WO2025067472A1

WO2025067472A1 - Methods of culturing gamma delta t cells

Info

Publication number: WO2025067472A1
Application number: PCT/CN2024/121884
Authority: WO
Inventors: Chenyu SHU; Fei Xu; Yi Xu; Ting GENG; Yafeng Zhang
Original assignee: Nanjing Legend Biotechnology Co Ltd; Legend Biotech Ireland Ltd
Current assignee: Nanjing Legend Biotechnology Co Ltd; Legend Biotech Ireland Ltd
Priority date: 2023-09-27
Filing date: 2024-09-27
Publication date: 2025-04-03
Anticipated expiration: 2026-03-27

Abstract

Provided are methods of preparing γδT cells and uses thereof. The γδT cells are useful in the treatment of various cancers, infectious diseases, and immune disorders. Also provided are methods for expanding γδT cell populations to therapeutically useful quantities. The γδT cells may be administered to a subject with any MHC haplotype.

Description

METHODS OF CULTURING GAMMA DELTA T CELLS

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority benefits of International Application No. PCT/CN2023/122040, filed on September 27, 2023, the contents of which are incorporated herein by reference in its entirety.

SEQUENCE STATEMENT

The content of the following submission on XML file is incorporated herein by reference in its entirety: a computer readable form (CRF) of the Sequence Listing (file name: IEC240436PCT SEQUENCE LISTING. xml, date recorded: September 27, 2024, size: 34,002 bytes) .

TECHNICAL FIELD

This disclosure generally relates to methods of preparing γδT cells and uses thereof.

BACKGROUND

αβT cells play fundamental roles in cancer immunotherapy. However, their dependence on major histocompatibility complex (MHC) , their ability to only recognize mutated peptides, as well as their low tropism to the tumor sites, especially in solid tumors, have put an obstacle on their way into the clinical settings. The γδT cells (e.g., Vδ1, Vδ2 and Vδ1-Vδ2-T cells) exhibit potent cancer antigen recognition independent of classical peptide MHC complexes, making it an attractive candidate for allogeneic cancer adoptive immunotherapy. γδT 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.

Making cell therapies based on γδT cells is challenging. One important factor that limits the clinical progress of the use of the γδT cells is the lack of robust, consistent, and GMP-compatible expansion protocols. A reliable and GMP-compatible expansion protocol is needed.

SUMMARY

This disclosure relates to methods of preparing γδT cells and uses thereof. In one aspect, the present disclosure provides a two-step protocol for γδT cell expansion from peripheral blood mononuclear cells (PBMCs) that is further compatible with high-efficiency gene engineering for immunotherapy purposes.

In one aspect, the disclosure is related to a method for culturing γδT cells comprising: (1) culturing cells from a sample in a first culture medium comprising interleukin-15 (IL-15) , interferon-γ (IFN-γ) , and interleukin-2 (IL-2) ; and (2) culturing the cells obtained in step (1) in a second culture medium comprising IL-15, IFN-γ, and IL-2.

In some embodiments, the γδT cells obtained after step (2) comprising Vδ1 T cells, Vδ2 T cells and Vδ1-Vδ2-T cells.

In some embodiments, at least about 5%of the γδT cells obtained after step (2) are Vδ1 T cells.

In some embodiments, at least about 5%of the γδT cells obtained after step (2) are Vδ2 T cells.

In some embodiments, at least about 1%of γδT cells obtained after step (2) are Vδ1-Vδ2-T cells.

In some embodiments, 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-κB inhibitor, BCL-2 inhibitor, ERK inhibitor, MEK inhibitor, Raf-1 inhibitor, EGFR inhibitor, DAC inhibitor, HDAC inhibitor or CDK46 inhibitor.

In some embodiments, 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.

In some embodiments, the AKT inhibitor is MK-2206 and is present in an amount of 0.1-10 μg/ml.

In some embodiments, 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.

In some embodiments, the IL-15 in the first culture medium is present in a concentration of about 1-30 ng/ml.

In some embodiments, the IL-15 in the second culture medium is present in a concentration of about 1-200 ng/ml.

In some embodiments, the IFN-γ in the first culture medium and/or the second culture medium is present in a concentration of about 1-500 ng/ml.

In some embodiments, the IFN-γ in the first culture medium is present in a concentration of about 50-150 ng/ml.

In some embodiments, the IFN-γ in the second culture medium is present in a concentration of about 1-100 ng/ml.

In some embodiments, 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.

In some embodiments, the IL-2 in the first culture medium is present in a concentration of about 50-150 IU/ml.

In some embodiments, the IL-2 in the second culture medium is present in a concentration of about 50-150 IU/ml.

In some embodiments, 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.

In some embodiments, the concentration of IFN-γ in the first culture medium is at least 1 or 2 times higher than the concentration of IFN-γ in the second culture medium.

In some embodiments, the first culture medium and/or the second culture medium further comprises IL-4 and IL-1β.

In some embodiments, the IL-4 in the first culture medium is present in a concentration of about 1-500 ng/ml.

In some embodiments, the IL-4 in the first culture medium is present in a concentration of about 50-150 ng/ml.

In some embodiments, the IL-1β in the first culture medium is present in a concentration of about 1-200 ng/ml.

In some embodiments, the IL-1β in the first culture medium is present in a concentration of about 1-30 ng/ml.

In some embodiments, the first culture medium and/or the second culture medium further comprises IL-21.

In some embodiments, the IL-21 in the first culture medium is present in a concentration of about 1-200 ng/ml.

In some embodiments, the IL-21 in the first culture medium is present in a concentration of about 1-30 ng/ml.

In some embodiments, the first culture medium and/or the second culture medium does not comprise IL-21.

In some embodiments, the first culture medium and/or the second culture medium does not comprise IL-7.

In some embodiments, step (1) further comprises stimulating the γδT cells by an anti-γδTCR antibody.

In some embodiments, the anti-γδTCR antibody specifically binds to the constant chain of TCR gamma or delta chain.

In some embodiments, the anti-γδTCR antibody is a V_HH 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.

In some embodiments, the V_HH 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.

In some embodiments, the anti-γδTCR 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.

In some embodiments, the anti-γδTCR 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.

In some embodiments, the anti-γδTCR antibody comprises an amino sequence that is at least 80%, 90%or 100%identical to SEQ ID NO: 4 and 7.

In some embodiments, the anti-γδTCR antibody is immobilized on a cell culture plate.

In some embodiments, the anti-γδTCR antibody is immobilized on the cell culture plate at 0.1-5 μg/ml per well.

In some embodiments, the first culture medium comprises the anti-γδTCR-specific antibody.

In some embodiments, the first culture medium and/or the second culture medium does not comprise anti-CD28 antibody.

In some embodiments, prior to step (1) , the sample is enriched for γδT cells.

In some embodiments, αβT cells are depleted prior to step (1) , between step (1) and step (2) , or after step (2) .

In some embodiments, NK cells are depleted prior to step (1) , between step (1) and step (2) , or after step (2) .

In some embodiments, the sample is selected from blood, peripheral blood, umbilical cord blood, lymphoid tissue, bone marrow, spleen, induced pluripotent stem cells or skin tissues.

In some embodiments, the sample comprises peripheral blood mononuclear cells (PBMCs) .

In some embodiments, the cells are cultured for 3-9 days during step (1) .

In some embodiments, the cells are cultured for 6-12 days during step (2) .

In some embodiments, the cells are collected prior to 35 days of culturing.

In some embodiments, the cells are collected prior to 21 days of culturing.

In some embodiments, the first and/or the second culture medium comprises L-glutamine, streptomycin sulfate, and gentamicin sulfate.

In some embodiments, the first and/or second culture media comprises serum.

In some embodiments, the serum is present in an amount from about 0.5 to about 25%by volume.

In some embodiments, the serum is human AB serum.

In some embodiments, the first and/or second culture media comprises human serum replacement.

In some embodiments, the human serum replacement is human platelet lysate (HPL) .

In some embodiments, the method further comprises introducing a heterologous nucleic acid into the cells prior to step (1) .

In some embodiments, the method further comprises introducing a heterologous nucleic acid into the cells between step (1) and step (2) .

In some embodiments, the method further comprises introducing a heterologous nucleic acid into the cells after step (2) .

In some embodiments, the heterologous nucleic acid encodes a chimeric antigen receptor (CAR) or a T cell receptor (TCR) .

In some embodiments, the heterologous nucleic acid is delivered with a lentiviral vector or retroviral vector.

In some embodiments, 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.

In one aspect, the disclosure is related to a method for preparing γδT cells, the method comprising: (1) culturing cells from a sample in a first culture medium comprising IL-15, IFN-γ, IL-2, IL-4 and IL-1β; and (2) culturing the cells obtained in step (1) in a second culture medium comprising IL-15, IFN-γ and IL-2.

In some embodiments, , the disclosure is related to a method for preparing γδT cells, the method comprising: (1) culturing cells from a sample in a first culture medium comprising IL-15, IFN-γ, IL-2, IL-4, IL-1β, and IL-21; and (2) culturing the cells obtained in step (1) in a second culture medium comprising IL-15, IFN-γ and IL-2.

In some embodiments, the first culture medium and/or the second culture medium further comprises an AKT inhibitor (e.g., MK-2206) .

In some embodiments, prior to step (1) , the sample is depleted of αβT cells and NK cells.

In some embodiments, during step (1) the cells are exposed to an anti-γδTCR-specific antibody.

In some embodiments, prior to step (2) , the cells are transfected with a vector encoding an engineered receptor (e.g., CAR) .

In some embodiments, the cells are cultured for 3-9 days during step (1) .

In some embodiments, the cells are cultured for 6-12 days during step (2) .

In some embodiments, the first culture medium and/or second culture medium comprises HPL.

In one aspect, the disclosure is related to a cell preparation prepared using the method described herein.

In one aspect, the disclosure is related to a pharmaceutical composition comprising the cell preparation described herein, and a pharmaceutically acceptable carrier.

In one aspect, the disclosure relates to methods of generating chimeric antigen receptor (CAR) modified polyclonal γδT cells with Vδ1+, Vδ2+ and Vδ1-Vδ2-sub-types. The methods may be feeder cell free, serum free, and thus suitable for clinically usage. The resulting polyclonal γδT cells may exhibit potent intrinsic cytotoxicity towards a variety of tumors, including hematologic tumors and solid tumors. The CAR modified polyclonal γδT cells may have better efficacy in vivo and in vitro, providing long-term protection from relapse. Also, the CAR-polyclonal γδT produced by the current methods exhibit better proliferation in a MLR system, revealing resistance to host versus graft disease (HvGD) .

The methods as described herein can provide an off-the-shelf CAR-T cell therapy method with a lower cost and more standardized production process, which can be applied to a wider range of hematological tumors or solid tumors disease patients, reducing remission rates and improving drug effect durability.

As used herein, the terms “approximately” and “about, ” as applied to one or more values of interest, refer to a value that is similar to a stated reference value. In certain embodiments, the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11 %, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1 %, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100%of a possible value) . For example, when used in the context of an amount of a given compound in a composition, “about” may mean +/-10%of the recited value. For instance, a culture medium including about 100 ng/ml of a given compound may include 90～110 ng/ml of the compound.

As used herein, the term “IL-15” refers to a polypeptide derived from a wild-type IL-15 or a functional variant thereof. The IL-15 may be a wildtype IL-15 (e.g., human IL-15) . The IL-15 may have one or more mutations (e.g., insertions, deletions, or substitutions) . The IL-15 may be a human IL-15. The IL-15 may be a recombinant IL-15.

As used herein, the term “IL-4” refers to a polypeptide derived from a wild-type IL-4 or a functional variant thereof. The IL-4 may be a wildtype IL-4 (e.g., human IL-4) . The IL-4 may have one or more mutations (e.g., insertions, deletions, or substitutions) . The IL-4 may be a human IL-4. The IL-4 may be a recombinant IL-4.

As used herein, the term “IL-1β” refers to a polypeptide derived from a wild-type IL-1β or a functional variant thereof. The IL-1β may be a wildtype IL-1β (e.g., human IL-1β) . The IL-1β may have one or more mutations (e.g., insertions, deletions, or substitutions) . The IL-1βmay be a human IL-1β. The IL-1β may be a recombinant IL-1β.

As used herein, the term “IFN-γ” refers to a polypeptide derived from a wild-type IFN-γ or a functional variant thereof. The IFN-γ may be a wildtype IFN-γ (e.g., human IFN-γ) . The IFN-γ may have one or more mutations (e.g., insertions, deletions, or substitutions) . The IFN-γmay be a human IFN-γ. The IFN-γ may be a recombinant IFN-γ.

As used herein, the term “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.

As used herein, the term “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.

As used herein, 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. Such a compound may, for example, act by inhibition of up-stream effectors/activators of AKT in both the PI3K pathway and the mTOR pathway. Yet further, the inhibitor of AKT may act to prevent or reduce the transcription, translation, post-translational processing and/or mobilization of AKT (i.e. reduce the expression of AKT) , or an upstream activator of the expression of AKT.

As used herein, the term “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. Also included are 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. 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. The term “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.

As used herein, 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) . In addition to humans, patients include but are not limited to mice, rats, hamsters, guinea-pigs, rabbits, ferrets, cats, dogs, and primates. Included are, for example, non-human primates (e.g., monkey, chimpanzee, gorilla, and the like) , rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits) , lagomorphs, swine (e.g., pig, miniature pig) , equine, canine, feline, bovine, and other domestic, farm, and zoo animals.

As used herein, the terms “polypeptide, ” “peptide, ” and “protein” are used interchangeably to refer to polymers of amino acids of any length of at least two amino acids.

As used herein, the term “chimeric antigen receptor” or “CAR” as used herein 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.

As used herein, the term “T-cell receptor” or “TCR” as used herein 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 TCRαpolypeptide chain and a TCRβ polypeptide chain. The TCR may comprise a TCRγ polypeptide chain and a TCRδ polypeptide chain. The TCR may specifically bind a tumor antigen. “TCR-T” refers to a T cell that expresses a recombinant TCR. Expression of a heterologous antigen receptor, such as a heterologous 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.

As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. 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. (1989) Nature 342: 878-883) , AbM numbering system (Martin, in Antibody Engineering, Vol. 2, Chapter 3, Springer Verlag) or IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003) . For a given antibody, those skilled in the art can easily identify the CDRs defined by each numbering system. Moreover, the correspondence between different numbering systems is well known to those skilled in the art (e.g., see Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003) . 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.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Methods and materials are described herein for use in the present disclosure; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

DESCRIPTION OF DRAWINGS

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 γδT cells prepared under different conditions (with or without IL-2 and/or IL-7) . CAR-expressing Vδ1 T cells ( “CAR-Vδ1” ) and CAR-expressing Vδ2 T cells ( “CAR-Vδ2” ) 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 γδT cells prepared under different conditions (with or without IL-4 and/or IL-1β) .

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 γδT 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 γδT 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 γδT 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 γδT 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 γδT cells (1.5×10⁴ cells per well) , NCI-H929 tumor cells (1.5×10⁴ cells per well) and allogeneic/autologous PBMCs (1.5×10⁶ 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 αβ T cells. FIG. 7B shows the cell number of CAR γδT 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 γδT cells.

FIGS. 9A-9D show the in vivo anti-tumor effects of the γδT 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.

[Corrected under Rule 26, 20.12.2024]
This application relevant amino acid sequences are as follows:

[Corrected under Rule 26, 20.12.2024]

[Corrected under Rule 26, 20.12.2024]
This application relevant complementarity determining region (CDR) amino acid sequences are as follows:

[Corrected under Rule 26, 20.12.2024]

DETAILED DESCRIPTION

In humans, γδT 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 γδT cell receptors (γδTCRs) , and γδT cells and can be directly activated via the recognition of an antigen by either the γδTCR or other, non-TCR proteins, acting independently or together to activate γδT cell effector functions. γδT 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. Unlike αβTCRs, which recognize antigens bound to major histocompatibility complex molecules (MHC) , γδTCRs 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.

γδT 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 αβT cells, the γδTCR recognizes antigens in a MHC-independent manner, thus paving the way for the use of γδT cells as allogeneic, “off-the-shelf” products to treat cancer, since it does not cause GvHD.

Human γδT cells consist of three major populations, according to their Vδ chains. There are Vδ1, Vδ2 and Vδ3 cells. Typically, Vδ2 T cells are paired with Vγ9 chains within γδTCR complex and are primarily distributed in peripheral blood, while Vδ1 T cells are paired with Vγ2/3/4/5/8/9 within γδTCR complex and can be found in peripheral blood, skin, gut, spleen and liver owing to its diversity. A detailed discussion of γδT cells and their application in cancer immunotherapy can be found in, e.g., Deng, Jiechu, and Hongna Yin. "Gamma delta (γδ) T cells in cancer immunotherapy; where it comes from, where it will go? . " European Journal of Pharmacology 919 (2022) : 174803; Ganapathy, Thamizhselvi, et al. "CAR γδT cells for cancer immunotherapy. Is the field more yellow than green? . " Cancer Immunology, Immunotherapy 72.2 (2023) : 277-286; Sebestyen, Zsolt, et al. "Translating gammadelta (γδ) T cells and their receptors into cancer cell therapies. " Nature reviews Drug discovery 19.3 (2020) : 169-184, each of which is incorporated herein in its entirety by reference.

