Method for culturing Vdelta1 T cells

Abstract

The present application provides methods of making engineered and non-engineered Vdelta1T cells and uses thereof. The Vdelta1T cells are useful for the treatment of various cancers, infectious diseases and immune disorders. The present application also provides methods for expanding engineered and non-engineered V [delta] 1T cell populations to therapeutically available amounts. The engineered V [delta] 1 T cells may be a universal donor and may be administered to a subject having any MHC haplotype.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority of international application No. PCT / CN2023 / 082247, filed on March 17, 2023, the contents of which are incorporated herein by reference in their entirety.

[0003] Sequence listings should be submitted as ASCII text files

[0004] The following content, submitted as an XML file, is incorporated herein by reference in its entirety: Sequence Listing in Computer Readable Form (CRF) (File name: IEC240138PCT_SequenceListing.xml, Record Date: March 14, 2024, Size: 3KB). Technical Field

[0005] The present disclosure relates to methods of culturing Vδ1 T cells and uses thereof. Background Art

[0006] γδ T cells in peripheral lymphocytes exhibit potent cancer antigen recognition independent of classical peptide-MHC complexes, making them attractive candidates for allogeneic adoptive cancer immunotherapy. Compared with their more prevalent counterparts expressing Vγ9Vδ2-TCR and αβ-TCR, the peripheral γδ T cell subset expressing the Vδ1-T cell receptor (TCR) remains enigmatic.

[0007] Developing γδT cell-based cell therapies is challenging. The first patient was dosed with an allogeneic adoptively transferred Vδ1 T cell product in 2021. A significant factor limiting the clinical use of Vδ1 T cells is the lack of robust, sustained, and GMP-compliant expansion protocols. Reliable and GMP-compliant expansion protocols are needed. Summary of the Invention

[0008] The present disclosure relates to methods for preparing engineered or non-engineered Vδ1 T cells and their uses. On the one hand, the present disclosure provides a two-step protocol for amplifying γδT cells (e.g., Vδ1 T cells) from peripheral blood mononuclear cells (PBMCs), which is further compatible with efficient genetic engineering for immunotherapy purposes. As described herein, the method can provide a ready-made CAR-T cell therapy method with a lower cost and a more standardized production process, which can be applied to a wider range of patients with hematological or solid tumor diseases, thereby reducing remission rates and improving drug efficacy durability.

[0009] In one aspect, the disclosure relates to a method for culturing Vδ1 T cells, the method comprising:

[0010] (1) culturing γδ T cells from a sample in a first culture medium comprising interleukin-4 (IL-4), interleukin-15 (IL-15), interleukin-1β (IL-1β), and interferon-γ (IFN-γ); and

[0011] (2) Expanding the cells obtained in step (1) in a second culture medium containing IL-15 and IFN-γ.

[0012] In some embodiments, the γδ T cells comprise Vδ1 T cells.

[0013] In some embodiments, the γδ T cells are a mixture of Vδ1, Vδ2, or Vδ3 T cells.

[0014] In some embodiments, after the two-step culture, the percentage of Vδ1 T cells is greater than 60%, 70%, 80%, or 90% of the total cells in culture.

[0015] In some embodiments, the present disclosure relates to a method for culturing Vδ1 T cells, the method comprising:

[0016] (1) culturing Vδ1 T cells from a sample in a first culture medium comprising interleukin-4 (IL-4), interleukin-15 (IL-15), interleukin-1β (IL-1β), and interferon-γ (IFN-γ); and

[0017] (2) Expanding the cells obtained in step (1) in a second culture medium containing IL-15 and IFN-γ.

[0018] In some embodiments, the IL-4 in the first culture medium has a concentration of about 1 to about 1000 ng / ml, about 10 to about 500 ng / ml, about 10 to about 300 ng / ml, about 20 to about 200 ng / ml, about 30 to 180 ng / ml, about 50 to about 150 ng / ml, about 60 to about 140 ng / ml, about 70 to about 130 ng / ml, about 80 to about 120 ng / ml, or about 90 to about 110 ng / ml.

