about 15 ng/ml IL-21, and about 75 IU/ml IL-2; and (2) culturing the cells obtained in step (1) in a second culture medium comprising about 75 ng/ml IL-15, about 75 ng/ml IFN- ⁇ , and about 100 IU/ml IL-2.
  • IL-4 interleukin-4
  • IL-15 interleukin-15
  • IL-1 ⁇ interleukin-1 ⁇
  • IFN- ⁇ interferon- ⁇
  • the method of preparing ⁇ T cells may comprise: (1) culturing cells in the sample in a first culture medium comprising about 100 ng/ml interleukin-4 (IL-4) , about 15 ng/ml interleukin-15 (IL-15) , about 15 ng/ml interleukin-1 ⁇ (IL-1 ⁇ ) , about 100 ng/ml interferon- ⁇ (IFN- ⁇ ) , about 15 ng/ml IL-21, and about 100 IU/ml IL-2; and (2) culturing the cells obtained in step (1) in a second culture medium comprising about 100 ng/ml IL-15, about 75 ng/ml IFN- ⁇ , and about 100 IU/ml IL-2.
  • IL-4 interleukin-4
  • IL-15 interleukin-15
  • IFN- ⁇ interferon- ⁇
  • the method of preparing ⁇ T cells may comprise: (1) culturing cells in the sample in a first culture medium comprising about 100 ng/ml interleukin-4 (IL-4) , about 15 ng/ml interleukin-15 (IL-15) , about 15 ng/ml interleukin-1 ⁇ (IL-1 ⁇ ) , about 100 ng/ml interferon- ⁇ (IFN- ⁇ ) , and about 100 IU/ml IL-2; and (2) culturing the cells obtained in step (1) in a second culture medium comprising about 100 ng/ml IL-15, about 75 ng/ml IFN- ⁇ , and about 100 IU/ml IL-2.
  • IL-4 interleukin-4
  • IL-15 interleukin-15
  • IL-1 ⁇ interleukin-1 ⁇
  • IFN- ⁇ interferon- ⁇
  • the method of preparing ⁇ T cells may comprise: (1) culturing cells in the sample in a first culture medium comprising about 100 ng/ml interleukin-4 (IL-4) , about 7 ng/ml interleukin-15 (IL-15) , about 7 ng/ml interleukin-1 ⁇ (IL-1 ⁇ ) , about 100 ng/ml interferon- ⁇ (IFN- ⁇ ) , about 7 ng/ml IL-21, and about 100 IU/ml IL-2; and (2) culturing the cells obtained in step (1) in a second culture medium comprising about 75 ng/ml IL-15, about 75 ng/ml IFN- ⁇ , and about 100 IU/ml IL-2.
  • IL-4 interleukin-4
  • IL-15 interleukin-15
  • IL-1 ⁇ interleukin-1 ⁇
  • IFN- ⁇ interferon- ⁇
  • the method of preparing ⁇ T cells may comprise: (1) culturing cells in the sample in a first culture medium comprising about 100 ng/ml interleukin-4 (IL-4) , about 10 ng/ml interleukin-15 (IL-15) , about 15 ng/ml interleukin-1 ⁇ (IL-1 ⁇ ) , about 70 ng/ml interferon- ⁇ (IFN- ⁇ ) , and about 50 IU/ml IL-2; and (2) culturing the cells obtained in step (1) in a second culture medium comprising about 70 ng/ml IL-15, about 30 ng/ml IFN- ⁇ , and about 50 IU/ml IL-2.
  • IL-4 interleukin-4
  • IL-15 interleukin-15
  • IFN- ⁇ interferon- ⁇
  • IL-2 interferon- ⁇
  • the method of preparing ⁇ T cells may comprise: (1) culturing cells in the sample in a first culture medium comprising about 100 ng/ml interleukin-4 (IL-4) , about 10 ng/ml interleukin-15 (IL-15) , about 15 ng/ml interleukin-1 ⁇ (IL-1 ⁇ ) , about 70 ng/ml interferon- ⁇ (IFN- ⁇ ) , about 7 ng/ml IL-21, and about 50 IU/ml IL-2; and (2) culturing the cells obtained in step (1) in a second culture medium comprising about 70 ng/ml IL-15, about 30 ng/ml IFN- ⁇ , and about 50 IU/ml IL-2.
  • IL-4 interleukin-4
  • IL-15 interleukin-15
  • IFN- ⁇ interferon- ⁇
  • the method of preparing ⁇ T cells may comprise: (1) culturing cells in the sample in a first culture medium comprising about 100 ng/ml interleukin-4 (IL-4) , about 7 ng/ml interleukin-15 (IL-15) , about 7 ng/ml interleukin-1 ⁇ (IL-1 ⁇ ) , about 75 ng/ml interferon- ⁇ (IFN- ⁇ ) , about 15 ng/ml IL-21, and about 100 IU/ml IL-2; and (2) culturing the cells obtained in step (1) in a second culture medium comprising about 75 ng/ml IL-15, about 75 ng/ml IFN- ⁇ , and about 100 IU/ml IL-2.
  • IL-4 interleukin-4
  • IL-15 interleukin-15
  • IFN- ⁇ interferon- ⁇
  • the method of preparing ⁇ T cells may comprise: (1) culturing cells in the sample in a first culture medium comprising about 100 ng/ml interleukin-4 (IL-4) , about 7 ng/ml interleukin-15 (IL-15) , about 15 ng/ml interleukin-1 ⁇ (IL-1 ⁇ ) , about 70 ng/ml interferon- ⁇ (IFN- ⁇ ) , about 7 ng/ml IL-21, and about 75 IU/ml IL-2; and (2) culturing the cells obtained in step (1) in a second culture medium comprising about 70 ng/ml IL-15, about 75 ng/ml IFN- ⁇ , and about 100 IU/ml IL-2.
  • IL-4 interleukin-4
  • IL-15 interleukin-15
  • IFN- ⁇ interferon- ⁇
  • the method of preparing ⁇ T cells may comprise: (1) culturing cells in the sample in a first culture medium comprising about 75 ng/ml interleukin-4 (IL-4) , about 7 ng/ml interleukin-15 (IL-15) , about 7 ng/ml interleukin-1 ⁇ (IL-1 ⁇ ) , about 70 ng/ml interferon- ⁇ (IFN- ⁇ ) , about 7 ng/ml IL-21, and about 50 IU/ml IL-2; and (2) culturing the cells obtained in step (1) in a second culture medium comprising about 70 ng/ml IL-15, about 30 ng/ml IFN- ⁇ , and about 50 IU/ml IL-2.
  • IL-4 interleukin-4
  • IL-15 interleukin-15
  • IFN- ⁇ interferon- ⁇
  • IFN- ⁇ interferon- ⁇
  • the method of preparing ⁇ T cells may comprise: (1) culturing cells in the sample in a first culture medium comprising about 75 ng/ml interleukin-4 (IL-4) , about 7 ng/ml interleukin-15 (IL-15) , about 7 ng/ml interleukin-1 ⁇ (IL-1 ⁇ ) , about 70 ng/ml interferon- ⁇ (IFN- ⁇ ) , and about 50 IU/ml IL-2; and (2) culturing the cells obtained in step (1) in a second culture medium comprising about 70 ng/ml IL-15, about 30 ng/ml IFN- ⁇ , and about 50 IU/ml IL-2.
  • IL-4 interleukin-4
  • IL-15 interleukin-15
  • IL-1 ⁇ interleukin-1 ⁇
  • IFN- ⁇ interferon- ⁇
  • IL-2 interferon- ⁇
  • the method of preparing ⁇ T cells may comprise: (1) culturing cells in the sample in a first culture medium comprising about 75 ng/ml interleukin-4 (IL-4) , about 10 ng/ml interleukin-15 (IL-15) , about 15 ng/ml interleukin-1 ⁇ (IL-1 ⁇ ) , about 100 ng/ml interferon- ⁇ (IFN- ⁇ ) , about 7 ng/ml IL-21, and about 50 IU/ml IL-2; and (2) culturing the cells obtained in step (1) in a second culture medium comprising about 70 ng/ml IL-15, about 30 ng/ml IFN- ⁇ , and about 75 IU/ml IL-2.
  • IL-4 interleukin-4
  • IL-15 interleukin-15
  • IFN- ⁇ interferon- ⁇
  • the method of preparing ⁇ T cells may comprise: (1) culturing cells in the sample in a first culture medium comprising about 100 ng/ml interleukin-4 (IL-4) , about 7 ng/ml interleukin-15 (IL-15) , about 7 ng/ml interleukin-1 ⁇ (IL-1 ⁇ ) , about 70 ng/ml interferon- ⁇ (IFN- ⁇ ) , about 7 ng/ml IL-21, and about 50 IU/ml IL-2; and (2) culturing the cells obtained in step (1) in a second culture medium comprising about 70 ng/ml IL-15, about 30 ng/ml IFN- ⁇ , and about 50 IU/ml IL-2.
  • IL-4 interleukin-4
  • IL-15 interleukin-15
  • IFN- ⁇ interferon- ⁇
  • IFN- ⁇ interferon- ⁇
  • the method of preparing ⁇ T cells may comprise: (1) culturing cells in the sample in a first culture medium comprising about 75 ng/ml interleukin-4 (IL-4) , about 7 ng/ml interleukin-15 (IL-15) , about 15 ng/ml interleukin-1 ⁇ (IL-1 ⁇ ) , about 70 ng/ml interferon- ⁇ (IFN- ⁇ ) , about 7 ng/ml IL-21, and about 50 IU/ml IL-2; and (2) culturing the cells obtained in step (1) in a second culture medium comprising about 70 ng/ml IL-15, about 50 ng/ml IFN- ⁇ , and about 75 IU/ml IL-2.