The present disclosure provides improved methods for culturing and expanding γδT cells, after testing multiple combinations of cytokines for their capacity to culture and expand peripheral blood γδT cells in culture. γδT cells can be selectively expanded in vitro by culturing these cells in two phases. In the first phase, these cells can be cultured in a first culture medium comprising interleukin-15 (IL-15) , interferon-γ (IFN-γ) , and interleukin-2 (IL-2) . In the second phase, these cells can be expanded in a second culture medium containing IL-15, IFN-γ, and IL-2. These cells can also be isolated, cultured and expanded in culture in the absence of feeder cells.

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 γδT cells for use in adoptive immunotherapy of cancer, and in a variety of other therapeutic applications.

The γδT cells obtained by the method described herein may be used in cell therapies. γδT cells are consider as a first line of defense against infectious pathogens. In addition, γδT cells possess intrinsic cytolytic activity against transformed cells of various origins including B-cell lymphomas, sarcomas and carcinomas. As a result, the γδT 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.

Overview of γδT cells

In the mid-1980s, the unexpected discovery of T lymphocytes bearing T cell receptors (TCRs) composed of γ and δ (rather than α and β) chains led to the identification of the distinct γδT cell lineage.

These γδT cell are infrequent and account for less 10%of human peripheral blood lymphocytes but are substantially enriched in epithelial tissues of healthy individuals. γδT cells develop alongside αβT cells from shared thymic progenitors, although functional activation of γδT cells at peripheral sites occurs more rapidly than that of conventional αβT cells. The reason is γδT 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, γδT cells are usually considered a first-line surveillance mechanism against infection and tumors. Recognition and subsequent killing of tumor is achieved upon ligation of antigens to heterodimers of g and d T-cell receptor (TCR) chains. The human TCR variable (V) region defines 14 unique Vγ alleles (TRGV) , 3 unique Vδ alleles (TRDV1, TRDV2, and TRDV3) , and 5 Vδ alleles that share a common nomenclature with Vαalleles (TRDV4/TRAV14, TRDV5/TRAV29, TRDV6/TRAV23, TRDV7/TRAV36, and TRDV8/TRAV38-2; ) . Of all eight Vδ variants, Vδ1, Vδ2 and Vδ3 are clearly the most used gene segments and are, therefore, used to classify γδT cell subtypes.

So far, much attention has been given to the predominant subset of circulating γδT cells, the Vδ2 T cells. Vδ2 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.

In the past several decades, various protocols have been developed and successfully used to generate clinical scale quantities of Vγ9Vδ2 T cells in vitro for clinical applications. Zoledronate plus IL-2 or other cytokines are the most general ways. Although remarkably safe have been proved, Vγ9Vδ2 T-cell–based trials have shown a very limited benefit in cancer patients. suggesting room for improvement in the therapeutical use of γδT cells.

Vδ1 T cells are not well study as Vδ2 T cells to date. Different from Vδ2 T cells, Vδ1 T cells account for only up to one-third of circulating γδT cells, but they preferentially reside in peripheral tissues including the gut epithelium, dermis, spleen and liver, where they are the predominant γδT cell subset and contribute to tissue homeostasis. Different ligands have been identified as being recognized by certain Vδ1 TCRs, such as the MHC-like proteins of the CD1 family, including the lipid-presenting proteins CD1c and CD1d. An interesting correlation was found between the increase in number of donor-derived peripheral blood Vδ1+ T cells and improved 5-10-year disease-free survival following bone marrow transplantation for acute lymphoblastic leukaemia. Also, γδ CAR-T cells may remain detectable for more than ten years.

Vδ3 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 γδT cell subset seems to share functional similarities with Vδ1 T cells, including the ability to recognize glycolipids presented by CD1d on target cells.

Samples

The present disclosure provides methods for selectively culturing and expanding γδT 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 γδT cells. The samples can be enriched for γδT cells.

The sample can be any sample that contains γδT 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. For example, 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.

αβT cells in the sample may be depleted. αβT cells in the sample may be depleted using antibodies targeting αβT cells. The antibodies targeting αβT cells may be linked to magnetic beads. αβT cells may be removed along with these magnetic beads. αβT cells in the sample may be depleted using a TCRαβ 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.

αβT cells in the sample may be depleted using a TCRαβ 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.

αβT 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.

Methods of preparing γδT cells

In one aspect, the present disclosure provided methods to produce CAR-γδT cells with high purity, high expansion rate and high transduction rate for clinical use and production. Such cells display high activation, low exhaustion and predominantphenotype. In vitro validation shows superior anti-tumor activity and safer profile than cells obtained from some other existing methods.

In one aspect, the methods for expanding human γδT cells have a culturing phase and an expanding phase. In the culturing phase, these cells can be cultured in a cell culture medium comprising one or more cytokines selected from interleukin-15 (IL-15) , interferon-γ (IFN-γ) , and interleukin-2 (IL-2) . The cells may be stimulated by a γδTCR-specific antibody. In the expanding phase, these cells can be expanded in a cell expansion culture medium containing one or more cytokines selected from IL-15, IFN-γ, 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.

Accordingly, in a first aspect, the method for culturing and expanding γδT cells in a sample comprising:

(1) culturing cells in the sample in a first culture medium comprising one or more cytokines selected from IL-15, IFN-γ, and IL-2; and

(2) culturing the cells obtained in step (1) in a second culture medium comprising one or more cytokines selected from IL-15, IFN-γ, and IL-2.

During step (1) , the cells may be stimulated by a γδTCR-specific antibody. The first culture medium may be located in a container (e.g., cell culture plate) that is coated with a γδTCR-specific antibody. The γδTCR-specific antibody may be AS287963, AS281850, AS287435 or AS288180 (an anti-γδTCR antibody, SEQ ID NO: 1, 3, 4 and 7) .

The first culture medium may comprise 1, 2, 3, 4, 5, 6, or more than 6 cytokines. The first culture medium may comprise only 1, only 2, only 3, only 4, only 5, or only 6 cytokines. The first culture medium may comprise IL-2. The first culture medium may comprise IL-15. The first culture medium may comprise IFN-γ. The first culture medium may comprise IL-4. The first culture medium may comprise IL-1β. The first culture medium may comprise IL-21. The first culture medium may comprise or consist of IL-2, IL-15, and IFN-γ. The first culture medium may comprise or consist of IL-15, IFN-γ, IL-2, and IL-1β. The first culture medium may comprise or consist of IL-15, IFN-γ, IL-2, IL-1β, and IL-4. The first culture medium may comprise or consist of IL-15, IFN-γ, IL-2, IL-1β, 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.

The second culture medium may comprise 1, 2, 3, 4, or more than 4 cytokines. The second culture medium comprises only 1, only 2, only 3, only 4, or only 5 cytokines. The second culture medium may comprise IL-2. The second culture medium may comprise IL-15. The second culture medium may comprise INF-γ. The second culture medium may comprise IL-4. The second culture medium may comprise IL-1β. The second culture medium may comprise IL-21. The second culture medium may comprise or consist of IL-2 and IL-15. The second culture medium may comprise or consist of IL-2, IL-15, and INF-γ. The second culture medium may lack IL-7, IL-18, and/or IL-12. The second culture medium may lack IL-21.

IL-4 may be present in an amount from about 1 to about 1000 ng/ml. Optionally, IL-4 may be present in an amount from about 2 to about 500 ng/ml. Optionally, IL-4 may be present in an amount from about 20 to about 200 ng/ml. Optionally, IL-4 may be present in the amount of about 50-150 ng/ml. Optionally, IL-4 may be present in an amount of about 50-150 ng/ml in the first culture medium. IL-4 may be present in an amount that is greater than 1 ng/ml, greater than 2 ng/ml, greater than 5 ng/ml, greater than 10 ng/ml, greater than 20 ng/ml, greater than 30 ng/ml, greater than 40 ng/ml, greater than 50 ng/ml, greater than 60 ng/ml, greater than 70 ng/ml, greater than 80 ng/ml, greater than 90 ng/ml, greater than 100 ng/ml, greater than 110 ng/ml, or greater than 120 ng/ml. IL-4 may be present in an amount that is less than 1 ng/ml, less than 2 ng/ml, less than 5 ng/ml, less than 10 ng/ml, less than 20 ng/ml, less than 30 ng/ml, less than 40 ng/ml, less than 50 ng/ml, less than 60 ng/ml, less than 70 ng/ml, less than 80 ng/ml, less than 90 ng/ml, less than 100 ng/ml, less than 110 ng/ml, or less than 120 ng/ml. IL-4 may be present in an amount that is about 1 ng/ml, about 2 ng/ml, about 5 ng/ml, about 10 ng/ml, about 20 ng/ml, about 30 ng/ml, about 40 ng/ml, about 50 ng/ml, about 60 ng/ml, about 70 ng/ml, about 80 ng/ml, about 90 ng/ml, about 100 ng/ml, about 110 ng/ml, or about 120 ng/ml. IL-4 may be present in an amount that is from about 1 to about 1000 ng/ml, from about 10 to 100 ng/ml, from about 20 to 200 ng/ml, from about 30 to 300 ng/ml, from about 40 to 400 ng/ml, from about 50 to 500 ng/ml, from about 50 to 150 ng/ml, from about 80 to 120 ng/ml, or from about 90 to 100 ng/ml.

IL-15 may be present in an amount from about 1 to about 500 ng/ml. Optionally, IL-15 may be present in an amount from about 2 to about 200 ng/ml. Optionally, IL-15 may be present in an amount from about 5 to about 100 ng/ml. Optionally, in the first culture medium, IL-15 may be present in an amount of about 1-30 ng/mL. Optionally, in the second culture medium, IL-15 may be present in an amount of about 1-200 ng/mL. IL-15 may be present in an amount that is greater than 1 ng/ml, greater than 2 ng/ml, greater than 5 ng/ml, greater than 10 ng/ml, greater than 20 ng/ml, greater than 30 ng/ml, greater than 40 ng/ml, greater than 50 ng/ml, greater than 60 ng/ml, greater than 70 ng/ml, greater than 80 ng/ml, greater than 90 ng/ml, greater than 100 ng/ml, greater than 110 ng/ml, or greater than 120 ng/ml. IL-15 may be present in an amount that is less than 1 ng/ml, less than 2 ng/ml, less than 5 ng/ml, less than 10 ng/ml, less than 20 ng/ml, less than 30 ng/ml, less than 40 ng/ml, less than 50 ng/ml, less than 60 ng/ml, less than 70 ng/ml, less than 80 ng/ml, less than 90 ng/ml, less than 100 ng/ml, less than 110 ng/ml, or less than 120 ng/ml. IL-15 may be present in an amount that is about 1 ng/ml, about 2 ng/ml, about 5 ng/ml, about 10 ng/ml, about 20 ng/ml, about 30 ng/ml, about 40 ng/ml, about 50 ng/ml, about 60 ng/ml, about 70 ng/ml, about 80 ng/ml, about 90 ng/ml, about 100 ng/ml, about 110 ng/ml, or about 120 ng/ml. IL-15 may be present in an amount that is from about 1 to about 1000 ng/ml, from about 10 to 100 ng/ml, from about 20 to 200 ng/ml, from about 30 to 300 ng/ml, from about 40 to 400 ng/ml, from about 50 to 500 ng/ml, from about 50 to 150 ng/ml, from about 50 to 90 ng/ml, or from about 60 to 80 ng/ml.

IFN-γ may be present in an amount from about 1 to about 1000 ng/ml. Optionally, IFN-γ may be present in an amount from about 2 to about 500 ng/ml. Optionally, IFN-γ may be present in an amount from about 20 to about 200 ng/ml. Optionally, in the first culture medium IFN-γ may be present in an amount of about 50-150 ng/mL. Optionally, in the second culture medium, IFN-γmay be present in an amount of about 1-100 ng/mL. IFN-γ may be present in an amount that is greater than 1 ng/ml, greater than 2 ng/ml, greater than 5 ng/ml, greater than 10 ng/ml, greater than 20 ng/ml, greater than 30 ng/ml, greater than 40 ng/ml, greater than 50 ng/ml, greater than 60 ng/ml, greater than 70 ng/ml, greater than 80 ng/ml, greater than 90 ng/ml, greater than 100 ng/ml, greater than 110 ng/ml, or greater than 120 ng/ml. IFN-γ may be present in an amount that is less than 1 ng/ml, less than 2 ng/ml, less than 5 ng/ml, less than 10 ng/ml, less than 20 ng/ml, less than 30 ng/ml, less than 40 ng/ml, less than 50 ng/ml, less than 60 ng/ml, less than 70 ng/ml, less than 80 ng/ml, less than 90 ng/ml, less than 100 ng/ml, less than 110 ng/ml, or less than 120 ng/ml. IFN-γ may be present in an amount that is about 1 ng/ml, about 2 ng/ml, about 5 ng/ml, about 10 ng/ml, about 20 ng/ml, about 30 ng/ml, about 40 ng/ml, about 50 ng/ml, about 60 ng/ml, about 70 ng/ml, about 80 ng/ml, about 90 ng/ml, about 100 ng/ml, about 110 ng/ml, or about 120 ng/ml. IFN-γ may be present in an amount that is from about 1 to about 1000 ng/ml, from about 10 to 100 ng/ml, from about 20 to 200 ng/ml, from about 30 to 300 ng/ml, from about 40 to 400 ng/ml, from about 50 to 500 ng/ml, from about 50 to 150 ng/ml, from about 50 to 90 ng/ml, or from about 60 to 80 ng/ml.

IL-1β may be present in an amount from about 1 to about 500 ng/ml. Optionally, IL-1β may be present in an amount from about 2 to about 200 ng/ml. Optionally, IL-1β may be present in an amount from about 5 to about 100 ng/ml. Optionally, IL-1β may be present in an amount of about 1-30 ng/ml. Optionally, IL-1β may be present in an amount of about 1-30 ng/ml in the first culture medium. IL-1β 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-1β may be present in an amount that is less than 1 ng/ml, less than 2 ng/ml, less than 3 ng/ml, less than 4 ng/ml, less than 5 ng/ml, less than 6 ng/ml, less than 7 ng/ml, less than 8 ng/ml, less than 9 ng/ml, less than 10 ng/ml, less than 11 ng/ml, less than 12 ng/ml, less than 13 ng/ml, less than 14 ng/ml, less than 15 ng/ml, less than 16 ng/ml, less than 17 ng/ml, less than 18 ng/ml, less than 19 ng/ml, less than 20 ng/ml, less than 25 ng/ml, or less than 30 ng/ml. IL-1β may be present in an amount that is about 1 ng/ml, about 2 ng/ml, about 3 ng/ml, about 4 ng/ml, about 5 ng/ml, about 6 ng/ml, about 7 ng/ml, about 8 ng/ml, about 9 ng/ml, about 10 ng/ml, about 11 ng/ml, about 12 ng/ml, about 13 ng/ml, about 14 ng/ml, about 15 ng/ml, about 16 ng/ml, about 17 ng/ml, about 18 ng/ml, about 19 ng/ml, about 20 ng/ml, about 25 ng/ml, or about 30 ng/ml. IL-1β 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.

IL-2 may be present in an amount from about 1 to about 500 IU/ml. Optionally, IL-2 may be present in an amount from about 2 to about 200 IU/ml. Optionally, IL-2 may be present in an amount from about 5 to about 100 IU/ml. Optionally, in the first culture medium, IL-2 may be present in an amount of about 50-150 IU/ml. Optionally, in the second culture medium, IL-2 may be present in an amount of about 50-150 IU/ml. IL-2 may be present in an amount that is greater than 1 IU/ml, greater than 2 IU/ml, greater than 5 IU/ml, greater than 10 IU/ml, greater than 20 IU/ml, greater than 30 IU/ml, greater than 40 IU/ml, greater than 50 IU/ml, greater than 60 IU/ml, greater than 70 IU/ml, greater than 80 IU/ml, greater than 90 IU/ml, greater than 100 IU/ml, greater than 110 IU/ml, or greater than 120 IU/ml. IL-2 may be present in an amount that is less than 1 IU/ml, less than 2 IU/ml, less than 5 IU/ml, less than 10 IU/ml, less than 20 IU/ml, less than 30 IU/ml, less than 40 IU/ml, less than 50 IU/ml, less than 60 IU/ml, less than 70 IU/ml, less than 80 IU/ml, less than 90 IU/ml, less than 100 IU/ml, less than 110 IU/ml, or less than 120 IU/ml. IL-2 may be present in an amount that is about 1 IU/ml, about 2 IU/ml, about 5 IU/ml, about 10 IU/ml, about 20 IU/ml, about 30 IU/ml, about 40 IU/ml, about 50 IU/ml, about 60 IU/ml, about 70 IU/ml, about 80 IU/ml, about 90 IU/ml, about 100 IU/ml, about 110 IU/ml, or about 120 IU/ml. IL-2 may be present in an amount that is from about 1 to about 1000 IU/ml, from about 10 to 100 IU/ml, from about 20 to 200 IU/ml, from about 30 to 300 IU/ml, from about 40 to 400 IU/ml, from about 50 to 500 IU/ml, from about 50 to 150 IU/ml, from about 50 to 90 IU/ml, or from about 50 to 60 IU/ml.