  • IL-4 interleukin-4
  • IL-15 interleukin-15
  • IFN- ⁇ interferon- ⁇
  • the method of preparing ⁇ T cells may comprise: (1) culturing cells in the sample in a first culture medium comprising about 75 ng/ml interleukin-4 (IL-4) , about 7 ng/ml interleukin-15 (IL-15) , about 15 ng/ml interleukin-1 ⁇ (IL-1 ⁇ ) , about 70 ng/ml interferon- ⁇ (IFN- ⁇ ) , and about 50 IU/ml IL-2; and (2) culturing the cells obtained in step (1) in a second culture medium comprising about 70 ng/ml IL-15, about 50 ng/ml IFN- ⁇ , and about 75 IU/ml IL-2.
  • IL-4 interleukin-4
  • IL-15 interleukin-15
  • IFN- ⁇ interferon- ⁇
  • IL-2 interferon- ⁇
  • the method of preparing ⁇ T cells may comprise: (1) culturing cells in the sample in a first culture medium comprising about 75 ng/ml interleukin-4 (IL-4) , about 10 ng/ml interleukin-15 (IL-15) , about 15 ng/ml interleukin-1 ⁇ (IL-1 ⁇ ) , about 100 ng/ml interferon- ⁇ (IFN- ⁇ ) , about 7 ng/ml IL-21, and about 50 IU/ml IL-2; and (2) culturing the cells obtained in step (1) in a second culture medium comprising about 70 ng/ml IL-15, about 30 ng/ml IFN- ⁇ , and about 100 IU/ml IL-2.
  • IL-4 interleukin-4
  • IL-15 interleukin-15
  • IFN- ⁇ interferon- ⁇
  • the method of preparing ⁇ T cells may comprise: (1) culturing cells in the sample in a first culture medium comprising about 100 ng/ml interleukin-4 (IL-4) , about 10 ng/ml interleukin-15 (IL-15) , about 15 ng/ml interleukin-1 ⁇ (IL-1 ⁇ ) , about 100 ng/ml interferon- ⁇ (IFN- ⁇ ) , about 7 ng/ml IL-21, and about 75 IU/ml IL-2; and (2) culturing the cells obtained in step (1) in a second culture medium comprising about 70 ng/ml IL-15, about 50 ng/ml IFN- ⁇ , and about 50 IU/ml IL-2.
  • IL-4 interleukin-4
  • IL-15 interleukin-15
  • IFN- ⁇ interferon- ⁇
  • the method of preparing ⁇ T cells may comprise: (1) culturing cells in the sample in a first culture medium comprising about 100 ng/ml interleukin-4 (IL-4) , about 10 ng/ml interleukin-15 (IL-15) , about 15 ng/ml interleukin-1 ⁇ (IL-1 ⁇ ) , about 100 ng/ml interferon- ⁇ (IFN- ⁇ ) , and about 75 IU/ml IL-2; and (2) culturing the cells obtained in step (1) in a second culture medium comprising about 70 ng/ml IL-15, about 50 ng/ml IFN- ⁇ , and about 50 IU/ml IL-2.
  • IL-4 interleukin-4
  • IL-15 interleukin-15
  • IL-1 ⁇ interleukin-1 ⁇
  • IFN- ⁇ interferon- ⁇
  • the method of preparing ⁇ T cells may comprise: (1) culturing cells in the sample in a first culture medium comprising about 100 ng/ml interleukin-4 (IL-4) , about 7 ng/ml interleukin-15 (IL-15) , about 15 ng/ml interleukin-1 ⁇ (IL-1 ⁇ ) , about 70 ng/ml interferon- ⁇ (IFN- ⁇ ) , about 7 ng/ml IL-21, and about 50 IU/ml IL-2; and (2) culturing the cells obtained in step (1) in a second culture medium comprising about 100 ng/ml IL-15, about 30 ng/ml IFN- ⁇ , and about 50 IU/ml IL-2.
  • IL-4 interleukin-4
  • IL-15 interleukin-15
  • IFN- ⁇ interferon- ⁇
  • the method of preparing ⁇ T cells may comprise: (1) culturing cells in the sample in a first culture medium comprising about 100 ng/ml interleukin-4 (IL-4) , about 7 ng/ml interleukin-15 (IL-15) , about 15 ng/ml interleukin-1 ⁇ (IL-1 ⁇ ) , about 70 ng/ml interferon- ⁇ (IFN- ⁇ ) , and about 50 IU/ml IL-2; and (2) culturing the cells obtained in step (1) in a second culture medium comprising about 100 ng/ml IL-15, about 30 ng/ml IFN- ⁇ , and about 50 IU/ml IL-2.
  • IL-4 interleukin-4
  • IL-15 interleukin-15
  • IFN- ⁇ interferon- ⁇
  • the method of preparing ⁇ T cells may comprise: (1) culturing cells in the sample in a first culture medium comprising about 75 ng/ml interleukin-4 (IL-4) , about 7 ng/ml interleukin-15 (IL-15) , about 15 ng/ml interleukin-1 ⁇ (IL-1 ⁇ ) , about 70 ng/ml interferon- ⁇ (IFN- ⁇ ) , about 7 ng/ml IL-21, and about 75 IU/ml IL-2; and (2) culturing the cells obtained in step (1) in a second culture medium comprising about 70 ng/ml IL-15, about 30 ng/ml IFN- ⁇ , and about 75 IU/ml IL-2.
  • IL-4 interleukin-4
  • IL-15 interleukin-15
  • IFN- ⁇ interferon- ⁇
  • the method of preparing ⁇ T cells may comprise: (1) culturing cells in the sample in a first culture medium comprising about 100 ng/ml interleukin-4 (IL-4) , about 7 ng/ml interleukin-15 (IL-15) , about 7 ng/ml interleukin-1 ⁇ (IL-1 ⁇ ) , about 70 ng/ml interferon- ⁇ (IFN- ⁇ ) , about 10 ng/ml IL-21, and about 50 IU/ml IL-2; and (2) culturing the cells obtained in step (1) in a second culture medium comprising about 70 ng/ml IL-15, about 30 ng/ml IFN- ⁇ , and about 50 IU/ml IL-2.
  • IL-4 interleukin-4
  • IL-15 interleukin-15
  • IFN- ⁇ interferon- ⁇
  • the method of preparing ⁇ T cells may comprise: (1) culturing cells in the sample in a first culture medium comprising about 100 ng/ml interleukin-4 (IL-4) , about 7 ng/ml interleukin-15 (IL-15) , about 7 ng/ml interleukin-1 ⁇ (IL-1 ⁇ ) , about 70 ng/ml interferon- ⁇ (IFN- ⁇ ) , and about 50 IU/ml IL-2; and (2) culturing the cells obtained in step (1) in a second culture medium comprising about 70 ng/ml IL-15, about 30 ng/ml IFN- ⁇ , and about 50 IU/ml IL-2.
  • IL-4 interleukin-4
  • IL-15 interleukin-15
  • IL-1 ⁇ interleukin-1 ⁇
  • IFN- ⁇ interferon- ⁇
  • IL-2 interferon- ⁇
  • the method of preparing ⁇ T cells may comprise: (1) culturing cells in the sample in a first culture medium comprising about 120 ng/ml interleukin-4 (IL-4) , about 7 ng/ml interleukin-15 (IL-15) , about 15 ng/ml interleukin-1 ⁇ (IL-1 ⁇ ) , about 70 ng/ml interferon- ⁇ (IFN- ⁇ ) , about 7 ng/ml IL-21, and about 50 IU/ml IL-2; and (2) culturing the cells obtained in step (1) in a second culture medium comprising about 100 ng/ml IL-15, about 50 ng/ml IFN- ⁇ , and about 100 IU/ml IL-2.
  • IL-4 interleukin-4
  • IL-15 interleukin-15
  • IFN- ⁇ interferon- ⁇
  • the method of preparing ⁇ T cells may comprise: (1) culturing cells in the sample in a first culture medium comprising about 75 ng/ml interleukin-4 (IL-4) , about 10 ng/ml interleukin-15 (IL-15) , about 15 ng/ml interleukin-1 ⁇ (IL-1 ⁇ ) , about 100 ng/ml interferon- ⁇ (IFN- ⁇ ) , about 10 ng/ml IL-21, and about 50 IU/ml IL-2; and (2) culturing the cells obtained in step (1) in a second culture medium comprising about 70 ng/ml IL-15, about 50 ng/ml IFN- ⁇ , and about 50 IU/ml IL-2.
  • IL-4 interleukin-4
  • IL-15 interleukin-15
  • IFN- ⁇ interferon- ⁇
  • the method of preparing ⁇ T cells may comprise: (1) culturing cells in the sample in a first culture medium comprising about 75 ng/ml interleukin-4 (IL-4) , about 10 ng/ml interleukin-15 (IL-15) , about 15 ng/ml interleukin-1 ⁇ (IL-1 ⁇ ) , about 100 ng/ml interferon- ⁇ (IFN- ⁇ ) , and about 50 IU/ml IL-2; and (2) culturing the cells obtained in step (1) in a second culture medium comprising about 70 ng/ml IL-15, about 50 ng/ml IFN- ⁇ , and about 50 IU/ml IL-2.
  • IL-4 interleukin-4
  • IL-15 interleukin-15
  • IL-1 ⁇ interleukin-1 ⁇
  • IFN- ⁇ interferon- ⁇
  • the method of preparing ⁇ T cells may comprise: (1) culturing cells in the sample in a first culture medium comprising about 120 ng/ml interleukin-4 (IL-4) , about 10 ng/ml interleukin-15 (IL-15) , about 15 ng/ml interleukin-1 ⁇ (IL-1 ⁇ ) , about 100 ng/ml interferon- ⁇ (IFN- ⁇ ) , about 7 ng/ml IL-21, and about 50 IU/ml IL-2; and (2) culturing the cells obtained in step (1) in a second culture medium comprising about 70 ng/ml IL-15, about 50 ng/ml IFN- ⁇ , and about 50 IU/ml IL-2.
  • IL-4 interleukin-4
  • IL-15 interleukin-15
  • IFN- ⁇ interferon- ⁇
  • the method of preparing ⁇ T cells may comprise: (1) culturing cells in the sample in a first culture medium comprising about 120 ng/ml interleukin-4 (IL-4) , about 10 ng/ml interleukin-15 (IL-15) , about 15 ng/ml interleukin-1 ⁇ (IL-1 ⁇ ) , about 100 ng/ml interferon- ⁇ (IFN- ⁇ ) , and about 50 IU/ml IL-2; and (2) culturing the cells obtained in step (1) in a second culture medium comprising about 70 ng/ml IL-15, about 50 ng/ml IFN- ⁇ , and about 50 IU/ml IL-2.
  • IL-4 interleukin-4
  • IL-15 interleukin-15
  • IL-1 ⁇ interleukin-1 ⁇
  • IFN- ⁇ interferon- ⁇
  • the method of preparing ⁇ T cells may comprise: (1) culturing cells in the sample in a first culture medium comprising about 75 ng/ml interleukin-4 (IL-4) , about 10 ng/ml interleukin-7 (IL-15) , about 15 ng/ml interleukin-1 ⁇ (IL-1 ⁇ ) , about 100 ng/ml interferon- ⁇ (IFN- ⁇ ) , about 7 ng/ml IL-21, and about 50 IU/ml IL-2; and (2) culturing the cells obtained in step (1) in a second culture medium comprising about 75 ng/ml IL-15, about 50 ng/ml IFN- ⁇ , and about 50 IU/ml IL-2.
  • IL-4 interleukin-4
  • IL-15 interleukin-7
  • IFN- ⁇ interferon- ⁇
  • the method of preparing ⁇ T cells may comprise: (1) culturing cells in the sample in a first culture medium comprising about 120 ng/ml interleukin-4 (IL-4) , about 7 ng/ml interleukin-15 (IL-15) , about 7 ng/ml interleukin-1 ⁇ (IL-1 ⁇ ) , about 100 ng/ml interferon- ⁇ (IFN- ⁇ ) , about 7 ng/ml IL-21, and about 50 IU/ml IL-2; and (2) culturing the cells obtained in step (1) in a second culture medium comprising about 70 ng/ml IL-15, about 50 ng/ml IFN- ⁇ , and about 50 IU/ml IL-2.
  • IL-4 interleukin-4
  • IL-15 interleukin-15
  • IL-1 ⁇ interleukin-1 ⁇
  • IFN- ⁇ interferon- ⁇
  • the method of preparing ⁇ T cells may comprise: (1) culturing cells in the sample in a first culture medium comprising about 120 ng/ml interleukin-4 (IL-4) , about 7 ng/ml interleukin-15 (IL-15) , about 7 ng/ml interleukin-1 ⁇ (IL-1 ⁇ ) , about 100 ng/ml interferon- ⁇ (IFN- ⁇ ) , and about 50 IU/ml IL-2; and (2) culturing the cells obtained in step (1) in a second culture medium comprising about 70 ng/ml IL-15, about 50 ng/ml IFN- ⁇ , and about 50 IU/ml IL-2.
  • IL-4 interleukin-4
  • IL-15 interleukin-15
  • IL-1 ⁇ interleukin-1 ⁇
  • IFN- ⁇ interferon- ⁇
  • IL-2 interferon- ⁇
  • the method of preparing ⁇ T cells may comprise: (1) culturing cells in the sample in a first culture medium comprising about 100 ng/ml interleukin-4 (IL-4) , about 7 ng/ml interleukin-15 (IL-15) , about 7 ng/ml interleukin-1 ⁇ (IL-1 ⁇ ) , about 70 ng/ml interferon- ⁇ (IFN- ⁇ ) , about 7 ng/ml IL-21, and about 50 IU/ml IL-2; and (2) culturing the cells obtained in step (1) in a second culture medium comprising about 100 ng/ml IL-15, about 30 ng/ml IFN- ⁇ , and about 75 IU/ml IL-2.
  • IL-4 interleukin-4
  • IL-15 interleukin-15
  • IL-1 ⁇ interleukin-1 ⁇
  • IFN- ⁇ interferon- ⁇
  • the method of preparing ⁇ T cells may comprise: (1) culturing cells in the sample in a first culture medium comprising about 100 ng/ml interleukin-4 (IL-4) , about 7 ng/ml interleukin-15 (IL-15) , about 7 ng/ml interleukin-1 ⁇ (IL-1 ⁇ ) , about 70 ng/ml interferon- ⁇ (IFN- ⁇ ) , and about 50 IU/ml IL-2; and (2) culturing the cells obtained in step (1) in a second culture medium comprising about 100 ng/ml IL-15, about 30 ng/ml IFN- ⁇ , and about 75 IU/ml IL-2.
  • IL-4 interleukin-4
  • IL-15 interleukin-15
  • IL-1 ⁇ interleukin-1 ⁇
  • IFN- ⁇ interferon- ⁇
  • the method of preparing ⁇ T cells may comprise: (1) culturing cells in the sample in a first culture medium comprising about 75 ng/ml interleukin-4 (IL-4) , about 7 ng/ml interleukin-15 (IL-15) , about 7 ng/ml interleukin-1 ⁇ (IL-1 ⁇ ) , about 100 ng/ml interferon- ⁇ (IFN- ⁇ ) , about 7 ng/ml IL-21, and about 50 IU/ml IL-2; and (2) culturing the cells obtained in step (1) in a second culture medium comprising about 70 ng/ml IL-15, about 50 ng/ml IFN- ⁇ , and about 75 IU/ml IL-2.
  • IL-4 interleukin-4
  • IL-15 interleukin-15
  • IL-1 ⁇ interleukin-1 ⁇
  • IFN- ⁇ interferon- ⁇
  • the method of preparing ⁇ T cells may comprise: (1) culturing cells in the sample in a first culture medium comprising about 75 ng/ml interleukin-4 (IL-4) , about 7 ng/ml interleukin-15 (IL-15) , about 15 ng/ml interleukin-1 ⁇ (IL-1 ⁇ ) , about 70 ng/ml interferon- ⁇ (IFN- ⁇ ) , about 7 ng/ml IL-21, and about 50 IU/ml IL-2; and (2) culturing the cells obtained in step (1) in a second culture medium comprising about 70 ng/ml IL-15, about 50 ng/ml IFN- ⁇ , and about 100 IU/ml IL-2.
  • IL-4 interleukin-4
  • IL-15 interleukin-15
  • IFN- ⁇ interferon- ⁇
  • the method of preparing ⁇ T cells may comprise: (1) culturing cells in the sample in a first culture medium comprising about 75 ng/ml interleukin-4 (IL-4) , about 7 ng/ml interleukin-15 (IL-15) , about 15 ng/ml interleukin-1 ⁇ (IL-1 ⁇ ) , about 70 ng/ml interferon- ⁇ (IFN- ⁇ ) , and about 50 IU/ml IL-2; and (2) culturing the cells obtained in step (1) in a second culture medium comprising about 70 ng/ml IL-15, about 50 ng/ml IFN- ⁇ , and about 100 IU/ml IL-2.
  • IL-4 interleukin-4
  • IL-15 interleukin-15
  • IFN- ⁇ interferon- ⁇
  • IL-2 interferon- ⁇
  • the method of preparing ⁇ T cells may comprise: (1) culturing cells in the sample in a first culture medium comprising about 75 ng/ml interleukin-4 (IL-4) , about 7 ng/ml interleukin-15 (IL-15) , about 7 ng/ml interleukin-1 ⁇ (IL-1 ⁇ ) , about 70 ng/ml interferon- ⁇ (IFN- ⁇ ) , about 7 ng/ml IL-21, and about 50 IU/ml IL-2; and (2) culturing the cells obtained in step (1) in a second culture medium comprising about 70 ng/ml IL-15, about 50 ng/ml IFN- ⁇ , and about 100 IU/ml IL-2.
  • IL-4 interleukin-4
  • IL-15 interleukin-15
  • IFN- ⁇ interferon- ⁇
  • IFN- ⁇ interferon- ⁇
  • the first culture medium and/or the second culture medium may further comprise 0.1-10 ⁇ g/ml AKT pathway inhibitor.
  • the first culture medium and/or the second culture medium may further comprise 1%-50%by volume HPL.
  • the sample Prior to step (1) , the sample may be depleted of ⁇ T cells and/or NK cells.
  • the cells may be exposed to a ⁇ TCR-specific antibody.
  • the cells Prior to step (2) , the cells may be transfected with a vector encoding an engineered receptor (e.g., CAR) .
  • an engineered receptor e.g., CAR
  • the cells may be cultured for about 3-9 days during step (1) .
  • the cells may be cultured for about 6-12 days during step (2) .
  • ⁇ T cells may be depleted at various stages of the method described herein. ⁇ T cells may be depleted prior to the first culture step. ⁇ T cells may be depleted prior to the second culture step. ⁇ T cells may be depleted after the second culture step. ⁇ T cells may be depleted by various means. ⁇ T cells may be depleted by using antibodies recognizing various ⁇ T cell surface markers (e.g., ⁇ TCR) . ⁇ T cells may be separated using techniques known in the art including fluorescence activated cell sorting, immunomagnetic separation, affinity column chromatography, density gradient centrifugation and cellular panning. ⁇ T cells in the sample may be depleted using antibodies targeting ⁇ T cells.
  • the antibodies targeting NK cells may specifically bind to ⁇ TCR.
  • the antibodies targeting ⁇ T cells may be linked to magnetic beads.
  • ⁇ T cells in the sample may be depleted using a TCR ⁇ cells depletion kit (e.g., Miltenyi Biotec, 170-070-416) .