IL-21 may be present in an amount from about 1 to about 500 ng/ml. Optionally, IL-21 may be present in an amount from about 2 to about 100 ng/ml. Optionally, IL-21 may be present in an amount from about 5 to about 15 ng/ml. Optionally, IL-21 may be present in an amount of about 1-30 ng/ml. Optionally, IL-21 may be present in an amount of about 1-30 ng/ml in the first culture medium. IL-21 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-21 may be present in an amount that is less than 1 ng/ml, less than 2 ng/ml, less than 3 ng/ml, less than 4 ng/ml, less than 5 ng/ml, less than 6 ng/ml, less than 7 ng/ml, less than 8 ng/ml, less than 9 ng/ml, less than 10 ng/ml, less than 11 ng/ml, less than 12 ng/ml, less than 13 ng/ml, less than 14 ng/ml, less than 15 ng/ml, less than 16 ng/ml, less than 17 ng/ml, less than 18 ng/ml, less than 19 ng/ml, less than 20 ng/ml, less than 25 ng/ml, or less than 30 ng/ml. IL-21 may be present in an amount that is about 1 ng/ml, about 2 ng/ml, about 3 ng/ml, about 4 ng/ml, about 5 ng/ml, about 6 ng/ml, about 7 ng/ml, about 8 ng/ml, about 9 ng/ml, about 10 ng/ml, about 11 ng/ml, about 12 ng/ml, about 13 ng/ml, about 14 ng/ml, about 15 ng/ml, about 16 ng/ml, about 17 ng/ml, about 18 ng/ml, about 19 ng/ml, about 20 ng/ml, about 25 ng/ml, or about 30 ng/ml. IL-21 may be present in an amount that is from about 1 to about 1000 ng/ml, from about 2 to 100 ng/ml, from 2 to 50 ng/ml, from 5 to 30 ng/ml, from about 5 to 15 ng/ml, from about 5 to 10 ng/ml, or from about 6 to 8 ng/ml.

Further, the first culture medium or the second culture medium may comprise an AKT pathway inhibitor (e.g., MK-2206) . The chemical structure of MK-2206 is shown below.

The AKT pathway inhibitor (e.g., MK-2206) may be present in an amount from about 1 to about 10,000 ng/ml. Optionally, the AKT pathway inhibitor (e.g., MK-2206) may be present in an amount from about 10 to about 10000 ng/ml. Optionally, the AKT pathway inhibitor (e.g., MK-2206) may be present in an amount from about 100 to about 5000 ng/ml., the AKT pathway inhibitor (e.g., MK-2206) may be present in an amount of about 100-10000 ng/ml in the first culture medium. Optionally, the AKT pathway inhibitor (e.g., MK-2206) may be present in an amount of about 100-10000 ng/ml in the second culture medium. The AKT pathway inhibitor (e.g., MK-2206) may be present in an amount that is greater than 1 ng/ml, greater than 2 ng/ml, greater than 5 ng/ml, greater than 10 ng/ml, greater than 20 ng/ml, greater than 50 ng/ml, greater than 100 ng/ml, greater than 200 ng/ml, greater than 500 ng/ml, greater than 750 ng/ml, greater than 1000 ng/ml, greater than 1250 ng/ml, greater than 1500 ng/ml, greater than 2000 ng/ml, or greater than 2500 ng/ml. The AKT pathway inhibitor (e.g., MK-2206) may be present in an amount that is less than 1 ng/ml, less than 2 ng/ml, less than 5 ng/ml, less than 10 ng/ml, less than 20 ng/ml, less than 50 ng/ml, less than 100 ng/ml, less than 200 ng/ml, less than 500 ng/ml, less than 750 ng/ml, less than 1000 ng/ml, less than 1250 ng/ml, less than 1500 ng/ml, less than 2000 ng/ml, or less than 2500 ng/ml. The AKT pathway inhibitor (e.g., MK-2206) may be present in an amount that is about 10 ng/ml, about 20 ng/ml, about 50 ng/ml, about 100 ng/ml, about 200 ng/ml, about 500 ng/ml, about 750 ng/ml, about 1000 ng/ml, about 1250 ng/ml, about 1500 ng/ml, about 2000 ng/ml, or about 2500 ng/ml. The AKT pathway inhibitor (e.g., MK-2206) may be present in an amount that is from about 1 to about 10,000 ng/ml, from about 10 to 5000 ng/ml, from about 100 to 5000 ng/ml, from about 500 to 5000 ng/ml, from about 500 to 2000 ng/ml, from about 750 to 2000 ng/ml, from about 750 to 1500 ng/ml, from about 750 to 1250 ng/ml, or from about 900 to 1100 ng/ml.

Further, the first culture medium or the second culture medium may comprise a serum replacement. The first culture medium or the second culture medium may comprise a human platelet lysate (HPL) . HPL may be present in an amount from about 1 to about 50% (v/v) . Optionally, HPL may be present in an amount from about 2 to about 40%. Optionally, HPL may be present in an amount from about 5 to about 20%. Optionally, HPL may be present in the amount of about 10%. Optionally, HPL may be present in an amount of about 10%in the first culture medium. Optionally, HPL may be present in an amount of about 10%in the second culture medium. 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%. HPL may be present in an amount that is 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 15%, about 20%, or about 25%. HPL may be present in an amount that is from about 1 to about 50%, from about 2 to 40%, from about 5 to 30%, from about 5 to 20%, from about 7 to 15%, from about 8 to 13%, from about 8 to 11 %, or from about 9 to 11%.

The first culture medium may further comprise a γδTCR-specific antibody (e.g., SEQ ID NO: 1, 3, 4 and 7) . The γδTCR-specific antibody may be present in an amount that is from about 0.1 to about 100 μg/ml. Optionally, the γδTCR-specific antibody may be present in an amount from about 0.1 to about 10 μg/ml. Optionally, the γδTCR-specific antibody may be present in an amount from about 0.1 to about 5 μg /ml. Optionally, in the first culture medium, the γδTCR-specific antibody may be present in an amount of about 0.1-5 μg/mL. The first culture medium may be in a container (e.g., cell culture plate) that is coated with a γδTCR-specific antibody. The γδTCR-specific antibody may be immobilized on the cell culture plate at from about 1 to about 5000 ng/ml. Optionally, the γδTCR-specific antibody may be immobilized on the cell culture plate at from about 50 to about 1000 ng/ml. Optionally, the γδTCR-specific antibody may be immobilized on the cell culture plate at from about 200 to about 1000 ng/ml. Optionally, the γδTCR-specific antibody may be immobilized on the cell culture plate at about 100-5000 ng/mL.

The concentration of IL-15 in the second culture medium may be 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-γ in the first culture medium may be at least 1, 2, or 3 times higher than the concentration of IFN-γ in the second culture medium.

The cells may be cultured in the first culture medium for a period ranging from about 2 days to about 21 days. Optionally, from about 3 days to about 14 days. Optionally, from about 4 days to 8 days. Optionally, the cells may be cultured in the first culture medium for 3-9 days. The cells may be cultured in the first culture medium for more than 1 day, more than 2 days, more than 3 days, more than 4 days, more than 5 days, more than 6 days, more than 7 days, more than 8 days, more than 9 days, more than 10 days, more than 11 days, more than 12 days, more than 13 days, or more than 14 days. The cells may be cultured in the first culture medium for less than 1 day, less than 2 days, less than 3 days, less than 4 days, less than 5 days, less than 6 days, less than 7 days, less than 8 days, less than 9 days, less than 10 days, less than 11 days, less than 12 days, less than 13 days, or less than 14 days. The cells may be cultured in the first culture medium for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, or about 14 days. The cells may be cultured in the first culture medium for a period of time ranging from about 2 days to about 21 days, from about 3 days to about 20 days, from about 3 days to about 10 days, from about 3 days to about 7 days, from about 2 days to about 7 days, from about 3 days to about 6 days, or from about 4 days to about 6 days.

The cells may be cultured in the second culture medium for a period ranging from about 2 days to about 21 days. Optionally, from about 3 days to about 14 days. Optionally, from about 6 days to 12 days. Optionally, the cells may be cultured in the second culture medium for 6-12 days. The cells may be cultured in the second culture medium for more than 1 day, more than 2 days, more than 3 days, more than 4 days, more than 5 days, more than 6 days, more than 7 days, more than 8 days, more than 9 days, more than 10 days, more than 11 days, more than 12 days, more than 13 days, or more than 14 days. The cells may be cultured in the second culture medium for less than 1 day, less than 2 days, less than 3 days, less than 4 days, less than 5 days, less than 6 days, less than 7 days, less than 8 days, less than 9 days, less than 10 days, less than 11 days, less than 12 days, less than 13 days, or less than 14 days. The cells may be cultured in the second culture medium for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, or about 14 days. The cells may be cultured in the second culture medium for a period of time ranging from about 2 days to about 21 days, from about 3 days to about 20 days, from about 3 days to about 15 days, from about 5 days to about 15 days, from about 5 days to about 12 days, from about 7 days to about 10 days, or from about 8 days to about 10 days.

The culture medium may be replenished as needed. This can be achieved through the addition of fresh culture medium to the first culture medium or the second culture medium, optionally after the removal of a fraction of the first culture medium or the second culture medium. This can be done by centrifuging the cells, removing (e.g., decanting) a fraction of the first culture medium or the second culture medium and resuspending the cells in the first culture medium or the second culture medium. The replenishment may involve the removal of at least 3/4 of the previous culture medium.

The first culture medium and the second culture medium may comprise a base medium. Any suitable mammalian cell culture medium such as AIM-V^TM, X-VIVO, TexMACS, RPMI 1640, OPTMIZER CTS^TM (Gibco, Life Technologies) , EXVIVO-10, EXVIVO-15 or EXVIVO-20 (Lonza) may be used as the base medium. The first cell culture medium and/or the second cell culture medium may comprise L-glutamine, streptomycin sulfate, and gentamicin sulfate. The first cell culture medium and/or the second cell culture medium may comprise L-glutamine, 50 μg/mL streptomycin sulfate, and 10 μg/mL gentamicin sulfate. The mammalian cell culture medium may comprise serum or plasma. The first cell culture medium and/or the second cell culture medium may contain a base medium in an amount that is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95%by volume. 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 γδT 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. from the same patient) may be used to avoid the introduction of hazardous products (e.g. viruses) into that patient. 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 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 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) ) . The 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.

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. In particular, the sample or fraction thereof may be enriched for T cells, or enriched for γδT cells, depleted of NK cells or depleted of aβT cells. Optionally, prior to culturing the sample in the first culture medium, the sample may be depleted of aβT 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 γδT cells. Examples of 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.

The first culture medium and/or second culture medium may also contain other growth factors, including cytokines that can further enhance the expansion of γδT cells. Examples of other growth factors that can be added include co-stimulatory molecules such as human anti-SLAM antibody, any soluble ligand of CD27, or any soluble ligand of CD7. Any combination of these growth factors can be included in the first or second culture medium, or in both media.

The method of preparing γδT 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-1β (IL-1β) , 50-150 ng/ml interferon-γ (IFN-γ) , 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-γ, and 50-150 IU/ml IL-2.

The method of preparing γδT cells may comprise: (1) culturing cells in the sample in a first culture medium comprising 1-30 ng/ml interleukin-15 (IL-15) , 50-150 ng/ml interferon-γ(IFN-γ) , 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-γ, and 50-150 IU/ml IL-2.

The method of preparing γδT 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-1β (IL-1β) , 50-150 ng/ml interferon-γ (IFN-γ) , 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-γ, and 50-150 IU/ml IL-2.

The method of preparing γδT 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-1β (IL-1β) , 50-150 ng/ml interferon-γ (IFN-γ) , 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-γ, and 50-150 IU/ml IL-2.

The method of preparing γδT cells may comprise: (1) culturing cells in the sample in a first culture medium comprising 1-30 ng/ml interleukin-15 (IL-15) , 50-150 ng/ml interferon-γ(IFN-γ) , 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-γ, and 50-150 IU/ml IL-2.

The method of preparing γδT 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-1β (IL-1β) , about 50 ng/ml interferon-γ (IFN-γ) , 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-γ, and about 25 IU/ml IL-2.

The method of preparing γδT 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-1β (IL-1β) , about 50 ng/ml interferon-γ (IFN-γ) , 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-γ, and about 25 IU/ml IL-2.

The method of preparing γδT 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-1β (IL-1β) , about 100 ng/ml interferon-γ (IFN-γ) , 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-γ, and about 100 IU/ml IL-2.

The method of preparing γδT 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-1β (IL-1β) , about 100 ng/ml interferon-γ(IFN-γ) , 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-γ, and about 100 IU/ml IL-2.

The method of preparing γδT 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-1β (IL-1β) , about 150 ng/ml interferon-γ (IFN-γ) , 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-γ, and about 100 IU/ml IL-2.

The method of preparing γδT 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-1β (IL-1β) , about 100 ng/ml interferon-γ (IFN-γ) , 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-γ, and about 100 IU/ml IL-2.

The method of preparing γδT 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-1β (IL-1β) , about 100 ng/ml interferon-γ(IFN-γ) , 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-γ, and about 100 IU/ml IL-2.

The method of preparing γδT 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-1β (IL-1β) , about 100 ng/ml interferon-γ (IFN-γ, 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-γ, and about 100 IU/ml IL-2.

The method of preparing γδT 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-1β (IL-1β) , about 120 ng/ml interferon-γ (IFN-γ) , 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-γ, and about 100 IU/ml IL-2.

The method of preparing γδT 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-1β (IL-1β) , about 120 ng/ml interferon-γ(IFN-γ) , 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-γ, and about 100 IU/ml IL-2.

The method of preparing γδT 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-1β (IL-1β) , about 100 ng/ml interferon-γ (IFN-γ) , 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-γ, and about 100 IU/ml IL-2.

The method of preparing γδT 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-1β (IL-1β) , about 75 ng/ml interferon-γ (IFN-γ) , 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-γ, and about 100 IU/ml IL-2.

The method of preparing γδT 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-1β (IL-1β) , about 75 ng/ml interferon-γ (IFN-γ) , 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-γ, and about 100 IU/ml IL-2.

The method of preparing γδT 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-1β (IL-1β) , about 75 ng/ml interferon-γ (IFN-γ) , 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-γ, and about 75 IU/ml IL-2.

The method of preparing γδT 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-1β (IL-1β) , about 120 ng/ml interferon-γ (IFN-γ) , 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-γ, and about 100 IU/ml IL-2.

The method of preparing γδT 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-1β (IL-1β) , about 120 ng/ml interferon-γ (IFN-γ) , 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-γ, and about 100 IU/ml IL-2.

The method of preparing γδT 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-1β (IL-1β) , about 75 ng/ml interferon-γ (IFN-γ) , 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-γ, and about 75 IU/ml IL-2.

The method of preparing γδT 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-1β (IL-1β) , about 100 ng/ml interferon-γ (IFN-γ) , 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-γ, and about 100 IU/ml IL-2.

The method of preparing γδT 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-1β (IL-1β) , about 100 ng/ml interferon-γ (IFN-γ) , 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-γ, and about 100 IU/ml IL-2.

The method of preparing γδT 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-1β (IL-1β) , about 100 ng/ml interferon-γ (IFN-γ) , 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-γ, and about 100 IU/ml IL-2.

The method of preparing γδT 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-1β (IL-1β) , about 100 ng/ml interferon-γ (IFN-γ) , 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-γ, and about 100 IU/ml IL-2.

The method of preparing γδT 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-1β (IL-1β) , about 100 ng/ml interferon-γ(IFN-γ) , 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-γ, and about 100 IU/ml IL-2.

The method of preparing γδT 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-1β (IL-1β) , about 100 ng/ml interferon-γ (IFN-γ) , 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-γ, about 100 IU/ml IL-2.

The method of preparing γδT 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-1β (IL-1β) , about 100 ng/ml interferon-γ (IFN-γ) , 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-γ, and about 100 IU/ml IL-2.

The method of preparing γδT 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-1β (IL-1β) , about 100 ng/ml interferon-γ (IFN-γ) , 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-γ, and about 100 IU/ml IL-2.

The method of preparing γδT 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-1β (IL-1β) , about 100 ng/ml interferon-γ (IFN-γ) , 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-γ, and about 100 IU/ml IL-2.

The method of preparing γδT 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-1β (IL-1β) , about 100 ng/ml interferon-γ (IFN-γ) , 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-γ, and about 100 IU/ml IL-2.

The method of preparing γδT 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-1β (IL-1β) , about 100 ng/ml interferon-γ(IFN-γ) , 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-γ, and about 100 IU/ml IL-2.

The method of preparing γδT 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-1β (IL-1β) , about 100 ng/ml interferon-γ (IFN-γ) , 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-γ, and about 100 IU/ml IL-2.

The method of preparing γδT 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-1β (IL-1β) , about 70 ng/ml interferon-γ (IFN-γ) , 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-γ, and about 50 IU/ml IL-2.

The method of preparing γδT 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-1β (IL-1β) , about 70 ng/ml interferon-γ (IFN-γ) , 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-γ, and about 50 IU/ml IL-2.

The method of preparing γδT 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-1β (IL-1β) , about 75 ng/ml interferon-γ (IFN-γ) , 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-γ, and about 100 IU/ml IL-2.

The method of preparing γδT 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-1β (IL-1β) , about 70 ng/ml interferon-γ (IFN-γ) , 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-γ, and about 100 IU/ml IL-2.

The method of preparing γδT 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-1β (IL-1β) , about 70 ng/ml interferon-γ (IFN-γ) , 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-γ, and about 50 IU/ml IL-2.

The method of preparing γδT 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-1β (IL-1β) , about 70 ng/ml interferon-γ (IFN-γ) , 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-γ, and about 50 IU/ml IL-2.

The method of preparing γδT 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-1β (IL-1β) , about 100 ng/ml interferon-γ (IFN-γ) , 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-γ, and about 75 IU/ml IL-2.

The method of preparing γδT 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-1β (IL-1β) , about 70 ng/ml interferon-γ (IFN-γ) , 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-γ, and about 50 IU/ml IL-2.

The method of preparing γδT 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-1β (IL-1β) , about 70 ng/ml interferon-γ (IFN-γ) , 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-γ, and about 75 IU/ml IL-2.

The method of preparing γδT 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-1β (IL-1β) , about 70 ng/ml interferon-γ (IFN-γ) , 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-γ, and about 75 IU/ml IL-2.

The method of preparing γδT 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-1β (IL-1β) , about 100 ng/ml interferon-γ (IFN-γ) , 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-γ, and about 100 IU/ml IL-2.

The method of preparing γδT 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-1β (IL-1β) , about 100 ng/ml interferon-γ (IFN-γ) , 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-γ, and about 50 IU/ml IL-2.

The method of preparing γδT 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-1β (IL-1β) , about 100 ng/ml interferon-γ(IFN-γ) , 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-γ, and about 50 IU/ml IL-2.

The method of preparing γδT 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-1β (IL-1β) , about 70 ng/ml interferon-γ (IFN-γ) , 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-γ, and about 50 IU/ml IL-2.

The method of preparing γδT 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-1β (IL-1β) , about 70 ng/ml interferon-γ (IFN-γ) , 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-γ, and about 50 IU/ml IL-2.

The method of preparing γδT 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-1β (IL-1β) , about 70 ng/ml interferon-γ (IFN-γ) , 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-γ, and about 75 IU/ml IL-2.

The method of preparing γδT 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-1β (IL-1β) , about 70 ng/ml interferon-γ (IFN-γ) , 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-γ, and about 50 IU/ml IL-2.

The method of preparing γδT 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-1β (IL-1β) , about 70 ng/ml interferon-γ (IFN-γ) , 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-γ, and about 50 IU/ml IL-2.

The method of preparing γδT 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-1β (IL-1β) , about 70 ng/ml interferon-γ (IFN-γ) , 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-γ, and about 100 IU/ml IL-2.

The method of preparing γδT 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-1β (IL-1β) , about 100 ng/ml interferon-γ (IFN-γ) , 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-γ, and about 50 IU/ml IL-2.

The method of preparing γδT 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-1β (IL-1β) , about 100 ng/ml interferon-γ(IFN-γ) , 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-γ, and about 50 IU/ml IL-2.

The method of preparing γδT 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-1β (IL-1β) , about 100 ng/ml interferon-γ (IFN-γ) , 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-γ, and about 50 IU/ml IL-2.

The method of preparing γδT 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-1β (IL-1β) , about 100 ng/ml interferon-γ(IFN-γ) , 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-γ, and about 50 IU/ml IL-2.

The method of preparing γδT 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-1β (IL-1β) , about 100 ng/ml interferon-γ (IFN-γ) , 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-γ, and about 50 IU/ml IL-2.

The method of preparing γδT 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-1β (IL-1β) , about 100 ng/ml interferon-γ (IFN-γ) , 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-γ, and about 50 IU/ml IL-2.

The method of preparing γδT 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-1β (IL-1β) , about 100 ng/ml interferon-γ (IFN-γ) , 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-γ, and about 50 IU/ml IL-2.

The method of preparing γδT 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-1β (IL-1β) , about 70 ng/ml interferon-γ (IFN-γ) , 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-γ, and about 75 IU/ml IL-2.

The method of preparing γδT 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-1β (IL-1β) , about 70 ng/ml interferon-γ (IFN-γ) , 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-γ, and about 75 IU/ml IL-2.

The method of preparing γδT 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-1β (IL-1β) , about 100 ng/ml interferon-γ (IFN-γ) , 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-γ, and about 75 IU/ml IL-2.

The method of preparing γδT 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-1β (IL-1β) , about 70 ng/ml interferon-γ (IFN-γ) , 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-γ, and about 100 IU/ml IL-2.

The method of preparing γδT 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-1β (IL-1β) , about 70 ng/ml interferon-γ (IFN-γ) , 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-γ, and about 100 IU/ml IL-2.

The method of preparing γδT 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-1β (IL-1β) , about 70 ng/ml interferon-γ (IFN-γ) , 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-γ, and about 100 IU/ml IL-2.

The first culture medium and/or the second culture medium may further comprise 0.1-10 μg/ml AKT pathway inhibitor. The first culture medium and/or the second culture medium may further comprise 1%-50%by volume HPL.

Prior to step (1) , the sample may be depleted of αβT cells and/or NK cells.

During step (1) , the cells may be exposed to a γδTCR-specific antibody.

Prior to step (2) , the cells may be transfected with a vector encoding 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) .

Depletion of αβT cells and/or NK cells, and enrichment of γδT cells

αβT cells may be depleted at various stages of the method described herein. αβT cells may be depleted prior to the first culture step. αβT cells may be depleted prior to the second culture step. αβT cells may be depleted after the second culture step. αβT cells may be depleted by various means. αβT cells may be depleted by using antibodies recognizing various αβT cell surface markers (e.g., αβTCR) . αβ 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. αβT cells in the sample may be depleted using antibodies targeting αβT cells. The antibodies targeting NK cells may specifically bind to αβTCR. The antibodies targeting αβT cells may be linked to magnetic beads. αβT cells in the sample may be depleted using a TCRαβ 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. The antibodies targeting NK cells may be linked to magnetic beads. NK cells in the sample may be depleted using a CD56+ cells depletion kit (e.g., Miltenyi, 130-050-401) . Both αβT cells and NK cells in the sample may be depleted using a TCRαβ cell depletion kit (e.g., Miltenyi Biotec, 170-070-416) and a CD56+ cells depletion kit (e.g., Miltenyi, 130-050-401) .

γδT cells may be enriched by various means. γδT cells may be directly enriched from a sample, for example, by sorting γδT cells that express one or more cell surface markers with flow cytometry techniques. γδT cells may be directly enriched by using antibodies recognizing various γδT cell surface markers (e.g., γδTCR) . Wild-type γδT cells exhibit numerous antigen recognition, antigen-presentation, co-stimulation, and adhesion molecules that can be associated with a γδT cells. One or more cell surface markers such as specific γδTCRs, antigen recognition, antigen-presentation, ligands, adhesion molecules, or co-stimulatory molecules may be used to isolate a wild-type γδT cell from a sample. Various molecules associated with, or expressed by, a γδT cell may be used to isolate a γδT cell from a sample. γδT 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. γδT cells, or a desired subpopulation of γδT 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 γδT cells can be used to directly isolate a population of γδT 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. For instance, γδT cells can be isolated from a complex sample based on positive or negative expression of CD2, CD3, CD4, CD8, CD24, CD25, CD44, Kit, TCRα, TCRβ, TCRγ, TCRδ, NKG2D, CD70, CD27, CD30, CD 16, CD337 (NKp30) , CD336 (NKp46) , OX40, CD46, CCR7, and other suitable cell surface markers.

γδT 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, αβT cells, B-cells, and NK cells, can be activated and expanded. Enriched γδT cell populations can be generated prior to their specific activation and expansion. Activation and expansion of γδT cells may be performed without the presence of native or engineered APCs. Isolation and expansion of γδT cells from tumor specimens may be performed using immobilized γδT cell mitogens, including antibodies specific to γδTCR, and other γδTCR activating agents, including lectins.

Characteristics of the resulting γδT cells

In another aspect, the present disclosure provides a cell preparation prepared according to the method described herein. The γδT 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) . Optionally, the resulting γδT cell preparation may have a purity that is greater than 80%, optionally greater than 90%, and optionally greater than 95%. The γδT 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 γδT 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 γδT 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 γδT cells can be transfected with a vector encoding an engineered receptor (e.g., CAR or TCR) . The γδT cells may be transfected with a vector encoding an engineered receptor (e.g., CAR or TCR) before the first culturing step. The γδT cells may be transfected with a vector encoding an engineered receptor (e.g., CAR or TCR) after the first culturing step. The γδT 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 γδT cells (e.g., un-transfected or transfected with a CAR expression vector) 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 γδT 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.

Cell viability can be measured by cellometer (e.g., following the methods described in Example 4) . The γδT cells (e.g., un-transfected or transfected with a CAR expression vector) may have a high cell viability. The γδT 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 γδT 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%.

CAR positive rate can be measured by flow cytometry (e.g., following the methods described in Example 4) . The expanded cell culture (e.g., transfected with a CAR expression vector) may comprise an amount of engineered γδT cells (e.g., CAR-T cells) , wherein the engineered γδT cells are engineered to express an antigen recognition moiety (e.g., CAR, TCR) . The expanded cell culture may comprise a percentage of engineered γδT 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 engineered γδT 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 engineered γδT 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 γδT cells prepared according to the method described herein may have a high CAR positive rate. The γδT cells prepared according to the method described herein may have a CAR positive rate of higher than 20%, higher than 30%, higher than 40%, higher than 50%, higher than 60%, higher than 70%, higher than 75%, higher than 80%, higher than 85%, or higher than 90%.

The ratio of γδT 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) may comprise a percentage of γδT 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 γδT 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 γδT 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 expanded cell culture (e.g., un-transfected or transfected with a CAR expression vector) may comprise a percentage of Vδ1 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 Vδ1 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 Vδ1 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 expanded cell culture (e.g., un-transfected or transfected with a CAR expression vector) may comprise a percentage of Vδ2 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 Vδ2 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 Vδ2 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 expanded cell culture (e.g., un-transfected or transfected with a CAR expression vector) may comprise a percentage of Vδ1 and Vδ2 double negative cells ( “DN 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 DN 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 DN 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 cytotoxicity of the γδT cells can be measured by a repetitive tumor challenge assay (e.g., following the methods described in Example 4) . The γδT cells prepared according to the method described herein (e.g., un-transfected or transfected with a CAR expression vector) may have a high cytotoxicity against tumor cells. To evaluate the cytotoxicity of the γδT cells in vitro, the γδT 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 long-term cytotoxicity can be measured by the percentage of target cell lysis. The γδT cells may lead to a target cell lysis percentage 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%, after 1 round, 2 rounds, 3 rounds, 4 rounds, 5 rounds, 6 rounds, 7 rounds, 8 rounds, 9 rounds, 10 rounds, 11 rounds, 12 rounds, 13 rounds, 14 rounds, 15 rounds, 16 rounds, 17 rounds, 18 rounds, 19 rounds, or 20 rounds of stimulation in a re-challenge assay. The γδT cells may lead to a target cell lysis percentage 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%, after 1 round, 2 rounds, 3 rounds, 4 rounds, 5 rounds, 6 rounds, 7 rounds, 8 rounds, 9 rounds, 10 rounds, 11 rounds, 12 rounds, 13 rounds, 14 rounds, 15 rounds, 16 rounds, 17 rounds, 18 rounds, 19 rounds, or 20 rounds of stimulation in a re-challenge assay.

The persistence of the γδT cells can be measured by a repetitive tumor challenge assay (e.g., following the methods described in Example 4) . The γδT cells prepared according to the method described herein (e.g., un-transfected or transfected with a CAR expression vector) may have a prolonged persistence in the presence of tumor cells. To evaluate the amplification and viability of the γδT cells in vitro, the γδT 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 γδT cells may be amplified by 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, greater than 1,000,000 fold, greater than 5,000,000 fold, or greater than 10,000,000 fold, after 1 round, 2 rounds, 3 rounds, 4 rounds, 5 rounds, 6 rounds, 7 rounds, 8 rounds, 9 rounds, 10 rounds, 11 rounds, 12 rounds, 13 rounds, 14 rounds, 15 rounds, 16 rounds, 17 rounds, 18 rounds, 19 rounds, or 20 rounds of stimulation in a re-challenge assay. The γδT cells may be amplified by 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, less than 1,000,000 fold, less than 5,000,000 fold, or less than 10,000,000 fold, after 1 round, 2 rounds, 3 rounds, 4 rounds, 5 rounds, 6 rounds, 7 rounds, 8 rounds, 9 rounds, 10 rounds, 11 rounds, 12 rounds, 13 rounds, 14 rounds, 15 rounds, 16 rounds, 17 rounds, 18 rounds, 19 rounds, or 20 rounds of stimulation in a re-challenge assay.

The cytokine secretion of the γδT cells can be measured by a repetitive tumor challenge assay (e.g., following the methods described in Example 4) . The γδT cells prepared according to the method described herein (e.g., un-transfected or transfected with a CAR expression vector) may secrete cytokines (e.g., IFN-γ and/or TNF-α) upon exposure to tumor cells. To evaluate the cytokine secretion of the γδT cells in vitro, the γδT 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 γδT cells may secrete an amount of IFN-γ that is more than 50 pg/ml, more than 100 pg/ml, more than 200 pg/ml, more than 300 pg/ml, more than 400 pg/ml, more than 500 pg/ml, more than 1000 pg/ml, more than 1500 pg/ml, more than 2000 pg/ml, more than 2500 pg/ml, more than 3000 pg/ml, more than 4000 pg/ml, more than 5000 pg/ml, more than 6000 pg/ml, more than 7000 pg/ml, more than 8000 pg/ml, more than 10,000 pg/ml, more than 15,000 pg/ml, more than 20,000 pg/ml, more than 25,000 pg/ml, or more than 50,000 pg/ml, after 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, or 12 days of co-culture with tumor cells. The γδT cells may secrete an amount of IFN-γ that is less than 50 pg/ml, less than 100 pg/ml, less than 200 pg/ml, less than 300 pg/ml, less than 400 pg/ml, less than 500 pg/ml, less than 1000 pg/ml, less than 1500 pg/ml, less than 2000 pg/ml, less than 2500 pg/ml, or less than 3000 pg/ml, less than 4000 pg/ml, less than 5000 pg/ml, less than 6000 pg/ml, less than 7000 pg/ml, less than 8000 pg/ml, less than 10,000 pg/ml, less than 15,000 pg/ml, less than 20,000 pg/ml, less than 25,000 pg/ml, or less than 50,000 pg/ml, after 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, or 12 days of co-culture with tumor cells. The γδT cells may secrete an amount of IFN-γ that is 500-5000 pg/ml, 1000-4000 pg/ml, 1000-3000 pg/ml, 1500-3000 pg/ml, 50-500 pg/ml, 50-400 pg/ml, 100-1000 pg/ml, 100-800 pg/ml, 100-600 pg/ml, 100-400 pg/ml, 200-400 pg/ml, 200-300 pg/ml, 200-1000 pg/ml, 200-800 pg/ml, 1500-3000 pg/ml, 1000-15000 pg/ml, 5000-15000 pg/ml, or 10000-20000 pg/ml, after 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, or 12 days of co-culture with tumor cells.