  • Natural killer (NK) cells may be depleted at various stages of the method described herein. NK cells may be depleted prior to the first culture step. NK cells may be depleted prior to the second culture step. NK cells may be depleted after the second culture step. NK cells may be depleted by various means. NK cells may be depleted by using antibodies recognizing various NK cell surface markers (e.g., CD56) . NK cells may be separated using techniques known in the art including fluorescence activated cell sorting, immunomagnetic separation, affinity column chromatography, density gradient centrifugation and cellular panning. NK cells in the sample may be depleted using antibodies targeting NK cells. The antibodies targeting NK cells may specifically bind to CD56.
  • NK cell surface markers e.g., CD56
  • NK cells in the sample may be depleted using a CD56+ cells depletion kit (e.g., Miltenyi, 130-050-401) .
  • a CD56+ cells depletion kit e.g., Miltenyi, 130-050-401
  • Both ⁇ T cells and NK cells in the sample may be depleted using a TCR ⁇ cell depletion kit (e.g., Miltenyi Biotec, 170-070-416) and a CD56+ cells depletion kit (e.g., Miltenyi, 130-050-401) .
  • ⁇ T cells may be enriched by various means. ⁇ T cells may be directly enriched from a sample, for example, by sorting ⁇ T cells that express one or more cell surface markers with flow cytometry techniques. ⁇ T cells may be directly enriched by using antibodies recognizing various ⁇ T cell surface markers (e.g., ⁇ TCR) . Wild-type ⁇ T cells exhibit numerous antigen recognition, antigen-presentation, co-stimulation, and adhesion molecules that can be associated with a ⁇ T cells. One or more cell surface markers such as specific ⁇ TCRs, antigen recognition, antigen-presentation, ligands, adhesion molecules, or co-stimulatory molecules may be used to isolate a wild-type ⁇ T cell from a sample.
  • ⁇ TCRs antigen recognition, antigen-presentation, ligands, adhesion molecules, or co-stimulatory molecules
  • ⁇ T cells may be used to isolate a ⁇ T cell from a sample.
  • ⁇ T cells may be separated using techniques known in the art including fluorescence activated cell sorting, immunomagnetic separation, affinity column chromatography, density gradient centrifugation and cellular panning.
  • Peripheral blood mononuclear cells can be collected from a subject, for example, with an apheresis machine, including the Ficoll-Paque TM PLUS (GE Healthcare) system, or another suitable device/system.
  • ⁇ T cells or a desired subpopulation of ⁇ T cells, can be purified from the collected sample with, for example, with flow cytometry techniques.
  • Cord blood cells can also be obtained from cord blood during the birth of a subject.
  • Positive and/or negative selection of cell surface markers expressed on the collected ⁇ T cells can be used to directly isolate a population of ⁇ T cells expressing similar cell surface markers from a peripheral blood sample, a cord blood sample, a tumor, a tumor biopsy, a tissue, a lymph, or from an epithelial sample of a subject.
  • ⁇ T cells can be isolated from a complex sample based on positive or negative expression of CD2, CD3, CD4, CD8, CD24, CD25, CD44, Kit, TCR ⁇ , TCR ⁇ , TCR ⁇ , TCR ⁇ , NKG2D, CD70, CD27, CD30, CD 16, CD337 (NKp30) , CD336 (NKp46) , OX40, CD46, CCR7, and other suitable cell surface markers.
  • ⁇ T cells can be isolated from a complex sample that is cultured in vitro.
  • Whole PBMC population without prior depletion of specific cell populations such as monocytes, ⁇ T cells, B-cells, and NK cells, can be activated and expanded.
  • Enriched ⁇ T cell populations can be generated prior to their specific activation and expansion.
  • Activation and expansion of ⁇ T cells may be performed without the presence of native or engineered APCs.
  • Isolation and expansion of ⁇ T cells from tumor specimens may be performed using immobilized ⁇ T cell mitogens, including antibodies specific to ⁇ TCR, and other ⁇ TCR activating agents, including lectins.
  • the present disclosure provides a cell preparation prepared according to the method described herein.
  • the ⁇ T cell preparation may have a purity that is greater than 80%.
  • the purity may be measured by the percentage of CD3 positive cells (e.g., according to the methods described in Example 8) .
  • the resulting ⁇ T cell preparation may have a purity that is greater than 80%, optionally greater than 90%, and optionally greater than 95%.
  • the ⁇ T cell preparation may have a purity that is greater than 5%, greater than 10%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99%.
  • the ⁇ T cell preparation may have a purity that is less than 5%, less than 10%, less than 20%, less than 30%, less than 40%, less than 50%, less than 60%, less than 70%, less than 75%, less than 80%, less than 85%, less than 90%, less than 91%, less than 92%, less than 93%, less than 94%, less than 95%, less than 96%, less than 97%, less than 98%, or less than 99%.
  • the ⁇ T cell preparation may have a purity that is about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%
  • the ⁇ T cells can be transfected with a vector encoding an engineered receptor (e.g., CAR or TCR) .
  • the ⁇ T cells may be transfected with a vector encoding an engineered receptor (e.g., CAR or TCR) before the first culturing step.
  • the ⁇ T cells may be transfected with a vector encoding an engineered receptor (e.g., CAR or TCR) after the first culturing step.
  • the ⁇ T cells may be transfected with a vector encoding an engineered receptor (e.g., CAR or TCR) after the second culturing step.
  • Cell number can be measured by cellometer (e.g., following the methods described in Example 4) .
  • the ⁇ T cells e.g., un-transfected or transfected with a CAR expression vector
  • the ⁇ T cells may proliferate during the preparation process, resulting in a greater than 10 fold, greater than 100 fold, greater than 200 fold, greater than 300 fold, greater than 400 fold, greater than 500 fold, greater than 1,000 fold, greater than 2,000 fold, greater than 5,000 fold, greater than 10,000 fold, greater than 20,000 fold, greater than 50,000 fold, greater than 100,000 fold, greater than 200,000 fold, greater than 500,000 fold, or greater than 1,000,000 fold expansion.
  • the ⁇ T cells may proliferate during the preparation process, resulting in a less than 10 fold, less than 100 fold, less than 200 fold, less than 300 fold, less than 400 fold, less than 500 fold, less than 1,000 fold, less than 2,000 fold, less than 5,000 fold, less than 10,000 fold, less than 20,000 fold, less than 50,000 fold, less than 100,000 fold, less than 200,000 fold, less than 500,000 fold, or less than 1,000,000 fold expansion.
  • the ⁇ T cells may have a high cell viability.
  • the ⁇ T cells may have a viability of more than 5%, more than 10%, more than 15%, more than 20%, more than 25%, more than 30%, more than 35%, more than 40%, more than 45%, more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, or more than 95%.
  • the ⁇ T cells may have a viability of less than 5%, less than 10%, less than 15%, less than 20%, less than 25%, less than 30%, less than 35%, less than 40%, less than 45%, less than 50%, less than 55%, less than 60%, less than 65%, less than 70%, less than 75%, less than 80%, less than 85%, less than 90%, or less than 95%.
  • the expanded cell culture (e.g., transfected with a CAR expression vector) may comprise an amount of engineered ⁇ T cells (e.g., CAR-T cells) , wherein the engineered ⁇ T cells are engineered to express an antigen recognition moiety (e.g., CAR, TCR) .
  • engineered ⁇ T cells e.g., CAR-T cells
  • an antigen recognition moiety e.g., CAR, TCR
  • the ratio of ⁇ T cell subtypes can be measured by flow cytometry (e.g., following the methods described in Example 4) .
  • the expanded cell culture e.g., un-transfected or transfected with a CAR expression vector
  • the expanded cell culture may comprise a percentage of ⁇ T cells that is greater than 5%, greater than 10%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99%.
  • the expanded cell culture (e.g., un-transfected or transfected with a CAR expression vector) may comprise a percentage of V ⁇ 2 positive cells that is greater than 5%, greater than 10%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99%.
  • the expanded cell culture may comprise a percentage of V ⁇ 2 positive cells that is less than 5%, less than 10%, less than 20%, less than 30%, less than 40%, less than 50%, less than 60%, less than 70%, less than 75%, less than 80%, less than 85%, less than 90%, less than 91%, less than 92%, less than 93%, less than 94%, less than 95%, less than 96%, less than 97%, less than 98%, or less than 99%.
  • the expanded cell culture may comprise a percentage of V ⁇ 2 positive cells that is about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%.
  • the ⁇ T cells may be repeatedly stimulated by tumor cells (e.g., H929 cells) for several rounds in a re-challenge assay.
  • the effector cell: target cell (E: T) ratio may be 0.5: 1, 1: 1, 2: 1, 2.5: 1, 5: 1, or 10: 1.
  • Each round of stimulation may last for 1 day, 2 days, 3 days, 4 days or 5 days.
  • the ⁇ T cells may be repeatedly stimulated by tumor cells (e.g., H929 cells) for several rounds in a re-challenge assay.
  • the effector cell: target cell (E: T) ratio may be 0.5: 1, 1: 1, 2: 1, 2.5: 1, 5: 1, or 10: 1.
  • Each round of stimulation may last for 1 day, 2 days, 3 days, 4 days or 5 days.
  • effector memory T cells refers to a subset of memory T cells with effector functions.
  • the memory T cells include stem cell memory T (Tscm) cells and central memory T (Tcm) cells and effector memory T (Tem) cells, which have different specific phenotypes.
  • Tcm cells and Tem cells are often distinguished by CCR7 expression and function.
  • Tcm cells characterized by the CD45RO+CCR7+CD27+CD28+CD62Lhi+phenotype
  • Tem cells are found in nonlymphoid tissues, have lytic activity and are CD62LloCCR7-.
  • Tem cells may express higher levels of receptors responsible for migration to inflamed tissues and have a stronger immediate effector function than Tcm cells.
  • Tumor suppression by the ⁇ T cells may be evaluated using an in vivo tumor suppression assay (e.g., following the methods described in Example 9) .
  • the ⁇ T cells prepared according to the method described herein may effectively suppress tumor growth.
  • the ⁇ T cells prepared according to the methods described herein may have a tumor growth inhibition percentage (TGI%) that is greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%.