To evaluate the cytokine secretion of the γδT cells in vitro, the γδT 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 γδT cells may secrete an amount of TNF-α that is more than 10 pg/ml, more than 20 pg/ml, more than 30 pg/ml, more than 40 pg/ml, more than 50 pg/ml, more than 60 pg/ml, more than 70 pg/ml, more than 80 pg/ml, more than 90 pg/ml, more than 100 pg/ml, more than 200 pg/ml, more than 300 pg/ml, more than 400 pg/ml, more than 500 pg/ml, more than 600 pg/ml, more than 700 pg/ml, more than 800 pg/ml, more than 900 pg/ml, more than 1000 pg/ml, more than 1500 pg/ml, more than 2000 pg/ml, more than 2500 pg/ml, more than 3000 pg/ml, more than 3500 pg/ml, more than 4000 pg/ml, more than 5000 pg/ml, or more than 10,000 pg/ml, after 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, or 12 days of co-culture with tumor cells. The γδT cells may secrete an amount of TNF-α that is less than 10 pg/ml, less than 20 pg/ml, less than 30 pg/ml, less than 40 pg/ml, less than 50 pg/ml, less than 60 pg/ml, less than 70 pg/ml, less than 80 pg/ml, less than 90 pg/ml, less than 100 pg/ml, less than 200 pg/ml, less than 300 pg/ml, less than 400 pg/ml, less than 500 pg/ml, less than 600 pg/ml, less than 700 pg/ml, less than 800 pg/ml, less than 900 pg/ml, less than 1000 pg/ml, less than 1500 pg/ml, less than 2000 pg/ml, less than 2500 pg/ml, less than 3000 pg/ml, less than 3500 pg/ml, less than 4000 pg/ml, less than 5000 pg/ml, or less than 10,000 pg/ml, after 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, or 12 days of co-culture with tumor cells. The γδT cells may secrete an amount of TNF-α that is 10-5000 pg/ml, 10-4000 pg/ml, 10-3000 pg/ml, 100-3000 pg/ml, 500-2000 pg/ml, 750-1250 pg/ml, 20-200 pg/ml, 20-300 pg/ml, 20-400 pg/ml, 50-1000 pg/ml, 50-800 pg/ml, 50-400 pg/ml, 50-200 pg/ml, 50-150 pg/ml, 100-800 pg/ml, 200-800 pg/ml, 200-600 pg/ml, 200-500 pg/ml, 200-400 pg/ml, 300-800 pg/ml, 400-800 pg/ml, 400-900 pg/ml 400-600 pg/ml, 300-600 pg/ml, or 350-600 pg/ml, after 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, or 12 days of co-culture with tumor cells.

To evaluate the cytokine secretion of the γδT cells in vitro, the γδT 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 γδT cells may secrete an amount of GM-CSF that is more than 50 pg/ml, more than 100 pg/ml, more than 200 pg/ml, more than 300 pg/ml, more than 400 pg/ml, more than 500 pg/ml, more than 1000 pg/ml, more than 1500 pg/ml, more than 2000 pg/ml, more than 2500 pg/ml, more than 3000 pg/ml, more than 4000 pg/ml, more than 5000 pg/ml, more than 6000 pg/ml, more than 7000 pg/ml, more than 8000 pg/ml, more than 10,000 pg/ml, more than 15,000 pg/ml, more than 20,000 pg/ml, more than 25,000 pg/ml, more than 50,000 pg/ml, after 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, or 12 days of co-culture with tumor cells. The γδT cells may secrete an amount of GM-CSF that is less than 50 pg/ml, less than 100 pg/ml, less than 200 pg/ml, less than 300 pg/ml, less than 400 pg/ml, less than 500 pg/ml, less than 1000 pg/ml, less than 1500 pg/ml, less than 2000 pg/ml, less than 2500 pg/ml, or less than 3000 pg/ml, less than 4000 pg/ml, less than 5000 pg/ml, less than 6000 pg/ml, less than 7000 pg/ml, less than 8000 pg/ml, less than 10,000 pg/ml, less than 15,000 pg/ml, less than 20,000 pg/ml, less than 25,000 pg/ml, or less than 50,000 pg/ml, after 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, or 12 days of co-culture with tumor cells. The γδT cells may secrete an amount of GM-CSF that is 500-5000 pg/ml, 1000-4000 pg/ml, 1000-3000 pg/ml, 1500-3000 pg/ml, 50-500 pg/ml, 50-400 pg/ml, 100-1000 pg/ml, 100-800 pg/ml, 100-600 pg/ml, 100-400 pg/ml, 200-400 pg/ml, 200-300 pg/ml, 200-1000 pg/ml, 200-800 pg/ml, 1500-3000 pg/ml, 1000-15000 pg/ml, 5000-15000 pg/ml, or 10000-20000 pg/ml, after 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, or 12 days of co-culture with tumor cells.

To evaluate the cytokine secretion of the γδT cells in vitro, the γδT 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 γδT cells may secrete an amount of IL-2 that is more than 10 pg/ml, more than 20 pg/ml, more than 30 pg/ml, more than 40 pg/ml, more than 50 pg/ml, more than 60 pg/ml, more than 70 pg/ml, more than 80 pg/ml, more than 90 pg/ml, more than 100 pg/ml, more than 200 pg/ml, more than 300 pg/ml, more than 400 pg/ml, more than 500 pg/ml, more than 600 pg/ml, more than 700 pg/ml, more than 800 pg/ml, or more than 900 pg/ml, after 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, or 12 days of co-culture with tumor cells. The γδT cells may secrete an amount of IL-2 that is less than 10 pg/ml, less than 20 pg/ml, less than 30 pg/ml, less than 40 pg/ml, less than 50 pg/ml, less than 60 pg/ml, less than 70 pg/ml, less than 80 pg/ml, less than 90 pg/ml, less than 100 pg/ml, less than 200 pg/ml, less than 300 pg/ml, less than 400 pg/ml, less than 500 pg/ml, less than 600 pg/ml, less than 700 pg/ml, less than 800 pg/ml, or less than 900 pg/ml, after 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, or 12 days of co-culture with tumor cells. The γδT cells may secrete an amount of IL-2 that is 10-1000 pg/ml, 10-800 pg/ml, 10-500 pg/ml, 10-400 pg/ml, 10-300 pg/ml, 10-200 pg/ml, 20-200 pg/ml, 20-300 pg/ml, 20-400 pg/ml, 50-1000 pg/ml, 50-800 pg/ml, 50-400 pg/ml, 50-200 pg/ml, 50-150 pg/ml, 100-800 pg/ml, 200-800 pg/ml, 200-600 pg/ml, 200-500 pg/ml, 200-400 pg/ml, 300-800 pg/ml, 400-800 pg/ml, 400-900 pg/ml 400-600 pg/ml, 300-600 pg/ml, or 350-600 pg/ml, after 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, or 12 days of co-culture with tumor cells.

To evaluate the cytokine secretion of the γδT cells in vitro, the γδT 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 γδT cells may secrete an amount of IL-17 that is more than 10 pg/ml, more than 20 pg/ml, more than 30 pg/ml, more than 40 pg/ml, more than 50 pg/ml, more than 60 pg/ml, more than 70 pg/ml, more than 80 pg/ml, more than 90 pg/ml, more than 100 pg/ml, more than 200 pg/ml, more than 300 pg/ml, more than 400 pg/ml, more than 500 pg/ml, more than 600 pg/ml, more than 700 pg/ml, more than 800 pg/ml, or more than 900 pg/ml, after 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, or 12 days of co-culture with tumor cells. The γδT cells may secrete an amount of IL-17 that is less than 10 pg/ml, less than 20 pg/ml, less than 30 pg/ml, less than 40 pg/ml, less than 50 pg/ml, less than 60 pg/ml, less than 70 pg/ml, less than 80 pg/ml, less than 90 pg/ml, less than 100 pg/ml, less than 200 pg/ml, less than 300 pg/ml, less than 400 pg/ml, less than 500 pg/ml, less than 600 pg/ml, less than 700 pg/ml, less than 800 pg/ml, or less than 900 pg/ml, after 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, or 12 days of co-culture with tumor cells. The γδT cells may secrete an amount of IL-17 that is 10-1000 pg/ml, 10-800 pg/ml, 10-500 pg/ml, 10-400 pg/ml, 10-300 pg/ml, 10-200 pg/ml, 20-200 pg/ml, 20-300 pg/ml, 20-400 pg/ml, 50-1000 pg/ml, 50-800 pg/ml, 50-400 pg/ml, 50-200 pg/ml, 50-150 pg/ml, 100-800 pg/ml, 200-800 pg/ml, 200-600 pg/ml, 200-500 pg/ml, 200-400 pg/ml, 300-800 pg/ml, 400-800 pg/ml, 400-900 pg/ml 400-600 pg/ml, 300-600 pg/ml, or 350-600 pg/ml, after 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, or 12 days of co-culture with tumor cells.

When the γδT cells derived from one subject are applied to a different subject (allogeneic cell therapy) , the allogeneic γδT cells may suffer from suppression by the host (HvG reactions) . HvG reactions against the γδT cells can be evaluated using an in vitro Mixed Lymphocyte Reaction (MLR) assay (e.g., following the methods described in Example 7) . The γδT cells may be co-cultured with allogeneic or autologous PBMC and tumor cells (e.g., H929 cells) . Cell proliferation can be measured after 7 days of co-culture. Cell number can be measured by cellometer (e.g., following the methods described in Example 4) . After 7 days of co-culture, the γδT cells (e.g., un-transfected or transfected with a CAR expression vector) may proliferate, 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, with or without exposure to tumor cells. The γδT cells may proliferate, 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, with or without exposure to tumor cells.

As used herein, a “T cell” “Tnaive, ” or “Tn” refers to a T cell that is antigen-inexperienced. An antigen-inexperienced T cell may have encountered its cognate antigen in the thymus but not in the periphery. T cells may be precursors of memory cells. T cells may express CCR7, but not CD45RO. T cells may express both CD45RA and CCR7, but not CD45RO. T cells may be characterized by expression of CD62L, CD27, CCR7, CD45RA, CD28, and CD127, and the absence of CD95 or CD45RO isoform. T cells may express CD62L, IL-7 receptor-α, IL-6 receptor, and CD132, but not CD25, CD44, CD69, or CD45RO. T cells may express CD45RA, CCR7, and CD62L and not CD95 or IL-2 receptor β.Surface expression levels of markers may be assessed using flow cytometry.

As used herein, the term “central memory T cells” or “Tcm” refers to a subset of T cells that in humans are CD45RO positive and express CCR7. Central memory T cells may express CD95. Central memory T cells may express IL-2R, IL-7R and/or IL-15R. Central memory T cells may express CD45RO, CD95, IL-2 receptor β, CCR7, and CD62L. Surface expression levels of markers may be assessed using flow cytometry.

As used herein, the term “effector memory T cells” or “Tem” refers to a subset of memory T cells with effector functions. In general, 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) generally reside in lymphoid organs and do not have an immediate lytic function, whereas 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.

As used herein, the term “effector T cells” or “Teff” refers to a subset of T cells with effector functions. AfterT cells differentiate into Teff cells, they may readily release cytotoxic granules and effector cytokines upon engagement of their TCR with the cognate antigen. Teff cells may be negative for CD27, CD28, and lymph node homing markers, but may express markers of terminal T cell activation such as Killer cell lectin-like receptor subfamily G, member 1 (KLGR-1) and the NK marker CD57.

The percentage of γδT cell phenotypes can be measured by flow cytometry (e.g., following the methods described in Example 8) . The expanded cell culture (e.g., un-transfected or transfected with a CAR expression vector) may comprise a percentage of Tnaive that is greater than 1%, greater than 2%, greater than 3%, greater than 4%, 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%or greater than 95%. The expanded cell culture may comprise a percentage of Tnaive that is less than 1%, less than 2%, less than 3%, less than 4%, 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%, or less than 95%. The expanded cell culture may comprise a percentage of Tnaive that is about 1%, about 2%, about 3%, about 4%, 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 percentage of γδT cell phenotypes can be measured by flow cytometry (e.g., following the methods described in Example 8) . The expanded cell culture (e.g., un-transfected or transfected with a CAR expression vector) may comprise a percentage of Tcm that is greater than 1%, greater than 2%, greater than 3%, greater than 4%, 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%or greater than 95%. The expanded cell culture may comprise a percentage of Tcm that is less than 1%, less than 2%, less than 3%, less than 4%, 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%, or less than 95%. The expanded cell culture may comprise a percentage of Tcm that is about 1%, about 2%, about 3%, about 4%, 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 percentage of γδT cell phenotypes can be measured by flow cytometry (e.g., following the methods described in Example 8) . The expanded cell culture (e.g., un-transfected or transfected with a CAR expression vector) may comprise a percentage of Tem that is greater than 1%, greater than 2%, greater than 3%, greater than 4%, 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%or greater than 95%. The expanded cell culture may comprise a percentage of Tem that is less than 1%, less than 2%, less than 3%, less than 4%, 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%, or less than 95%. The expanded cell culture may comprise a percentage of Tem that is about 1%, about 2%, about 3%, about 4%, 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 percentage of γδT cell phenotypes can be measured by flow cytometry (e.g., following the methods described in Example 8) . The expanded cell culture (e.g., un-transfected or transfected with a CAR expression vector) may comprise a percentage of Teff that is greater than 1%, greater than 2%, greater than 3%, greater than 4%, 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%or greater than 95%. The expanded cell culture may comprise a percentage of Teff that is less than 1%, less than 2%, less than 3%, less than 4%, 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%, or less than 95%. The expanded cell culture may comprise a percentage of Teff that is about 1%, about 2%, about 3%, about 4%, 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%.

Tumor suppression by the γδT cells may be evaluated using an in vivo tumor suppression assay (e.g., following the methods described in Example 9) . The γδT cells prepared according to the method described herein may effectively suppress tumor growth. The γδT 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%. The γδT cells prepared according to the method described herein may have a tumor growth inhibition percentage that is less than 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. The TGI%can be determined, e.g., at 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days after the treatment starts, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after the treatment starts. As used herein, the tumor growth inhibition percentage (TGI%) is calculated using the following formula: TGI (%) = [1- (Ti-T0) / (Vi-V0)] ×100, wherein Ti is the average tumor volume in the treatment group on day i. T0 is the average tumor volume in the treatment group on day zero. Vi is the average tumor volume in the control group on day i. V0 is the average tumor volume in the control group on day zero. The tumor suppression effect of γδT cells prepared according to the methods described herein may be stronger comparing to γδT cells prepared according to other methods. The γδT cells prepared according to the methods described herein may inhibit tumor growth by 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%.

Further, the amount of the γδT cells in the peripheral blood of the animals may be quantified (e.g., following the methods described in Example 9) . The percentage of γδT cells prepared according to the method described herein in total peripheral blood may be 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%, at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days after the treatment starts. The percentage of γδT cells prepared according to the method described herein in total peripheral blood may be less than 0.5%, 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 13%, less than 14%, less than 15%, less than 20%, less than 25%, or less than 30%, at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days after the treatment starts.

Comparing to γδT cells prepared according to other methods, γδT cells prepared according to the methods described herein may have higher purity, yield and persistence with better anti-tumor cytotoxicity.

Engineered receptor (e.g., CAR and TCR)

One aspect of the present disclosure provides γδT cells that express an engineered receptor. The engineered receptor can comprise an extracellular ligand binding domain or an extracellular antigen binding domain, and optionally an intracellular signaling domain. Exemplary engineered receptors include, but are not limited to, chimeric antigen receptor (CAR) , engineered T-cell receptor (TCR) , and T-cell antigen coupler (TAC) receptor. The engineered receptor can comprise an extracellular antigen binding domain that specifically binds to an antigen (e.g., a tumor antigen) , a transmembrane domain, and an intracellular signaling domain. The intracellular signaling domain can comprise a primary intracellular signaling domain and/or a co-stimulatory signaling domain. The intracellular signaling domain can comprise an intracellular signaling domain of a TCR co-receptor. The engineered receptor can be encoded by a heterologous polynucleotide operably linked to a promoter (such as a constitutive promoter or an inducible promoter) .

The engineered receptor can comprise one or more specific binding domains that target at least one tumor antigen, and one or more intracellular effector domains, such as one or more primary intracellular signaling domains and/or co-stimulatory signaling domains.

The engineered receptor can be a chimeric antigen receptor (CAR) . Many chimeric antigen receptors are known in the art and can be suitable for the γδT cells described herein. CARs can also be constructed with a specificity for any cell surface marker by utilizing antigen binding fragments or antibody variable domains of, for example, antibody molecules.

CARs of the present disclosure may comprise an extracellular domain comprising at least one antigen binding domain that specifically binds at least one tumor antigen, a transmembrane domain, and an intracellular signaling domain.

The intracellular signaling domain may generate a signal that promotes an immune effector function of the CAR-containing cell, e.g., a CAR-T cell. Immune effector function or immune effector response refers to function or response, e.g., of an immune effector cell, that enhances or promotes an immune attack of a target cell. For example, an immune effector function or response can refer to a property of a T or NK cell that promotes killing or the inhibition of growth or proliferation, of a target cell. Examples of immune effector function, e.g., in a CAR-T cell, include cytolytic activity (such as antibody-dependent cellular toxicity, or ADCC) and helper activity (such as the secretion of cytokines) . The intracellular signaling domain may generate a signal that promotes proliferation and/or survival of the CAR containing cell. The CAR may comprise one or more intracellular signaling domains selected from the signaling domains of CD28, CD137, CD3, CD27, CD40, ICOS, GITR, and OX40. The signaling domain of a naturally occurring molecule can comprise the entire intracellular or cytoplasmic portion, or the entire native intracellular signaling domain, of the molecule, or a fragment or derivative thereof.

The intracellular signaling domain of a CAR can comprise a primary intracellular signaling domain. “Primary intracellular signaling domain” refers to cytoplasmic signaling sequence that acts in a stimulatory manner to induce immune effector functions. The primary intracellular signaling domain may contain a signaling motif known as Immunoreceptor Tyrosine-based Activation Motif, or ITAM. The primary intracellular signaling domain may comprise a functional signaling domain of a protein selected from the group consisting of CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, common FcR gamma (FCER1G) , FcR beta (Fc Epsilon RIb) , CD79a, CD79b, Fcgamma R IIa, DAP10, and DAP 12. The primary intracellular signaling domain may comprise a nonfunctional or attenuated signaling domain of a protein selected from the group consisting of CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, common FcR gamma (FCER1G) , FcR beta (Fc Epsilon RIb) , CD79a, CD79b, Fcgamma R IIa, DAP10, and DAP12. The nonfunctional or attenuated signaling domain can be a mutant signaling domain having a point mutation, insertion or deletion that attenuates or abolishes one or more immune effector functions, such as cytolytic activity or helper activity, including antibody-dependent cellular toxicity (ADCC) . The CAR may comprise a nonfunctional or attenuated CD3 zeta (i.e. CD3ζ or CD3z) signaling domain. The intracellular signaling domain may not comprise a primary intracellular signaling domain. An attenuated primary intracellular signaling domain may induce no more than about any of 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%or less of an immune effector function (such as cytolytic function against target cells) compared to CARs having the same construct, but with the wild-type primary intracellular signaling domain.

The intracellular signaling domain of a CAR can comprise one or more (such as any of 1, 2, 3, or more) co-stimulatory signaling domains. “Co-stimulatory signaling domain” can be the intracellular portion of a co-stimulatory molecule. The term “co-stimulatory molecule” refers to a cognate binding partner on an immune cell (such as T cell) that specifically binds with a co-stimulatory ligand, thereby mediating a co-stimulatory response by the immune cell, such as, but not limited to, proliferation and survival. Co-stimulatory molecules are cell surface molecules other than antigen receptors or their ligands that contribute to an efficient immune response. A co-stimulatory molecule can be represented in the following protein families: TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins) , and activating NK cell receptors. Co-stimulatory molecules include but are not limited to an MHC class I molecule, BTLA and a Toll ligand receptor, as well as OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18) , ICOS (CD278) , and 4-1BB (CD137) . Further examples of such co-stimulatory molecules include CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR) , SLAMF7, NKp80 (KLRF1) , NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8alpha, CD8beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49d, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226) , SLAMF4 (CD244, 2B4) , CD84, CD96 (Tactile) , CEACAM1, CRTAM, Ly9 (CD229) , CD160 (BY55) , PSGL1, CD100 (SEMA4D) , CD69, SLAMF6 (NTB-A, Ly108) , SLAM (SLAMF1, CD150, IPO-3) , BLAME (SLAMF8) , SELPLG (CD162) , LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, and a ligand that specifically binds with CD83.

The CAR can comprise a single co-stimulatory signaling domain. The CAR can comprise two or more co-stimulatory signaling domains. The intracellular signaling domain can comprise a functional primary intracellular signaling domain and one or more co-stimulatory signaling domains. The CAR may not comprise a functional primary intracellular signaling domain (such as CD3ζ) . The CAR can comprise an intracellular signaling domain consisting of or consisting essentially of one or more co-stimulatory signaling domains. The CAR can comprise an intracellular signaling domain consisting of or consisting essentially of a nonfunctional or attenuated primary intracellular signaling domain (such as a mutant CD3ζ) and one or more co-stimulatory signaling domains. Upon binding of the antigen binding domain to tumor antigen, the co-stimulatory signaling domains of the CAR can transduce signals for enhanced proliferation, survival and differentiation of the modified immune cells having the CAR (such as T cells) , and inhibit activation induced cell death. The one or more co-stimulatory signaling domains can be derived from one or more molecules selected from the group consisting of CD27, CD28, 4-1BB (i.e., CD137) , OX40, CD30, CD40, CD3, lymphocyte function-associated antigen-1 (LFA-1) , CD2, CD7, LIGHT, NKG2C, B7-H3 and ligands that specially bind to CD83.

The intracellular signaling domain of a CAR can comprise a co-stimulatory signaling domain derived from CD28. The intracellular signaling domain can comprise a primary intracellular signaling domain of CD3ζ and a co-stimulatory signaling domain of CD28. The intracellular signaling domain in the chimeric receptor of the present application can comprise a co-stimulatory signaling domain derived from 4-1BB (i.e., CD137) . The intracellular signaling domain can comprise a primary intracellular signaling domain of CD3ζ and a co-stimulatory signaling domain of 4-1BB. The intracellular signaling domain can comprise a polypeptide comprising from the N-terminus to the C-terminus: a co-stimulatory signaling domain of 4-1BB and a primary intracellular signaling domain of CD3ζ.

The intracellular signaling domain of the CAR can comprise a co-stimulatory signaling domain of CD28 and a co-stimulatory signaling domain of 4-1BB. The intracellular signaling domain can comprise a primary intracellular signaling domain of CD3ζ, a co-stimulatory signaling domain of CD28, and a co-stimulatory signaling domain of 4-1BB. The intracellular signaling domain can comprise a polypeptide comprising from the N-terminus to the C-terminus: a co-stimulatory signaling domain of CD28, a co-stimulatory signaling domain of 4-1BB, and a primary intracellular signaling domain of CD3ζ.

The antigen binding domain of a CAR may comprise one or more (such as any one of 1, 2, 3, 4, 5, 6 or more) antibodies or antibody fragments, which can be selected from an scFv, a Fv, a Fab, a (Fab′) ₂, a minibody, a diabody, a single domain antibody (sdAb) , or a V_HH domain. The antigen binding domain of a CAR can comprise a ligand or an extracellular portion of a receptor that specifically binds to a tumor antigen. The CAR can be a monospecific, bispecific or multispecific CAR. The antigen binding domain of a CAR can specifically bind to a single tumor antigen. The antigen binding domain of a CAR can bind to two or more tumor antigens. The engineered receptor (e.g., CAR) may redirect the specificity of the γδT cells through the expression of a chimeric antigen receptor (CAR) or TCR on these cells.

The antigen may be a tumor antigen selected from the group consisting of BCMA, CLL1, CD4, GPC3, GPRC5D, GU2CYC, CD19, MUC16, MUC1, CAIX, CEA, CD8, CD7, CD10, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, EGP-2, EGP-40, EpCAM, ERBB2, ERBB3, ERBB4, FBP, fetal acetylcholine receptor, folate receptor-α, GD2, GD3, hTERT, IL-13Rα2, κ-light chain, KDR, LeY, L1 cell adhesion molecule, MAGE-A1, mesothelin, MAGEA3, p53, MART1, GP100, proteinase-3 (PR3) , tyrosinase, survivin, hTERT, EphA2, NY-ESO-1, h5T4, PSCA, PSMA, ROR1, TAG-72, VEGF-R2, WT-1, CD123, CD44V6, NKCS1, IGF1R, EGFR, EGFR-VIII, Claudin 18.2, Claudin 6, NKG2D, Delta-like 3 (DLL3) , CD70, CS-1, c-Met, Glycolipid F77, PD-L1, PD-L2, and other tumor antigens with clinical significance, and combinations thereof. The antigen may be GPC3. The antigen may be DLL3. The antigen may be BCMA.

The tumor antigen can be derived from an intracellular protein of tumor cells. The tumor antigen can be expressed on the surface of tumor cells. Many TCRs specific for tumor antigens (including tumor-associated antigens) have been described, including, for example, NY-ESO-1 cancer-testis antigen, the p53 tumor suppressor antigens, TCRs for tumor antigens in melanoma (e.g., MARTI, gp 100) , leukemia (e.g., WT1, minor histocompatibility antigens) , and breast cancer (e.g., HER2, NY-BR1) .

The transmembrane domain of a CAR can be selected from the transmembrane domain of an alpha, beta or zeta chain of a T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18) , ICOS (CD278) , 4-1BB (CD137) , GITR, CD40, BAFFR, HVEM (LIGHTR) , SLAMF7, NKp80 (KLRF1) , CD160, CD19, IL-2R beta, IL-2R gamma, IL-7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226) , SLAMF4 (CD244, 2B4) , CD84, CD96 (Tactile) , CEACAM1, CRT AM, Ly9 (CD229) , CD160 (BY55) , PSGL1, CD100 (SEMA4D) , SLAMF6 (NTB-A, Ly108) , SLAM (SLAMF1, CD150, IPO-3) , BLAME (SLAMF8) , SELPLG (CD162) , LTBR, PAG/Cbp, NKp44, NKp30, NKp46, NKG2D, and/or NKG2C. The transmembrane domain of the CAR can be a CD4, CD3, CD8α, or CD28 transmembrane domain. The transmembrane domain of the CAR can comprise a CD8α transmembrane domain. The transmembrane domain can be derived from a molecule selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152 and PD1.

The extracellular domain can be connected to the transmembrane domain by a hinge domain. The hinge domain can be a hinge domain of CD8α.

The CAR can also comprise a signal peptide (SP) , such as a CD8α signal peptide.

Many CARs targeting different tumor antigens have been widely disclosed in the field, such as CD19 CARs or BCMA CARs. The extracellular antigen binding domain of CD19 CARs can be or include the CD19 binding fragment (e.g., FMC63, SJ25C1, or those disclosed in different patents such as WO 2022/012683, etc) . BCMA CARs also have been well described, related patents include but not limited to WO 2016/014789, WO 2016/014565, WO 2013/154760, and WO 2018/028647, etc. The extracellular antigen binding domain of BCMA CARs may be or include BCMA binding fragment. The BCMA binding fragment may bind to one or more epitopes on BCMA. The BCMA CARs may be bivalent CARs comprising two anti-BCMA sdAbs targeting same or different BCMA epitopes.

The engineered receptor can be a modified T-cell receptor or engineered T-cell receptor. The engineered TCR can be specific for a tumor antigen. The tumor antigen can be selected from the group consisting of BCMA, CLL1, CD4, GPC3, GPRC5D, GU2CYC, CD19, MUC16, MUC1, CAIX, CEA, CD8, CD7, CD10, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, EGP-2, EGP-40, EpCAM, ERBB2, ERBB3, ERBB4, FBP, fetal acetylcholine receptor, folate receptor-α, GD2, GD3, HER-2, hTERT, IL-13R-α2, κ-light chain, KDR, LeY, L1 cell adhesion molecule, MAGE-A1, mesothelin, MAGEA3, p53, MART1, GP100, proteinase-3 (PR3) , tyrosinase, survivin, hTERT, EphA2, NY-ESO-1, h5T4, PSCA, PSMA, ROR1, TAG-72, VEGF-R2, WT-1, CD123, CD44V6, NKCS1, IGF1R, EGFR, EGFR-VIII, Claudin 18.2, Claudin 6, NKG2D, Delta-like 3 (DLL3) , CD70, CS-1, c-Met, Glycolipid F77, PD-L1, PD-L2, and other tumor antigens with clinical significance, and combinations thereof. The tumor antigen can be derived from an intracellular protein of tumor cells. The tumor antigen can be expressed on the surface of tumor cells. Many TCRs specific for tumor antigens (including tumor-associated antigens) have been described, including, for example, NY-ESO-1 cancer-testis antigen, the p53 tumor suppressor antigens, TCRs for tumor antigens in melanoma (e.g., MARTI, gp 100) , leukemia (e.g., WT1, minor histocompatibility antigens) , and breast cancer (e.g., HER2, NY-BR1) . Any of the TCRs known in the art can be used. The TCR can have an enhanced affinity to the tumor antigen. Exemplary TCRs and methods for introducing the TCRs to immune cells have been described, for example, in U.S. Pat. No. 5,830,755, and Kessels et al. Immunotherapy through TCR gene transfer. Nat. Immunol. 2, 957-961 (2001) , which are incorporated herein by reference in the entirety.

The TCR receptor complex is an octameric complex formed by variable TCR receptor α and β chains (or γ and δ chains on case of γδT cells) with three dimeric signaling modules CD3δ/ε, CD3γ/ε and CD247 (T-cell surface glycoprotein CD3 zeta chain) ζ/ζ or ζ/η. Ionizable residues in the transmembrane domain of each subunit form a polar network of interactions that hold the complex together. TCR complex has the function of activating signaling cascades in T cells.

The engineered receptor can be an engineered TCR comprising one or more T-cell receptor (TCR) fusion proteins (TFPs) . Exemplary TFPs have been described, for example, in US20170166622A1, which is incorporated herein by reference in its entirety. The TFP can comprise an extracellular domain of a TCR subunit that comprises an extracellular domain or portion thereof of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications. The TFP can comprise a transmembrane domain that comprises a transmembrane domain of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications. The TFP can comprise a transmembrane domain that comprises a transmembrane domain of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, a TCR zeta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.

The TFP can comprise a TCR subunit comprising at least a portion of a TCR extracellular domain, and a TCR intracellular domain comprising a stimulatory domain from an intracellular signaling domain of CD3 epsilon; and an antigen binding domain, wherein the TCR subunit and the antigen binding domain are operatively linked, and wherein the TFP incorporates into a TCR when expressed in a T cell.

The engineered receptor can be a T-cell antigen coupler (TAC) receptor. Exemplary TAC receptors have been described, for example, in US20160368964A1, which is incorporated herein by reference. The TAC can comprise an antigen binding domain, a TCR-binding domain that specifically binds a protein associated with the TCR complex, and a T-cell receptor signaling domain. The antigen binding domain can be an antibody fragment, such as scFv or V_HH, which specifically binds to a tumor antigen. The antigen binding domain can be a designed Ankyrin repeat (DARPin) polypeptide. The tumor antigen can be selected from the group consisting of BCMA, CLL1, CD4, GPC3, GPRC5D, GU2CYC, CD19, MUC16, MUC1, CAIX, CEA, CD8, CD7, CD10, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, EGP-2, EGP-40, EpCAM, ERBB2, ERBB3, ERBB4, FBP, fetal acetylcholine receptor, folate receptor-α, GD2, GD3, HER-2, hTERT, IL-13R-α2, κ-light chain, KDR, LeY, L1 cell adhesion molecule, MAGE-A1, mesothelin, MAGEA3, p53, MART1, GP100, proteinase-3 (PR3) , tyrosinase, survivin, hTERT, EphA2, NY-ESO-1, h5T4, PSCA, PSMA, ROR1, TAG-72, VEGF-R2, WT-1, CD123, CD44V6, NKCS1, IGF1R, EGFR, EGFR-VIII, Claudin 18.2, Claudin 6, NKG2D, Delta-like 3 (DLL3) , CD70, CS-1, c-Met, Glycolipid F77, PD-L1, PD-L2, and other tumor antigens with clinical significance, and combinations thereof. The tumor antigen can be derived from an intracellular protein of tumor cells. The tumor antigen can be expressed on the surface of tumor cells. The protein associated with the TCR complex can be CD3, such as CD3 epsilon. The TCR-binding domain can be a single chain antibody, such as scFv, or a V_HH. The TCR-binding domain can be derived from UCHT1. The TAC receptor can comprise a cytosolic domain and a transmembrane domain. The T-cell receptor signaling domain can comprise a cytosolic domain derived from a TCR co-receptor. 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 CD8α) .

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 two subtypes of T cell co-receptor, CD4 and CD8, display strong specificity for particular MHC classes. 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.

The engineered receptor (such as CAR, TCR, or TAC) can target one or more tumor antigens. Tumor antigens are proteins that are produced by tumor cells that can elicit an immune response, particularly T-cell mediated immune responses. The selection of the targeted antigen will depend on the particular type of cancer to be treated. Exemplary tumor antigens include, for example, a glioma-associated antigen, carcinoembryonic antigen (CEA) , β-human chorionic gonadotropin, alpha-fetoprotein (AFP) , lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CAIX, human telomerase reverse transcriptase, RU1, RU2 (AS) , intestinal carboxyl esterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA) , PAP, NY-ESO-1, LAGE-1a, p53, prostein, PSMA, HER2/neu, survivin, telomerase, prostate-carcinoma tumor antigen-1 (PCTA-1) , MAGE, ELF2M, neutrophil elastase, ephrinB2, CD22, insulin growth factor (IGF) -I, IGF-II, IGF-I receptor and mesothelin.

The tumor antigen can comprise one or more antigenic cancer epitopes associated with a malignant tumor. Malignant tumors express a number of proteins that can serve as target antigens for an immune attack. These molecules include but are not limited to tissue-specific antigens such as MART-1, tyrosinase and gp100 in melanoma and prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to the group of transformation-related molecules such as the oncogene HER2/Neu/ErbB-2. Yet another group of target antigens are onco-fetal antigens such as carcinoembryonic antigen (CEA) . In 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) . 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.