  • TGI% tumor growth inhibition percentage
  • Exemplary TCR co-receptors include, but are not limited to, CD4, CD8, CD28, CD45, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154.
  • the TAC receptor can comprise a transmembrane domain and a cytosolic domain derived from CD4.
  • the TAC receptor can comprise a transmembrane domain and a cytosolic domain derived from CD8 (such as CD8 ⁇ ) .
  • T cell co-receptors are expressed as membrane proteins on T cells. They can provide stabilization of the TCR: peptide: MHC complex and facilitate signal transduction.
  • the CD4 co-receptor can only stabilize TCR: MHC II complexes while the CD8 co-receptor can only stabilize the TCR: MHC I complex.
  • the differential expression of CD4 and CD8 on different T cell types results in distinct T cell functional subpopulations.
  • CD8+ T cells are cytotoxic T cells.
  • B-cell lymphoma the tumor-specific idiotype immunoglobulin constitutes a truly tumor-specific immunoglobulin antigen that is unique to the individual tumor.
  • B cell differentiation antigens such as CD19, CD20 and CD37 are other candidates for target antigens in B-cell lymphoma.
  • the tumor antigen can be a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA) .
  • TSA tumor-specific antigen
  • TAA tumor-associated antigen
  • a TSA is unique to tumor cells and does not occur on other cells in the body.
  • a TAA associated antigen is not unique to a tumor cell, and instead is also expressed on a normal cell under conditions that fail to induce a state of immunologic tolerance to the antigen.
  • the expression of the antigen on the tumor can occur under conditions that enable the immune system to respond to the antigen.
  • TAAs can be antigens that are expressed on normal cells during fetal development, when the immune system is immature, and unable to respond or they can be antigens that are normally present at extremely low levels on normal cells, but which are expressed at much higher levels on tumor cells.
  • TSA or TAA antigens include the following: Differentiation antigens such as MART-1/MelanA, gp 100 (Pmel 17) , tyrosinase, TRP-1, TRP-2 and tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor-suppressor genes such as p53, Ras, HER2/neu; unique tumor antigens resulting from chromosomal translocations; such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such as the Epstein Barr virus antigens EBVA and the human papillomavirus (HPV) antigens E6 and E7.
  • Differentiation antigens such as MART-1/MelanA, gp 100 (Pmel
  • ⁇ T cells of the disclosure can be designed to home to a specific physical location in the body of a subject and hence target an antigen at a particular tissue, organ or body site. Endogenous T-cells have distinct repertoires of trafficking ligands and receptors that influence their patterns of migration. ⁇ T cells of the disclosure can be designed to express, from the expression cassette comprising the tumor recognition moiety or from a separate expression cassette, one or more trafficking ligand (s) , or receptor (s) that guide the migration of the ⁇ T cells to a particular tissue, organ, or body site.
  • the ⁇ T cells may be tumor-specific allogeneic ⁇ T cells.
  • the ⁇ T cells may be derived from tumor infiltrating lymphocytes (TIL) isolated from a tumor. Different TILs can be isolated from different tumor types.
  • TILs tumor infiltrating lymphocytes
  • An expression cassette encoding a tumor recognition moiety, and activation domain, or another engineered featured can be inserted into the genome of TILs isolated from various tumors.
  • Such ⁇ T cells can infiltrate solid tumors, weaken and kill tumors cells expressing one or more target antigens, and they can provide an effective treatment for various malignancies.
  • Tumor specific allogeneic ⁇ T cells can be engineered to express at least one tumor recognition moiety that recognizes an epitope of choice.
  • Some tumor specific allogeneic ⁇ T cells may be designed to express at least two different tumor recognition moieties, and each different tumor recognition moiety is designed to recognize a different epitope of the same antigen, distinct antigens, an antigen and an activating or inactivating co-stimulatory/immune modulation receptor (s) , an antigen in complex with an MHC molecule, or a homing receptor.
  • each different tumor recognition moiety is designed to recognize a different epitope of the same antigen, distinct antigens, an antigen and an activating or inactivating co-stimulatory/immune modulation receptor (s) , an antigen in complex with an MHC molecule, or a homing receptor.
  • the disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising the ⁇ T cells made by the methods described herein and a pharmaceutically acceptable carrier.
  • the disclosure provides a method of treating a disease or disorder in a subject (e.g., human subject) , the method comprising administering to the subject, an effective amount of the ⁇ T cells described herein, or the pharmaceutical composition described herein.
  • the disease or disorder may be cancer, an autoimmune disease, a tumor, or an infection.
  • Solid tumor refers to an abnormal mass of tissue that usually does not contain cysts or liquid areas. Solid tumors may be benign (not cancer) , or malignant (cancer) . Different types of solid tumors are named for the type of cells that form them. Examples of solid tumors are sarcomas, carcinomas, and lymphomas. Leukemias (cancers of the blood) generally do not form solid tumors.
  • the disclosure provides a method of modulating an immune response comprising administering an effective amount of ⁇ T cells described herein to a subject in need thereof.
  • the present disclosure provides a method for treating a disease or disorder by administering an effective amount of ⁇ T cells described herein to a subject in need thereof.
  • the disease or disorder may be infectious disease, autoimmune disease, or tumor.
  • the cancer may be hematological cancer or solid tumor.
  • cancer examples include, but are not limited to, acute myeloid leukemia (AML) , B-cell acute lymphoid leukemia (BALL) , T-cell acute lymphoid leukemia (TALL) , acute lymphoid leukemia (ALL) , chronic myelogenous leukemia (CML) , chronic lymphocytic leukemia (CLL) , multiple myeloma (MM) , myelodysplastic syndrome (MDS) , myeloproliferative neoplasms (MPNs) , chronic myeloid leukemia (CML) , and blastic plasmacytoid dendritic cell neoplasm (BPDCN) , breast cancer, lung cancer, pancreatic cancer, melanoma, oral cancer, mesothelioma, ovarian cancer, colorectal cancer, gastric cancer, cervical cancer, brain cancer, skin cancer, lymphoma, epithelial neoplasms
  • AML
  • the disclosure further includes the use of the ⁇ T cells described herein in the manufacture of a medicament or pharmaceutical composition to modulate an immune response, to treat an infection or to treat cancer as described hereinabove.
  • the ⁇ T cells can also be used in experimental models, for example, to further study and elucidate the function of the cells.
  • One or more of the ⁇ T cells described herein can be administered to a subject in a single, unified form, such as an intravenous injection, or in multiple forms, for example, as multiple intravenous infusions or injections, or subcutaneous injections.
  • the ⁇ T cells may expand within a subject's body, in vivo, after administration to a subject.
  • the ⁇ T cells can be frozen to provide cells for multiple treatments with the same cell preparation.
  • the ⁇ T cells of the disclosure, and pharmaceutical compositions comprising the same can be packaged as a kit.
  • a kit can include instructions (e.g., written instructions) on the use of the ⁇ T cells and compositions comprising the same.
  • a method of treatment can comprise administering to a subject a therapeutically effective amount of the ⁇ T cells.
  • the therapeutically effective amount of the ⁇ T cells may be administered for at least 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or 1 year.
  • the therapeutically effective amount of the ⁇ T cells may be administered for at least one week.
  • the therapeutically effective amount of the ⁇ T cells may be administered for at least two weeks.
  • the ⁇ T cells described herein can be administered before, during, or after the occurrence of a disease or condition, and the timing of administering the ⁇ T cells can vary.
  • the ⁇ T cells can be used as a prophylactic and can be administered continuously to subjects with a propensity to conditions or diseases in order to lessen a likelihood of the occurrence of the disease or condition.
  • the ⁇ T cells can be administered to a subject during or as soon as possible after the onset of the symptoms.
  • the administration of the ⁇ T cells can be initiated immediately within the onset of symptoms, within the first 3 hours of the onset of the symptoms, within the first 6 hours of the onset of the symptoms, within the first 24 hours of the onset of the symptoms, within 48 hours of the onset of the symptoms, or within any period of time from the onset of symptoms.
  • the initial administration can be via any route practical (e.g., intravenous infusions or injections) , such as by any route described herein using any formulation described herein.
  • the administration of the ⁇ T cells of the disclosure may be an intravenous administration.
  • One or multiple dosages of the ⁇ T cells can be administered as soon as is practicable after the onset of a cancer or an infectious disease, and for a length of time necessary for the treatment of the disease, such as, for example, from about 24 hours to about 48 hours, from about 48 hours to about 1 week, from about 1 week to about 2 weeks, from about 2 weeks to about 1 month, from about 1 month to about 3 months.
  • one or multiple dosages of the ⁇ T cells can be administered years after onset of the cancer and before or after other treatments.
  • the ⁇ T cells can be administered for at least about 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 1 year, at least 2 years at least 3 years, at least 4 years, or at least 5 years.
  • the length of treatment can vary for each subject.
  • ⁇ T cells for adoptive cell therapy are known and can be used in connection with the provided methods and compositions.
  • adoptive T cell therapy methods are described, e.g., in US Patent Application Publication No. 2003/0170238 to Gruenberg et al; US Patent No. 4, 690, 915 to Rosenberg; Rosenberg (2011) Nat Rev Clin Oncol. 8(10) : 577-85) . See, e.g., Themeli et al. (2013) Nat Biotechnol. 31 (10) : 928-933; Tsukahara et al. (2013) Biochem Biophys Res Commun 438 (1) : 84-9; Davila et al.
  • the cell therapy e.g., adoptive T cell therapy can be carried out by autologous transfer, in which the cells are isolated and/or otherwise prepared from the subject who is to receive the cell therapy, or from a sample derived from such a subject.
  • the cells may be derived from a subject, e.g., patient, in need of a treatment and the cells, following isolation and processing are administered to the same subject.
  • the cell therapy (e.g., adoptive T cell therapy) can be carried out by allogeneic transfer, in which the cells are isolated and/or otherwise prepared from a subject other than a subject who is to receive or who ultimately receives the cell therapy, e.g., a first subject.
  • the cells then may be administered to a different subject, e.g., a second subject, of the same species.
  • the first and second subjects may be genetically identical.
  • the first and second subjects may be genetically similar.
  • the second subject may express the same HLA class or supertype as the first subject.
  • the subject may have been treated with a therapeutic agent targeting the disease or condition, e.g., the tumor, prior to administration of the cells or composition containing the cells.
  • the subject may be refractory or non-responsive to the other therapeutic agent.
  • the subject may have persistent or relapsed disease, e.g., following treatment with another therapeutic intervention, including chemotherapy, radiation, and/or hematopoietic stem cell transplantation (HSCT) , e.g., allogenic HSCT.
  • HSCT hematopoietic stem cell transplantation
  • the administration may effectively treat the subject despite the subject having become resistant to another therapy.
  • the subject may be responsive to the other therapeutic agent, and treatment with the therapeutic agent reduces disease burden.
  • the subject may be initially responsive to the therapeutic agent, but exhibits a relapse of the disease or condition over time.
  • the subject may have not relapsed.
  • the subject may be determined to be at risk for relapse, such as at a high risk of relapse, and thus the cells are administered prophylactically, e.g., to reduce the likelihood of or prevent relapse.
  • the subject may has not received prior treatment with another therapeutic agent.
  • the subject may have persistent or relapsed disease, e.g., following treatment with another therapeutic intervention, including chemotherapy, radiation, and/or hematopoietic stem cell transplantation (HSCT) , e.g., allogenic HSCT.
  • HSCT hematopoietic stem cell transplantation
  • the administration may effectively treat the subject despite the subject having become resistant to another therapy.
  • the ⁇ T cells described herein can be administered to an animal, such as a mammal, even more a human, to treat a cancer.
  • the ⁇ T cells can be used for the treatment of any condition related to a cancer, especially a cell-mediated immune response against a tumor cell (s) , where it is desirable to treat or alleviate the disease.
  • the ⁇ T cells (e.g., immune cells, T cells, or NK cells) described herein can be included in a composition for immunotherapy.
  • the composition can include a pharmaceutical composition and further include a pharmaceutically acceptable carrier.
  • a therapeutically effective amount of the pharmaceutical composition comprising the ⁇ T cells can be administered.
  • the ⁇ T cells can be immediately used in the above therapeutic, experimental or commercial applications or the cells can be cryopreserved for use at a later date.
  • the pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.
  • the ⁇ T cells disclosed herein can be formulated in unit dosage forms suitable for single administration of precise dosages.
  • the unit dosage forms may comprise additional lymphocytes.
  • the formulation is divided into unit doses containing appropriate quantities of one or more compounds.
  • the unit dosage can be in the form of a package containing discrete quantities of the formulation. Non-limiting examples are packaged tablets or capsules, and powders in vials or ampoules.
  • Aqueous suspension compositions can be packaged in single-dose non-reclosable containers. Multiple-dose reclosable containers can be used, for example, in combination with a preservative or without a preservative.
  • the pharmaceutical composition may not comprise a preservative.
  • Formulations for parenteral injection can be presented in unit dosage form, for example, in ampoules, or in multi-dose containers with a preservative.
  • T cells were isolated from healthy donor PBMCs using TCR ⁇ / ⁇ -Biotin (Miltenyi Biotec, 170-070-416) and anti-Biotin Reagent and CD56 MicroBeads (Miltenyi Biotec, 130-050-401) .
  • TCR delta Monoclonal Antibody (TS-1) (ThermoFisher) was used as an activating agent, and was immobilized on a 24 well plate at a final concentration of 1 ⁇ g/ml. The supernatant was discarded and the wells were washed with PBS.
  • T cells were seeded in the 24 well plate at a cell density of 0.5-5 ⁇ 10 6 cell/ml and cultured in based medium supplement with human IL-15 (10ng/mL) for 2-6 days.
  • T cells were transduced with lentivirus or retrovirus encoding BCMA CAR (SEQ ID NO: 2) at proper multiplicity of infection (MOI) .
  • Fresh medium was supplemented 24 hours post lentivirus or retrovirus infection.
  • CAR-V ⁇ 1 T cells were harvested 10-12 days after transduction.
  • PBMCs Human peripheral blood mononuclear cells
  • PBMCs Human peripheral blood mononuclear cells
  • the cells were cultured in RPMI 1640 medium supplemented with 10%FBS and antibiotics.
  • ZOL zoledronic acid
  • Recombinant human IL-2 100 IU/mL
  • Recombinant human IL-15 100 IU/mL
  • vitamin C 70 ⁇ M
  • CAR-V ⁇ 2 T Cells were harvested 10 days post-transduction and the total number, purity and transduction efficiency were determined. Cells were further enriched with a negative TCR ⁇ / ⁇ + T cell isolation kit (Miltenyi Biotec) before future applications or cryopreserved.
  • Soluble ⁇ TCR proteins were generated in house for animal immunization.
  • a pair of charge-complementary leucine zipper (LZ) sequences were connected to the C-terminus of TCR ⁇ and TCR ⁇ chain through a flexible (G4S) 3 linker.
  • G4S flexible
  • 6xHis-tag was added to ⁇ -chain and a flag-tag to ⁇ -chain C-terminus for the purposes of both purification and detection.
  • TCR ⁇ chain and TCR ⁇ chain plasmids were prepared and used for soluble ⁇ TCR production.
  • Soluble TCRs (sTCRs) were produced by FreeStyle 293-F cells and purified by Anti-DYKDDDDK G1 Affinity Resin (Genscript, Cat. #L00432) .
  • ⁇ 9 ⁇ 2 TCR protein was formulated as an emulsion with complete Freund's adjuvant (CFA) for primary immunization or with incomplete Freund's adjuvant (IFA) for boosting immunization.
  • CFA complete Freund's adjuvant
  • IFA incomplete Freund's adjuvant
  • the antigen emulsion was administered by double-spot injections intramuscularly at the neck.
  • the animal received 3 injections of 100-200 ⁇ g of human ⁇ 9 ⁇ 2 TCR protein at a 2-week interval and subsequently 2 injections of 100 ⁇ g of cynomolgus ⁇ 9 ⁇ 2 TCR protein at a weekly interval.
  • a final boost was given to the animal with 50 ⁇ g of human ⁇ 9 ⁇ 2 TCR protein.
  • PBLs peripheral blood lymphocytes
  • DNAs encoding V H H (variable region of heavy chain-only antibody, also known as single domain antibody, sdAb) , VH and VL were amplified from camel cDNA, purified and ligated in an in-house phagemid vector (see Patent No. US20170089914A1) .
  • the ligation product was used to transform SS320 electrocompetent cells (Lucigen, Cat. #60512-1) .
  • the resulting sdAb and scFv libraries were supplemented with 20%glycerol and stored at -80°C.
  • Binders were isolated from the above-mentioned phage libraries using protein-based panning as well as cell-based panning. One round of panning was carried out for both protein-and cell-based panning approaches. Percentage of ⁇ 9 ⁇ 2 TCR positive clones identified by ELISA reached at least 50%and the sequence diversity of ⁇ 9 ⁇ 2 TCR-specific clones was high for all output phages. These outputs were used for subsequent high-throughput screening.
  • the selected output phages were used to infect exponentially growing E. coli cells.
  • the double-strand DNA of the output was extracted.
  • the sdAb/scFv inserts were cut from the phagemid vector and inserted into an antibody fragment expression vector for high-throughput screening.
  • the resulting plasmid was used to transform exponentially growing E. coli cells, which were then plated and grown overnight at 37°C. Thousands of colonies were picked individually and grown in 96-deep-well plates containing 1 mL 2YT medium. The expression of antibody fragment was induced by adding 1.0 mM IPTG.
  • the sdAb/scFv proteins in the supernatant were analyzed for their ability to bind to human ⁇ 9 ⁇ 2 TCR proteins by ELISA and human ⁇ 9 ⁇ 2 T cells by flow cytometry.
  • Four antibodies were chosen to activate ⁇ T cells as AS287963, AS281850, AS287435 and AS288180.
  • the full-length V H H, VH, VL and CDR sequences are listed in SEQUENCE LISTING.
  • ⁇ T cells and NK cells were depleted from healthy donor PBMCs using TCR ⁇ / ⁇ -Biotin (Miltenyi Biotec, 170-070-416) and anti-Biotin Reagent and CD56 MicroBeads (Miltenyi Biotec, 130-050-401) .
  • An activating agent called AS287963 an anti- ⁇ TCR constant chain antibody, SEQ ID NO: 1 was immobilized on 24 well plate at a final concentration of 0.1-5 ⁇ g/ml. The supernatant was discarded, and the wells were washed with PBS twice.
  • the ⁇ T/NK depleted T cells were transferred to the plates and cultured in Medium I for 1-5 days for activation.
  • Medium I contains base medium, serum (e.g. human AB serum) or human serum replacement (e.g. human platelet lysate) and different cytokines with or without inhibitor.
  • Cells were transduced with lentivirus or retrovirus at proper multiplicity of infection (MOI) , to express a BCMA CAR (SEQ ID NO: 2) .
  • Fresh Medium I was supplemented 24 hours post lentivirus or retrovirus infection.
  • Medium II was prepared and after 1-5 days of transduction, the cells were cultured for 6-12 days in Medium II.
  • Medium II containing base medium, serum (e.g. human AB serum) or human serum replacement (e.g. human platelet lysate) and different cytokines with or without inhibitor.
  • serum e.g. human AB serum
  • human serum replacement e.g. human platelet lysate
  • the medium components were optimized to boost the expansion of ⁇ T cells.
  • IL-2 and IL-7 are cytokines of common gamma chain family and have been reported to promote the expansion of T cells and have been broadly used in cell therapies. Based on this, the effects of IL-2 and IL-7 were evaluated to see whether they can affect the proliferation and efficacy of CAR-polyclonal ⁇ T cells.