Non-limiting examples of 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. Other large, protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB3, c-Met, nm-23HI, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-Catenin, CDK4, Mum-1, p15, p16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\KP1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOv18, NB/70K, NY-CO-1, RCAS 1, SDCCAG16, 90K\Mac-2 binding protein\cyclophilin C-associated protein, TAAL6, TAG72, TLP, and TPS.

γδT 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. γδT 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 γδT cells to a particular tissue, organ, or body site.

The γδT cells may be tumor-specific allogeneic γδT cells. The γδT cells may be derived from tumor infiltrating lymphocytes (TIL) isolated from a tumor. Different TILs can be isolated from different tumor types. 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 γδT 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 γδT cells can be engineered to express at least one tumor recognition moiety that recognizes an epitope of choice. Some tumor specific allogeneic γδT 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.

Therapeutic use

The methods for making γδT cells described herein, and the resulting γδT cells described herein can be used in a variety of experimental, therapeutic and commercial applications.

In one aspect, the disclosure provides a pharmaceutical composition comprising the γδT cells made by the methods described herein and a pharmaceutically acceptable carrier.

In one aspect, 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 γδT cells described herein, or the pharmaceutical composition described herein. The disease or disorder may be cancer, an autoimmune disease, a tumor, or an infection.

The disease or disorder may be solid tumor. “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.

In one aspect, the disclosure provides a method of modulating an immune response comprising administering an effective amount of γδT cells described herein to a subject in need thereof.

The term “effective amount” as used herein means an amount effective, at dosages and for periods of time necessary to achieve the desired results.

In another aspect, the present disclosure provides a method for treating a disease or disorder by administering an effective amount of γδT 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. Examples of cancer that can be treated 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, soft tissue sarcoma, esophageal cancers, or CNS tumors.

The disclosure further includes the use of the γδT 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 γδT 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 γδT 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 γδT cells may expand within a subject's body, in vivo, after administration to a subject. The γδT cells can be frozen to provide cells for multiple treatments with the same cell preparation. The γδT 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 γδT cells and compositions comprising the same.

A method of treatment can comprise administering to a subject a therapeutically effective amount of the γδT cells. The therapeutically effective amount of the γδT 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 γδT cells may be administered for at least one week. The therapeutically effective amount of the γδT cells may be administered for at least two weeks.

The γδT cells described herein can be administered before, during, or after the occurrence of a disease or condition, and the timing of administering the γδT cells can vary. For example, the γδT 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 γδT cells can be administered to a subject during or as soon as possible after the onset of the symptoms. The administration of the γδT 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 γδT cells of the disclosure may be an intravenous administration. One or multiple dosages of the γδT 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. For the treatment of cancer, one or multiple dosages of the γδT cells can be administered years after onset of the cancer and before or after other treatments. The γδT 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.

Methods for administration of γδT cells for adoptive cell therapy are known and can be used in connection with the provided methods and compositions. For example, 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. (2013) PLoS ONE 8 (4) : e61338. 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. Thus, 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 (e.g., human 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. 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. The administration may effectively treat the subject despite the subject having become resistant to another therapy.

The γδT cells described herein can be administered to an animal, such as a mammal, even more a human, to treat a cancer. In addition, the γδT 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 γδT 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 γδT cells can be administered.

The γδT 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 γδT 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. In unit dosage form, 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.

EXAMPLES

The disclosure is further described in the following examples, which do not limit the scope of the disclosure described in the claims.

Example 1: Generation of CAR-Vδ1 T cells

T cells were isolated from healthy donor PBMCs using TCR α/β-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 μg/ml. The supernatant was discarded and the wells were washed with PBS. The isolated T cells were seeded in the 24 well plate at a cell density of 0.5-5×10⁶ 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-Vδ1 T cells were harvested 10-12 days after transduction.

Example 2: Generation of CAR-Vδ2 T cells

Human peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors using a Ficoll-Paque-based density gradient centrifugation protocol. The cells were cultured in RPMI 1640 medium supplemented with 10%FBS and antibiotics. To activate cells, zoledronic acid (ZOL) (50 μM working concentration, Sigma) was added to the culture medium on day 0. Recombinant human IL-2 (100 IU/mL) (Beijing Four Rings Bio-Pharm Co. ) , recombinant human IL-15 (100 IU/mL) , and vitamin C (70 μM) (Sigma) were included in the medium as well. After 96 hours post-activation, cells were transduced with lentivirus or retrovirus encoding BCMA CAR (SEQ ID NO: 2) at an MOI of 5. CAR-Vδ2 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 TCRγ/δ+ T cell isolation kit (Miltenyi Biotec) before future applications or cryopreserved.

Example 3: Generation of CAR-polyclonal γδT cells

Generation of anti-γδTCR antibody

Soluble γδTCR proteins were generated in house for animal immunization. To facilitate TCR heterodimerization, a pair of charge-complementary leucine zipper (LZ) sequences were connected to the C-terminus of TCRγ and TCRδ chain through a flexible (G4S) ₃ linker. Additionally, a 6xHis-tag was added to γ-chain and a flag-tag to δ-chain C-terminus for the purposes of both purification and detection. TCRγ chain and TCRδ chain plasmids were prepared and used for soluble γδTCR production. Soluble TCRs (sTCRs) were produced by FreeStyle 293-F cells and purified by Anti-DYKDDDDK G1 Affinity Resin (Genscript, Cat. #L00432) .

One camel was immunized with γ9δ2 TCR protein under all current animal welfare regulations. γ9δ2 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. The antigen emulsion was administered by double-spot injections intramuscularly at the neck. The animal received 3 injections of 100-200 μg of human γ9δ2 TCR protein at a 2-week interval and subsequently 2 injections of 100 μg of cynomolgus γ9δ2 TCR protein at a weekly interval. A final boost was given to the animal with 50 μg of human γ9δ2 TCR protein. Five days afterwards, 150 mL blood sample was collected and ～ 3×10⁸ peripheral blood lymphocytes (PBLs) were isolated as the genetic source of the conventional and heavy chain immunoglobulins.

Total RNA was extracted from lymphocytes of the immunized camel usingReagent (Invitrogen^TM, Cat. #15596026) . cDNA was synthesized based on RNA template using PRIMESCRIPT^TM 1st Strand cDNA Synthesis Kit with an oligo (dT) 20 primer (Takara, Cat. #6110A) . DNAs encoding V_HH (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℃.

Both sdAb and scFv phage libraries were rescued and stored after filter sterilization at 4℃ for further use. 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 γ9δ2 TCR positive clones identified by ELISA reached at least 50%and the sequence diversity of γ9δ2 TCR-specific clones was high for all output phages. These outputs were used for subsequent high-throughput screening.

[Corrected under Rule 26, 20.12.2024]
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℃. 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 γ9δ2 TCR proteins by ELISA and human γ9δ2 T cells by flow cytometry. Four antibodies were chosen to activate γδT cells as AS287963, AS281850, AS287435 and AS288180. The full-length V_HH, VH, VL and CDR sequences are listed in SEQUENCE LISTING.

Polyclonal γδT cell activation

αβ T cells and NK cells were depleted from healthy donor PBMCs using TCR α/β-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-γδTCR constant chain antibody, SEQ ID NO: 1) was immobilized on 24 well plate at a final concentration of 0.1-5 μg/ml. The supernatant was discarded, and the wells were washed with PBS twice. The αβT/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.

Polyclonal γδT cell transduction and proliferation

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.

Finally, CAR-polyclonal γδT cells were harvested.

Example 4: Optimization of the medium components

To expand CAR-polyclonal γδT to clinical-relevant numbers, the medium components were optimized to boost the expansion of γδT cells.

Optimizing cytokines combination to improve proliferation and efficacy of CAR-polyclonal γδT cells (IL-2 and IL-7)

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 γδT cells.

Table 1

Polyclonal γδT 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 γδT cells were harvested. Cell number and viability were analyzed by Cellometer (Nexcelom, K2) . For phenotype and CAR positive rate detection, the harvested cells were stained with fluorescent-labeled antibodies (CD3-BV785 [344842, Clone: SK7, BioLegend] , TCRVδ1-APC [17-5679-42, Clone: TS8.2, Invitrogen] , TCRVδ2-BV421 [331428, Clone: B6, Biolegend] and Alexa Fluor 488-labeled anti-mouse sdAb antibodies [GenScript] ) and analyzed by FACS.

Efficacy of CAR-γδT cells were evaluated in a repetitive tumor challenge assay. Briefly, 2×10⁵ CAR+ γδT cells were co-cultured with 2×10⁵ 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+ γδT was quantified and re-plated with fresh NCI-H929 cells at a ratio of 1: 1 for the next round. IFN-γ, GM-CSF and TNF-α release in the supernatant was determined at the end of first round. The cytokine release analysis was performed usingreagents (Human IFN gamma kit, Cisbio, Cat#62HIFNGPEH, Human TNF alpha kit, Cisbio, Cat#62HTNFAPEH, Human GM-CSF kit, Cisbio, Cat#62HGMCSFPEH) .

As shown in FIG. 1, adding IL-2 or IL-7 had no significant effects on proliferation, viability, CAR positive rate and cell sub-type ratio (FIGS. 1A-1D) . However, in the long-term killing assay towards NCI-H929 cells, IL-2 stimulated CAR-polyclonal γδT showed improved efficacy and improved proliferation capacity (FIGS. 1E-1F) . Further, adding IL-2 or IL-7 had no significant effects on cytokines release (FIGS. 1G-1K) . Thus, the data indicated that IL-2 addition can improve the efficacy and proliferation capacity.

Optimizing cytokines combination to improve proliferation and efficacy of CAR-polyclonal γδT cells (IL-4 and IL-1β)

In addition to IL-2 and IL-7, IL-4 and IL-1β were also used in T cell culture mediums. Based on this, the effects of IL-4 and IL-1β were evaluated to see whether they can affect the proliferation and efficacy of CAR-polyclonal γδT cells.

Table 2

Polyclonal γδT cells were cultured in Medium Ⅰ with or without IL-4 and IL-1β 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 γδT cells were harvested. Cell number and viability were analyzed by Cellometer (Nexcelom, K2) . For phenotype and CAR positive rate detection, the harvested cells were stained with fluorescent-labeled antibodies (CD3-BV785 [344842, Clone: SK7, BioLegend] , TCRVδ1-APC [17-5679-42, Clone: TS8.2, Invitrogen] , TCRVδ2-BV421 [331428, Clone: B6, Biolegend] and Alexa Fluor 488-labeled anti-mouse sdAb antibodies [GenScript] ) and analyzed by FACS.

As shown in FIG. 2, adding IL-4 or IL-1β had no significant effects on proliferation, viability and CAR positive rate (FIGS. 2A-2C) . However, adding IL-4 or IL-1β affected the percentage of different cell sub-types (FIG. 2D) , leading to increased Vδ1 T ratio in total γδT cells. In the long-term killing assay, CAR-polyclonal γδT cells cultured with IL-4 and IL-1β showed improved efficacy and improved proliferation capacity (FIGS. 2E-2F) . For Cytokine profiles, adding IL-4 or IL-1β could slightly decrease IFN-γ release and show no difference in TNF-α, GM-CSF, IL-2 and IL-17 (FIGS. 2G-2K) . Thus, the data indicated that IL-4 and IL-1β addition can improve the efficacy and proliferation capacity.

Using an AKT inhibitor compound to boost antitumor activity of CAR-polyclonal γδT cells.

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 γδT cells were generated with or without MK-2206 (Table 3) , and cell performance were detected and analyzed.

Table 3

Polyclonal γδT 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 γδT cells were harvested. Cell number and viability were analyzed by Cellometer (Nexcelom, K2) . For phenotype and CAR positive rate detection, the harvested cells were stained with fluorescent-labeled antibodies (CD3-BV785 [344842, Clone: SK7, BioLegend] , TCRVδ1-APC [17-5679-42, Clone: TS8.2, Invitrogen] , TCRVδ2-BV421 [331428, Clone: B6, Biolegend] and Alexa Fluor 488-labeled anti-mouse sdAb antibodies [GenScript] ) and analyzed by FACS.

As shown in FIG. 3, adding MK-2206 slightly decreased the cell number of CAR-polyclonal γδT cells (FIG. 3A) , but had no side effect on viability and transduction (FIGS. 3B-3C) . MK-2206 increased the Vδ2 T cell ratio in total T cells (FIG. 3D) . In the long-term killing assay, CAR-polyclonal γδT cells treated with MK-2206 exhibited improved efficacy, which indicates greater cell fitness (FIGS. 3E-3F) . Cytokines data also showed that adding MK-2206 led to higher cytokine release (FIGS. 3G-3I) . Based on these results, MK-2206 is a good supplement to improve cell fitness.

Cytokine release decline by sub-type ratio adjusting of CAR-polyclonal γδT cells with IL-21

In order to find a way to adjust cytokine release to obtain safety profiles, IL-21 was added to adjust sub-type ratio of expanded CAR-polyclonal γδT cells. CAR-polyclonal γδT cells were generated with or without IL-21 (Table 4) , and cell performance were detected and analyzed.

Table 4

Polyclonal γδT 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 γδT cells were harvested. Cell number and viability were analyzed by Cellometer (Nexcelom, K2) . For phenotype and CAR positive rate detection, the harvested cells were stained with fluorescent-labeled antibodies (CD3-BV785 [344842, Clone: SK7, BioLegend] , TCRVδ1-APC [17-5679-42, Clone: TS8.2, Invitrogen] , TCRVδ2-BV421 [331428, Clone: B6, Biolegend] and Alexa Fluor 488-labeled anti-mouse sdAb antibodies [GenScript] ) and analyzed by FACS.

As shown in FIG. 4, adding IL-21 did not significantly affect the viability (FIG. 4B) , CAR expression (FIG. 4C) and cytotoxicity (FIGS. 4E-4F) of CAR γδT cells. IL-21 slightly reduced proliferation (FIG. 4A) . IL-21 could raise the ratio of Vδ1 or double negative (Vδ1-Vδ2-) T cells (FIG. 4D) . Importantly, CAR-γδT cells which were cultured with IL-21 had lower cytokines level than those cultured without IL-21 (FIGS. 4G-4I) .

Example 5: Optimization of polyclonal γδT cells with different stimulator

To optimize the methods to activate polyclonal γδT cells, three activation conditions were tested, including an antibody which specific binds γδTCR constant chain called AS287963, an anti-CD3 antibody OKT3 (Invitrogen, 16-0037-85) , and anti-CD3/CD28 beads (Miltenyi Biotec, 130091441) . These antibodies were coated onto the cell culture plates. The anti-CD3/CD28 beads were directly added to the cell culture medium.

Table 5

Polyclonal γδT 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 γδT cells were harvested. Cell number and viability were analyzed by Cellometer (Nexcelom, K2) . For phenotype and CAR positive rate detection, the harvested cells were stained with fluorescent-labeled antibodies (CD3-BV785 [344842, Clone: SK7, BioLegend] , TCRVδ1-APC [17-5679-42, Clone: TS8.2, Invitrogen] , TCRVδ2-BV421 [331428, Clone: B6, Biolegend] and Alexa Fluor 488-labeled anti-mouse sdAb antibodies [GenScript] ) and analyzed by FACS.

The results of expansion and viability were shown in FIGS. 5A-5B. All γδT 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 γδT cells was higher in the AS287963 group than the OKT3 and CD3/CD28 groups (FIG. 5D) .

To further evaluate the correlation between different stimulators and killing potency, the expanded CAR-γδT cells were evaluated in a repetitive tumor challenge assay. In briefly, 2×10⁵ CAR+ γδT cells were co-cultured with 2×10⁵ 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+ γδT cells were quantified and re-plated with fresh NCI-H929 cells at a ratio of 1: 1 for the next round. IFN-γ, GM-CSF and TNF-α release in the supernatant was determined at the end of first round. The cytokine release analysis was performed withreagents (Human IFN gamma kit, Cisbio, Cat#62HIFNGPEH, Human TNF alpha kit, Cisbio, Cat#62HTNFAPEH, Human GM-CSF kit, Cisbio, Cat#62HGMCSFPEH) .

As shown in FIGS. 5E-5F, polyclonal γδT cells activated by AS287963 mediated effective elimination of H929 cells until round 12 (E: T=1: 1 each round) . However, in other groups, H929 cell challenges resulted in a lower level of lysis of tumor cells and lower CAR γδT cell expansion, indicating decreased cytotoxicity. The AS287963 group showed the lowest IFN-γsecretion and moderate secretion of GM-CSF and TNF-α compared with other groups (FIGS. 5G-5I) .

Example 6: Intrinsic killing assay

To test intrinsic anti-tumor responses without CAR expression, an in vitro cytotoxic assay was performed. 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 γδT cells were generated without transduction step by using the medium with IL-21 as described in Table 4. The untransducted Vδ1 T cells and Vδ2 T cells were produced without transduction step as the method described in Example 1 and Example 2. The γδT cells (polyclonal γδT cells, Vδ1 T cells or Vδ2 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, 100μl ONE-Glo reagent (E6110, Promega) was added to the 100μl 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 γδT cells (without CAR expression) against 6 tumor cell lines were comparable to Vδ1 T cells and significantly better than Vδ2 T cells. These results demonstrated that polyclonal γδT cells provide better intrinsic cytotoxicity than Vδ2 T cells.