  • Polyclonal ⁇ T cells were cultured in Medium I with or without IL-2 or IL-7 as described in Table 1 for 3-9 days. After 1-5 days of transduction, the cells were cultured in Medium II as described in Table 1 for another 6-12 days. CAR-polyclonal ⁇ T cells were harvested. Cell number and viability were analyzed by Cellometer (Nexcelom, K2) .
  • the harvested cells were stained with fluorescent-labeled antibodies (CD3-BV785 [344842, Clone: SK7, BioLegend] , TCRV ⁇ 1-APC [17-5679-42, Clone: TS8.2, Invitrogen] , TCRV ⁇ 2-BV421 [331428, Clone: B6, Biolegend] and Alexa Fluor 488-labeled anti-mouse sdAb antibodies [GenScript] ) and analyzed by FACS.
  • fluorescent-labeled antibodies CD3-BV785 [344842, Clone: SK7, BioLegend]
  • TCRV ⁇ 1-APC 17-5679-42, Clone: TS8.2, Invitrogen
  • TCRV ⁇ 2-BV421 [331428, Clone: B6, Biolegend]
  • Alexa Fluor 488-labeled anti-mouse sdAb antibodies [GenScript]
  • CAR- ⁇ T cells were evaluated in a repetitive tumor challenge assay. Briefly, 2 ⁇ 10 5 CAR+ ⁇ T cells were co-cultured with 2 ⁇ 10 5 NCI-H929 cells in a 24 well. Two days later, cells were harvested to determine the relative ratio of viable T cells and tumor cells. CAR+ ⁇ T was quantified and re-plated with fresh NCI-H929 cells at a ratio of 1: 1 for the next round. IFN- ⁇ , GM-CSF and TNF- ⁇ release in the supernatant was determined at the end of first round.
  • the cytokine release analysis was performed using reagents (Human IFN gamma kit, Cisbio, Cat#62HIFNGPEH, Human TNF alpha kit, Cisbio, Cat#62HTNFAPEH, Human GM-CSF kit, Cisbio, Cat#62HGMCSFPEH) .
  • IL-2 or IL-7 had no significant effects on proliferation, viability, CAR positive rate and cell sub-type ratio (FIGS. 1A-1D) .
  • IL-2 stimulated CAR-polyclonal ⁇ T showed improved efficacy and improved proliferation capacity (FIGS. 1E-1F) .
  • adding IL-2 or IL-7 had no significant effects on cytokines release (FIGS. 1G-1K) .
  • IL-4 and IL-1 ⁇ were also used in T cell culture mediums. Based on this, the effects of IL-4 and IL-1 ⁇ were evaluated to see whether they can affect the proliferation and efficacy of CAR-polyclonal ⁇ T cells.
  • Polyclonal ⁇ T cells were cultured in Medium I with or without IL-4 and IL-1 ⁇ as described in Table 2 for 3-9 days. After 1-5 days of transduction, the cells were cultured in Medium II as described in Table 2 for another 6-12 days. CAR-polyclonal ⁇ T cells were harvested. Cell number and viability were analyzed by Cellometer (Nexcelom, K2) .
  • the harvested cells were stained with fluorescent-labeled antibodies (CD3-BV785 [344842, Clone: SK7, BioLegend] , TCRV ⁇ 1-APC [17-5679-42, Clone: TS8.2, Invitrogen] , TCRV ⁇ 2-BV421 [331428, Clone: B6, Biolegend] and Alexa Fluor 488-labeled anti-mouse sdAb antibodies [GenScript] ) and analyzed by FACS.
  • fluorescent-labeled antibodies CD3-BV785 [344842, Clone: SK7, BioLegend]
  • TCRV ⁇ 1-APC 17-5679-42, Clone: TS8.2, Invitrogen
  • TCRV ⁇ 2-BV421 [331428, Clone: B6, Biolegend]
  • Alexa Fluor 488-labeled anti-mouse sdAb antibodies [GenScript]
  • CAR- ⁇ T cells were evaluated in a repetitive tumor challenge assay. Briefly, 2 ⁇ 10 5 CAR+ ⁇ T cells were co-cultured with 2 ⁇ 10 5 NCI-H929 cells in a 24 well. Two days later, cells were harvested to determine the relative ratio of viable T cells and tumor cells. CAR+ ⁇ T was quantified and re-plated with fresh NCI-H929 cells at a ratio of 1: 1 for the next round. IFN- ⁇ , GM-CSF and TNF- ⁇ release in the supernatant was determined at the end of first round.
  • the cytokine release analysis was performed using reagents (Human IFN gamma kit, Cisbio, Cat#62HIFNGPEH, Human TNF alpha kit, Cisbio, Cat#62HTNFAPEH, Human GM-CSF kit, Cisbio, Cat#62HGMCSFPEH) .
  • adding IL-4 or IL-1 ⁇ had no significant effects on proliferation, viability and CAR positive rate (FIGS. 2A-2C) .
  • adding IL-4 or IL-1 ⁇ affected the percentage of different cell sub-types (FIG. 2D) , leading to increased V ⁇ 1 T ratio in total ⁇ T cells.
  • CAR-polyclonal ⁇ T cells cultured with IL-4 and IL-1 ⁇ showed improved efficacy and improved proliferation capacity (FIGS. 2E-2F) .
  • adding IL-4 or IL-1 ⁇ could slightly decrease IFN- ⁇ release and show no difference in TNF- ⁇ , GM-CSF, IL-2 and IL-17 (FIGS. 2G-2K) .
  • the data indicated that IL-4 and IL-1 ⁇ addition can improve the efficacy and proliferation capacity.
  • the PI3K-AKT-mTOR signaling pathway is crucial for T cell activation, survival, expansion, migration, function, and differentiation. Inhibitors of this pathway can lead to a higher number of T memory cells, and increased expression of the lymph node homing marker CD62L and CCR7.
  • MK-2206 dihydrochloride was used as an AKT inhibitor to optimize T cell fitness and improve efficacy.
  • CAR-polyclonal ⁇ T cells were generated with or without MK-2206 (Table 3) , and cell performance were detected and analyzed.
  • Polyclonal ⁇ T cells were cultured in Medium I as described in Table 3 for 3-9 days. After 1-5 days of transduction, the cells were cultured in Medium II as described in Table 3 for another 6-12 days. CAR-polyclonal ⁇ T cells were harvested. Cell number and viability were analyzed by Cellometer (Nexcelom, K2) .
  • the harvested cells were stained with fluorescent-labeled antibodies (CD3-BV785 [344842, Clone: SK7, BioLegend] , TCRV ⁇ 1-APC [17-5679-42, Clone: TS8.2, Invitrogen] , TCRV ⁇ 2-BV421 [331428, Clone: B6, Biolegend] and Alexa Fluor 488-labeled anti-mouse sdAb antibodies [GenScript] ) and analyzed by FACS.
  • fluorescent-labeled antibodies CD3-BV785 [344842, Clone: SK7, BioLegend]
  • TCRV ⁇ 1-APC 17-5679-42, Clone: TS8.2, Invitrogen
  • TCRV ⁇ 2-BV421 [331428, Clone: B6, Biolegend]
  • Alexa Fluor 488-labeled anti-mouse sdAb antibodies [GenScript]
  • CAR- ⁇ T cells were evaluated in a repetitive tumor challenge assay. Briefly, 2 ⁇ 10 5 CAR+ ⁇ T cells were co-cultured with 2 ⁇ 10 5 NCI-H929 cells in a 24 well. Two days later, cells were harvested to determine the relative ratio of viable T cells and tumor cells. CAR+ ⁇ T was quantified and re-plated with fresh NCI-H929 cells at a ratio of 1: 1 for the next round. IFN- ⁇ , GM-CSF and TNF- ⁇ release in the supernatant was determined at the end of first round.
  • the cytokine release analysis was performed using reagents (Human IFN gamma kit, Cisbio, Cat#62HIFNGPEH, Human TNF alpha kit, Cisbio, Cat#62HTNFAPEH, Human GM-CSF kit, Cisbio, Cat#62HGMCSFPEH) .
  • IL-21 was added to adjust sub-type ratio of expanded CAR-polyclonal ⁇ T cells.
  • CAR-polyclonal ⁇ T cells were generated with or without IL-21 (Table 4) , and cell performance were detected and analyzed.
  • Polyclonal ⁇ T cells were cultured in Medium I as described in Table 4 for 3-9 days. After 1-5 days of transduction, the cells were cultured in Medium II as described in Table 4 for another 6-12 days. CAR-polyclonal ⁇ T cells were harvested. Cell number and viability were analyzed by Cellometer (Nexcelom, K2) .
  • the harvested cells were stained with fluorescent-labeled antibodies (CD3-BV785 [344842, Clone: SK7, BioLegend] , TCRV ⁇ 1-APC [17-5679-42, Clone: TS8.2, Invitrogen] , TCRV ⁇ 2-BV421 [331428, Clone: B6, Biolegend] and Alexa Fluor 488-labeled anti-mouse sdAb antibodies [GenScript] ) and analyzed by FACS.
  • fluorescent-labeled antibodies CD3-BV785 [344842, Clone: SK7, BioLegend]
  • TCRV ⁇ 1-APC 17-5679-42, Clone: TS8.2, Invitrogen
  • TCRV ⁇ 2-BV421 [331428, Clone: B6, Biolegend]
  • Alexa Fluor 488-labeled anti-mouse sdAb antibodies [GenScript]
  • CAR- ⁇ T cells were evaluated in a repetitive tumor challenge assay. Briefly, 2 ⁇ 10 5 CAR+ ⁇ T cells were co-cultured with 2 ⁇ 10 5 NCI-H929 cells in a 24 well. Two days later, cells were harvested to determine the relative ratio of viable T cells and tumor cells. CAR+ ⁇ T was quantified and re-plated with fresh NCI-H929 cells at a ratio of 1: 1 for the next round. IFN- ⁇ , GM-CSF and TNF- ⁇ release in the supernatant was determined at the end of first round.