Example 7: Three-Way Mixed Lymphocyte Reaction (MLR) Assay

To test allogeneic immune responses, a Three-Way MLR assay was performed. CAR-γδT cells (1.5×10⁴ cells per well) , NCI-H929 tumor cells (1.5×10⁴ cells per well) and allogeneic/autologous PBMCs (1.5×10⁶ 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. Thereafter, cells were harvested, stained with fluorescent-labeled antibodies (CD3-APC/Cy7 [300426, Clone: UCHT1, BioLegend] , TCRαβ-PE [130-113-529, Clone: BW242/412, Militenyi] , TCRVδ1-APC [17-5679-42, Clone: TS8.2, Invitrogen] , TCRVδ2-BV605 [331430, Clone: B6, Biolegend] , Alexa Fluor 488-labeled anti-mouse sdAb antibody [GenScript] and CD56-PerCP/Cy5.5 [362506, Clone: 5.1H11, Biolegend] ) and analyzed in a BD CELESTA flow cytometer. Proliferated αβT cells and CAR-γδT cells were discriminated by fluorescence signals. As shown in FIG. 7B, CAR-polyclonal γδT cells generated by using the medium without IL-21 as described in Table 4 exhibited better proliferation than CAR-Vδ2 T cells, in the presence of human PBMCs. As shown in FIG. 7A, proliferation of αβT cells from PBMCs was not significantly stimulated by CAR-γδT cells.

Example 8: CAR-polyclonal γδT cells demonstrated superior anti-tumor efficacy in vitro

The production and anti-tumor effects of CAR-polyclonal γδT, CAR-Vδ1T and CAR-Vδ2T cells in multiple myeloma (MM) indications were evaluated. Specifically, according to the culture method in Example 4 that using the medium without IL-21 as described in Table 4, γδT cells were activated (Medium I) and cultured for 11 days (Medium II) . Thereafter, cells were harvested. Cell number and viability were analyzed in Cellometer (Nexcelom, K2) . The cells were stained with fluorescent-labeled antibodies (TCRVδ1-APC, TCRVδ2-BV605, sdAb Alexa488, CD45RA-FITC, CD62L-Alexa647) and γδT cell subtypes and phenotype were determined by FACS. Persistence of CAR-γδT cells were evaluated in a repetitive tumor challenge assay. In briefly, 2×10⁵ CAR+ γδT cells were co-cultured with 2×10⁵ 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+ γδT cells were quantified and re-plated with fresh NCI-H929 cells at a ratio of 1: 1 for the next round. IFN-γ, GM-CSF and TNF-α release in the supernatant was determined at the end of first round.

The proliferation (FIG. 8A) , viability (FIG. 8B) , CD3 purity (FIG. 8D) and phenotype (FIG. 8F) of CAR-polyclonal γδT cells were not significantly different from CAR-Vδ1T and CAR-Vδ2T cells, but CAR expression (FIG. 8C) of CAR-polyclonal γδT cells was significantly higher than CAR-Vδ1T and CAR-Vδ2T cells. Compared with CAR-Vδ1T and CAR-Vδ2T cells, which have a single cell subtype, CAR-polyclonal γδT cell subtype species show diversity (FIG. 8E) . As shown in FIGS. 8G-8H, CAR-polyclonal γδT cells mediated effective elimination of H929 cells until round 19. By comparison, repeated H929 tumor challenges resulted in decreased CAR-Vδ1T and CAR-Vδ2T cells cytotoxicity, as reflected by lower levels of lysis of tumor cell and lower CAR γδT cell expansion fold. In addition, IFN-γ (FIG. 8I) release was lower in CAR-polyclonal γδT cells than in CAR-Vδ2T cells and much higher than in CAR-Vδ1T cells. GM-CSF (FIG. 8J) release and TNF-α (FIG. 8K) release were both higher in CAR-polyclonal γδT cells than in CAR-Vδ1T and CAR-Vδ2T cells. In conclusion, CAR-polyclonal γδT cells demonstrated superior anti-tumor efficacy in vitro.

Example 9: CAR-polyclonal γδT cells demonstrated superior anti-tumor efficacy in vivo

In vivo anti-tumor effects of CAR-polyclonal γδT, CAR-Vδ1T and CAR-Vδ2T cells in multiple myeloma (MM) and Non-Hodgkin's lymphoma (NHL) indications were evaluated. CAR-polyclonal γδT cells were cultured by following the method in Example 4 that using the medium without IL-21 as described in Table 4, CAR-Vδ1T and CAR-Vδ2T cells were generated by following the method in Example 1 and Example 2. Specifically, the anti-tumor activity of exemplary anti-BCMA CAR-T cells were assessed in vivo in an RPMI-8226 xenograft model. Briefly, five million (5×10⁶) RPMI-8226 cells stably expressing the firefly luciferase reporter were implanted subcutaneously/intravenously at day 0 in NOD/SCID IL-2RγCnull (NSG) mice. Fourteen days after tumor inoculation, mice were treated with intravenous injection of 1×10⁶ CAR-Vδ1T, CAR-Vδ2T and CAR-polyclonal γδT cells or phosphate-buffered saline (PBS) . Tumor progression was monitored by bioluminescent imaging (BLI) once a week. In addition, T cell proliferation was monitored via FACS analysis of blood plasma. As shown in FIGS. 9A-9B, CAR-polyclonal γδT showed better antitumor efficacy and expansion than the CAR-Vδ1T and CAR-Vδ2T cells in MM indications. To further evaluate the anti-tumor efficacy of CAR-polyclonal γδT, NCG mice were subcutaneously injected with z-138 cells to construct a xenotransplantation model. Fourteen days after tumor inoculation, mice were treated with intravenous injection of 1×10⁶ CAR-Vδ1T, CAR-Vδ2T and CAR-polyclonal γδT or phosphate-buffered saline (PBS) . Tumor length (L) and width (W) were measured by caliper every 3-4 days after CAR-T cells treatment. Tumor volume was estimated using the following formula: V = (W2× L) /2. In addition, T cell proliferation is monitored via FACS analysis of blood serum. As shown in FIGS. 9C-9D, CAR-polyclonal γδT showed better antitumor efficacy and expansion than the un-polyclonal CAR-Vδ1T and CAR-Vδ2T cells in NHL indications. In conclusion, CAR-polyclonal γδT cells demonstrated superior anti-tumor efficacy in vivo.

Example 10: CAR-polyclonal γδT cells stimulated with different antibodies

To optimize the methods to activate polyclonal γδT cells, four activation antibodies specific binds γδTCR were tested, including AS281850, AS287435, AS288180 and AS287963. These antibodies were coated onto the cell culture plates, and CAR-polyclonal γδT cells were generated (Table 6) , and cell performance were detected and analyzed.

Table 6

Polyclonal γδT 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 γδT cells were harvested. Cell number and viability were analyzed by Cellometer (Nexcelom, K2) . For phenotype and CAR positive rate detection, the harvested cells were stained with fluorescent-labeled antibodies (CD3-BV785 [344842, Clone: SK7, BioLegend] , TCRVδ1-APC [17-5679-42, Clone: TS8.2, Invitrogen] , TCRVδ2-BV421 [331428, Clone: B6, Biolegend] and Alexa Fluor 488-labeled anti-mouse sdAb antibodies [GenScript] ) and analyzed by FACS.

To further evaluate the correlation between different stimulators and killing potency, the expanded CAR-γδT cells were evaluated in a repetitive tumor challenge assay. In briefly, 2×10⁵ CAR+ γδT cells were co-cultured with 2×10⁵ 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+ γδT cells were quantified and re-plated with fresh NCI-H929 cells at a ratio of 1: 1 for the next round.

With all 4 heavy chain antibodies, γδT cells expanded well with at least 900-fold expansion and > 90%cell viability (FIGS. 10A-B) . The purity of γδT cells produced using all 4 heavy chain antibodies is high, > 98% (FIG. 10C) , and the composition of different γδT cell subtypes are similar: the percentage of Vδ2 T cells is the highest (～ 65%) and Vδ1-Vδ2-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. 10F, polyclonal γδT cells which activated by different antibodies mediated effective elimination of H929 cells until round 7, the total γδT expansion fold and CAR γδT expansion fold of all groups during long term killing were similar (FIGS. 10G-H) .

OTHER EMBODIMENTS

It is to be understood that while the disclosure has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the disclosure, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

A method for culturing γδT cells comprising:

(1) culturing cells from a sample in a first culture medium comprising interleukin-15 (IL-15) , interferon-γ (IFN-γ) , and interleukin-2 (IL-2) ; and

(2) culturing the cells obtained in step (1) in a second culture medium comprising IL-15, IFN-γ, and IL-2.
The method of claim 1, wherein the γδT cells obtained after step (2) comprising Vδ1 T cells, Vδ2 T cells and Vδ1-Vδ2-T cells.
The method of claim 1 or claim 2, wherein at least about 5%of the γδT cells obtained after step (2) are Vδ1 T cells.
The method of claim 1 or claim 2, wherein at least about 5%of the γδT cells obtained after step (2) are Vδ2 T cells.
The method of claim 1 or claim 2, wherein at least about 1%of γδT cells obtained after step (2) are Vδ1-Vδ2-T cells.
The method of any one of claims 1-5, wherein 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-κB inhibitor, BCL-2 inhibitor, ERK inhibitor, MEK inhibitor, Raf-1 inhibitor, EGFR inhibitor, DAC inhibitor, HDAC inhibitor or CDK46 inhibitor.
The method of claim 6, wherein 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 method of claim 7, wherein the AKT inhibitor is MK-2206 and is present in an amount of 0.1-10 μg/ml.
The method of any one of claims 1-8, wherein 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 method of any one of claims 1-9, wherein the IL-15 in the first culture medium is present in a concentration of about 1-30 ng/ml.
The method of any one of claims 1-10, wherein the IL-15 in the second culture medium is present in a concentration of about 1-200 ng/ml.
The method of any one of claims 1-11, wherein the IFN-γ in the first culture medium and/or the second culture medium is present in a concentration of about 1-500 ng/ml.
The method of any one of claims 1-12, wherein the IFN-γ in the first culture medium is present in a concentration of about 50-150 ng/ml.
The method of any one of claims 1-13, wherein the IFN-γ in the second culture medium is present in a concentration of about 1-100 ng/ml.
The method of any one of claims 1-14, wherein 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 method of any one of claims 1-15, wherein the IL-2 in the first culture medium is present in a concentration of about 50-150 IU/ml.
The method of any one of claims 1-16, wherein the IL-2 in the second culture medium is present in a concentration of about 50-150 IU/ml.
The method of any one of claims 1-17, wherein 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 method of any one of claims 1-18, wherein the concentration of IFN-γ in the first culture medium is at least 1 or 2 times higher than the concentration of IFN-γ in the second culture medium.
The method of any one of claims 1-19, wherein the first culture medium and/or the second culture medium further comprises IL-4 and/or IL-1β.
The method of claim 20, wherein the IL-4 in the first culture medium is present in a concentration of about 1-500 ng/ml.
The method of claim 20, wherein the IL-4 in the first culture medium is present in a concentration of about 50-150 ng/ml.
The method of claim 20, wherein the IL-1β in the first culture medium is present in a concentration of about 1-200 ng/ml.
The method of claim 20, wherein the IL-1β in the first culture medium is present in a concentration of about 1-30 ng/ml.
The method of any one of claims 20-24, wherein the first culture medium and/or the second culture medium further comprises IL-21.
The method of claim 25, wherein the IL-21 in the first culture medium is present in a concentration of about 1-200 ng/ml.
The method of claim 25, wherein the IL-21 in the first culture medium is present in a concentration of about 1-30 ng/ml.
The method of any one of claims 20-24, wherein the first culture medium and/or the second culture medium does not comprise IL-21.
The method of any one of claims 1-28, wherein the first culture medium and/or the second culture medium does not comprise IL-7.
The method of any one of claims 1-29, wherein step (1) further comprises stimulating the γδT cells by an anti-γδTCR antibody.
The method of claim 30, wherein the anti-γδTCR antibody specifically binds to the constant chain of TCR gamma or delta chain.
The method of claim 31, wherein the anti-γδTCR antibody is a V_HH 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 method of claim 32, wherein the V_HH 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 method of claim 31, wherein the anti-γδTCR 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 method of claim 34, wherein the anti-γδTCR 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 method of claim 34 or claim 35, wherein the anti-γδTCR antibody comprises an amino sequence that is at least 80%, 90%or 100%identical to SEQ ID NO: 4 and 7.
The method of any one of claims 30-36, wherein the anti-γδTCR antibody is immobilized on a cell culture plate.
The method of claim 37, wherein the anti-γδTCR antibody is immobilized on the cell culture plate at 0.1-5 μg/ml per well.
The method of any one of claims 30-38, wherein the first culture medium comprises the anti-γδTCR-specific antibody.
The method of any one of claims 30-39, wherein the first culture medium and/or the second culture medium does not comprise an anti-CD28 antibody.
The method of any one of claims 1-40, wherein prior to step (1) , the sample is enriched for γδT cells.
The method of any one of claims 1-41, wherein αβT cells are depleted prior to step (1) , between step (1) and step (2) , or after step (2) .
The method of any one of claims 1-42, wherein NK cells are depleted prior to step (1) , between step (1) and step (2) , or after step (2) .
The method of any one of claims 1-43, wherein the sample is selected from blood, peripheral blood, umbilical cord blood, lymphoid tissue, bone marrow, spleen, induced pluripotent stem cells or skin tissues.
The method of any one of claims 1-44, wherein the sample comprises peripheral blood mononuclear cells (PBMCs) .
The method of any one of claims 1-45, wherein the cells are cultured for 3-9 days during step (1) .
The method of any one of claims 1-46, wherein the cells are cultured for 6-12 days during step (2) .
The method of any one of claims 1-47, wherein the cells are collected prior to 35 days of culturing.
The method of claim 48, wherein the cells are collected prior to 21 days of culturing.
The method of any one of claims 1-49, wherein the first and/or the second culture medium comprises L-glutamine, streptomycin sulfate, and gentamicin sulfate.
The method of any one of claims 1-50, wherein the first and/or second culture media comprises serum.
The method of claim 51, wherein the serum is present in an amount from about 0.5 to about 25%by volume.
The method of claim 51 or claim 52, wherein the serum is human AB serum.
The method of any one of claims 1-50, wherein the first and/or second culture media comprises a human serum replacement.
The method of claim 54, wherein the human serum replacement is human platelet lysate (HPL) .
The method of any one of claims 1-55, wherein the method further comprises introducing a heterologous nucleic acid into the cells prior to step (1) .
The method of any one of claims 1-55, wherein the method further comprises introducing a heterologous nucleic acid into the cells between step (1) and step (2) .
The method of any one of claims 1-55, wherein the method further comprises introducing a heterologous nucleic acid into the cells after step (2) .
The method of any one of claims 56-58, wherein the heterologous nucleic acid encodes a chimeric antigen receptor (CAR) or a T cell receptor (TCR) .
The method of any one of claims 56-59, wherein the heterologous nucleic acid is delivered with a lentiviral vector or retroviral vector.
The method of any one of claims 56-60, wherein 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.
A method for preparing γδT cells, the method comprising:

(1) culturing cells from a sample in a first culture medium comprising IL-15, IFN-γ, IL-2, IL-4 and IL-1β; and

(2) culturing the cells obtained in step (1) in a second culture medium comprising IL-15, IFN-γ and IL-2.
A method for preparing γδT cells, the method comprising:

(1) culturing cells from a sample in a first culture medium comprising IL-15, IFN-γ, IL-2, IL-4, IL-1β, and IL-21; and

(2) culturing the cells obtained in step (1) in a second culture medium comprising IL-15, IFN-γ and IL-2.
The method of claim 62 or 63, wherein the first culture medium and/or the second culture medium further comprises an AKT inhibitor (e.g., MK-2206) .
The method of any one of claims 62-64, where prior to step (1) , the sample is depleted of αβ T cells and NK cells.
The method of any one of claims 62-65, where during step (1) the cells are exposed to an anti-γδTCR-specific antibody.
The method of any one of claims 62-66, wherein prior to step (2) , the cells are transfected with a vector encoding an engineered receptor (e.g., CAR) .
The method of any one of claims 62-67, wherein the cells are cultured for 3-9 days during step (1) .
The method of any one of claims 62-68, wherein the cells are cultured for 6-12 days during step (2) .
The method of any one of claims 62-69, wherein the first culture medium and/or second culture medium comprises HPL.
A cell preparation prepared using the method of any one of the preceding claims.
A pharmaceutical composition comprising the cell preparation of claim 71, and a pharmaceutically acceptable carrier.