  • the cytokine release analysis was performed using reagents (Human IFN gamma kit, Cisbio, Cat#62HIFNGPEH, Human TNF alpha kit, Cisbio, Cat#62HTNFAPEH, Human GM-CSF kit, Cisbio, Cat#62HGMCSFPEH) .
  • IL-21 did not significantly affect the viability (FIG. 4B) , CAR expression (FIG. 4C) and cytotoxicity (FIGS. 4E-4F) of CAR ⁇ T cells.
  • IL-21 slightly reduced proliferation (FIG. 4A) .
  • IL-21 could raise the ratio of V ⁇ 1 or double negative (V ⁇ 1-V ⁇ 2-) T cells (FIG. 4D) .
  • CAR- ⁇ T cells which were cultured with IL-21 had lower cytokines level than those cultured without IL-21 (FIGS. 4G-4I) .
  • Polyclonal ⁇ T cells were cultured in Medium I as described in Table 5 for 3-9 days. After 1-5 days of transduction, the cells were cultured in Medium II as described in Table 5 for another 6-12 days. CAR-polyclonal ⁇ T cells were harvested. Cell number and viability were analyzed by Cellometer (Nexcelom, K2) .
  • the harvested cells were stained with fluorescent-labeled antibodies (CD3-BV785 [344842, Clone: SK7, BioLegend] , TCRV ⁇ 1-APC [17-5679-42, Clone: TS8.2, Invitrogen] , TCRV ⁇ 2-BV421 [331428, Clone: B6, Biolegend] and Alexa Fluor 488-labeled anti-mouse sdAb antibodies [GenScript] ) and analyzed by FACS.
  • fluorescent-labeled antibodies CD3-BV785 [344842, Clone: SK7, BioLegend]
  • TCRV ⁇ 1-APC 17-5679-42, Clone: TS8.2, Invitrogen
  • TCRV ⁇ 2-BV421 [331428, Clone: B6, Biolegend]
  • Alexa Fluor 488-labeled anti-mouse sdAb antibodies [GenScript]
  • CAR- ⁇ T cells were evaluated in a repetitive tumor challenge assay. Briefly, 2 ⁇ 10 5 CAR+ ⁇ T cells were co-cultured with 2 ⁇ 10 5 NCI-H929 cells in a 24 well. Two days later, cells were harvested to determine the relative ratio of viable T cells and tumor cells. CAR+ ⁇ T was quantified and re-plated with fresh NCI-H929 cells at a ratio of 1: 1 for the next round. IFN- ⁇ , GM-CSF and TNF- ⁇ release in the supernatant was determined at the end of first round.
  • the cytokine release analysis was performed using reagents (Human IFN gamma kit, Cisbio, Cat#62HIFNGPEH, Human TNF alpha kit, Cisbio, Cat#62HTNFAPEH, Human GM-CSF kit, Cisbio, Cat#62HGMCSFPEH) .
  • FIGS. 5A-5B The results of expansion and viability were shown in FIGS. 5A-5B. All ⁇ T cells from different activators could achieve distinct clonal or polyclonal population subsets.
  • the OKT3 group exhibited the greatest cell proliferation potential. However, the CAR expression of OKT3 group was the lowest (FIG. 5C) .
  • the ratio of ⁇ T cells was higher in the AS287963 group than the OKT3 and CD3/CD28 groups (FIG. 5D) .
  • the expanded CAR- ⁇ T cells were evaluated in a repetitive tumor challenge assay.
  • 2 ⁇ 10 5 CAR+ ⁇ T cells were co-cultured with 2 ⁇ 10 5 NCI-H929 cells in a 24 well plate. Two days later, cells were harvested to determine the relative ratio of viable T cells and tumor cells.
  • CAR+ ⁇ T cells were quantified and re-plated with fresh NCI-H929 cells at a ratio of 1: 1 for the next round. IFN- ⁇ , GM-CSF and TNF- ⁇ release in the supernatant was determined at the end of first round.
  • the cytokine release analysis was performed with reagents (Human IFN gamma kit, Cisbio, Cat#62HIFNGPEH, Human TNF alpha kit, Cisbio, Cat#62HTNFAPEH, Human GM-CSF kit, Cisbio, Cat#62HGMCSFPEH) .
  • polyclonal ⁇ T cells activated by AS287963 mediated effective elimination of H929 cells until round 12 (E: T 1: 1 each round) .
  • H929 cell challenges resulted in a lower level of lysis of tumor cells and lower CAR ⁇ T cell expansion, indicating decreased cytotoxicity.
  • the AS287963 group showed the lowest IFN- ⁇ secretion and moderate secretion of GM-CSF and TNF- ⁇ compared with other groups (FIGS. 5G-5I) .
  • RPMI-8226, K562, NCI-H929, U937, Huh7 and SK-Hep-1 cells stably expressing firefly luciferase were used as target tumor cells.
  • the untransduced polyclonal ⁇ T cells were generated without transduction step by using the medium with IL-21 as described in Table 4.
  • the untransducted V ⁇ 1 T cells and V ⁇ 2 T cells were produced without transduction step as the method described in Example 1 and Example 2.
  • the ⁇ T cells (polyclonal ⁇ T cells, V ⁇ 1 T cells or V ⁇ 2 T cells) and target cells were co-cultured in 96-well plates (3610, Corning) at E: T ratios of 10: 1 and 2: 1. After 24 hours, cells were harvested, 100 ⁇ l ONE-Glo reagent (E6110, Promega) was added to the 100 ⁇ l culture medium in each well, mixed well, incubated for at least 3 minutes to allow complete cell lysis. The plates were measured in a luminometer. As shown in FIGS. 6A-6F, cytotoxicity of polyclonal ⁇ T cells (without CAR expression) against 6 tumor cell lines were comparable to V ⁇ 1 T cells and significantly better than V ⁇ 2 T cells. These results demonstrated that polyclonal ⁇ T cells provide better intrinsic cytotoxicity than V ⁇ 2 T cells.
  • CAR- ⁇ T cells 1.5 ⁇ 10 4 cells per well
  • NCI-H929 tumor cells 1.5 ⁇ 10 4 cells per well
  • allogeneic/autologous PBMCs 1.5 ⁇ 10 6 cells per well, stain with CellTrace Violet Reagent [C34557, Thermofisher] ) were co-cultured in a final volume of 4 mL medium (base medium+10%Hi-FBS) per well within 12-well Clear TC-treated Multiple Well Plates (3513, Costar) for 7 days.
  • ⁇ T cells and CAR- ⁇ T cells were discriminated by fluorescence signals.
  • FIG. 7B CAR-polyclonal ⁇ T cells generated by using the medium without IL-21 as described in Table 4 exhibited better proliferation than CAR-V ⁇ 2 T cells, in the presence of human PBMCs.
  • FIG. 7A proliferation of ⁇ T cells from PBMCs was not significantly stimulated by CAR- ⁇ T cells.
  • Example 9 CAR-polyclonal ⁇ T cells demonstrated superior anti-tumor efficacy in vivo
  • Polyclonal ⁇ T cells were cultured in Medium I as described in Table 6 for 3-9 days. After 1-5 days of transduction, the cells were cultured in Medium II as described in Table 6 for another 6-12 days. CAR-polyclonal ⁇ T cells were harvested. Cell number and viability were analyzed by Cellometer (Nexcelom, K2) .
  • the harvested cells were stained with fluorescent-labeled antibodies (CD3-BV785 [344842, Clone: SK7, BioLegend] , TCRV ⁇ 1-APC [17-5679-42, Clone: TS8.2, Invitrogen] , TCRV ⁇ 2-BV421 [331428, Clone: B6, Biolegend] and Alexa Fluor 488-labeled anti-mouse sdAb antibodies [GenScript] ) and analyzed by FACS.
  • fluorescent-labeled antibodies CD3-BV785 [344842, Clone: SK7, BioLegend]
  • TCRV ⁇ 1-APC 17-5679-42, Clone: TS8.2, Invitrogen
  • TCRV ⁇ 2-BV421 [331428, Clone: B6, Biolegend]
  • Alexa Fluor 488-labeled anti-mouse sdAb antibodies [GenScript]
  • the expanded CAR- ⁇ T cells were evaluated in a repetitive tumor challenge assay.
  • 2 ⁇ 10 5 CAR+ ⁇ T cells were co-cultured with 2 ⁇ 10 5 NCI-H929 cells in a 24 well plate. Two days later, cells were harvested to determine the relative ratio of viable T cells and tumor cells.
  • CAR+ ⁇ T cells were quantified and re-plated with fresh NCI-H929 cells at a ratio of 1: 1 for the next round.
  • ⁇ T cells expanded well with at least 900-fold expansion and > 90%cell viability (FIGS. 10A-B) .
  • the purity of ⁇ T cells produced using all 4 heavy chain antibodies is high, > 98% (FIG. 10C) , and the composition of different ⁇ T cell subtypes are similar: the percentage of V ⁇ 2 T cells is the highest ( ⁇ 65%) and V ⁇ 1-V ⁇ 2-T cells, the lowest ( ⁇ 10%) (FIG. 10D) .
  • the percentages of CAR-positive cells were also similar amongst 4 groups the same (FIG. 10E) . As depicted in FIG.

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Abstract

La présente proposition concerne des procédés de préparation de lymphocytes γδT et leurs utilisations. Les lymphocytes γδT sont utiles pour le traitement de divers cancers, maladies infectieuses et troubles immunitaires. La présente invention concerne également des procédés permettant de multiplier les populations de lymphocytes γδT en quantités utiles d'un point de vue thérapeutique. Les cellules γδT peuvent être administrées à un sujet ayant n'importe quel haplotype du complexe majeur d'histocompatibilité (CMH).
PCT/CN2024/121884 2023-09-27 2024-09-27 Procédés pour la culture de lymphocytes t gamma delta Pending WO2025067472A1 (fr)

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