[0019] In some embodiments, the IL-4 in the first culture medium has a concentration of about 80 to about 120 ng / ml.

[0020] In some embodiments, the IL-4 in the first culture medium has a concentration of about 100 ng / ml.

[0021] In some embodiments, the IL-15 in the first culture medium has a concentration of about 1 to about 1000 ng / ml, about 1 to about 500 ng / ml, about 1 to about 300 ng / ml, about 5 to about 200 ng / ml, about 5 to 150 ng / ml, about 5 to about 100 ng / ml, about 5 to about 50 ng / ml, about 5 to about 25 ng / ml, or about 5 to about 15 ng / ml.

[0022] In some embodiments, the IL-15 in the first culture medium has a concentration of about 5 to about 15 ng / ml.

[0023] In some embodiments, the IL-15 in the first culture medium has a concentration of about 10 ng / ml.

[0024] In some embodiments, the IL-1β in the first culture medium has a concentration of about 1 to about 1000 ng / ml, about 5 to about 500 ng / ml, about 5 to about 300 ng / ml, about 5 to about 200 ng / ml, about 5 to 180 ng / ml, about 5 to about 150 ng / ml, about 5 to about 140 ng / ml, about 5 to about 100 ng / ml, about 10 to about 50 ng / ml, or about 10 to about 20 ng / ml.

[0025] In some embodiments, the IL-1β in the first culture medium has a concentration of about 10 to about 20 ng / ml.

[0026] In some embodiments, the IL-1β in the first culture medium has a concentration of about 15 ng / ml.

[0027] In some embodiments, the IFN-γ in the first culture medium has a concentration of about 1 to about 1000 ng / ml, about 10 to about 500 ng / ml, about 10 to about 300 ng / ml, about 20 to about 200 ng / ml, about 30 to 150 ng / ml, about 50 to about 150 ng / ml, about 50 to about 140 ng / ml, about 60 to about 120 ng / ml, about 60 to about 100 ng / ml, or about 60 to about 80 ng / ml.

[0028] In some embodiments, the IFN-γ in the first culture medium has a concentration of about 60 to about 80 ng / ml.

[0029] In some embodiments, the IFN-γ in the first culture medium has a concentration of about 70 ng / ml.

[0030] In some embodiments, the IL-15 in the second culture medium has a concentration of about 1 to about 1000 ng / ml, about 10 to about 500 ng / ml, about 10 to about 300 ng / ml, about 20 to about 200 ng / ml, about 30 to 180 ng / ml, about 50 to about 150 ng / ml, about 60 to about 140 ng / ml, about 60 to about 120 ng / ml, about 60 to about 100 ng / ml, or about 60 to about 80 ng / ml.

[0031] In some embodiments, the IL-15 in the second culture medium has a concentration of about 60 to about 80 ng / ml.

[0032] In some embodiments, the IL-15 in the second culture medium has a concentration of about 70 ng / ml.

[0033] In some embodiments, the IFN-γ in the second culture medium has a concentration of about 1 to about 1000 ng / ml, about 5 to about 500 ng / ml, about 5 to about 300 ng / ml, about 10 to about 200 ng / ml, about 10 to 150 ng / ml, about 15 to about 120 ng / ml, about 15 to about 100 ng / ml, about 15 to about 50 ng / ml, or about 20 to about 40 ng / ml.

[0034] In some embodiments, the IFN-γ in the second culture medium has a concentration of about 20 to about 40 ng / ml.

[0035] In some embodiments, the IFN-γ in the second culture medium has a concentration of about 30 ng / ml.

[0036] In some embodiments, the concentration of IL-15 in the second culture medium is at least 1, 2, 3, 4, or 5 times greater than the concentration of IL-15 in the first culture medium.

[0037] In some embodiments, the concentration of IFN-γ in the first culture medium is at least 1 or 2 times greater than the concentration of IFN-γ in the second culture medium.

[0038] In some embodiments, step (1) further comprises stimulating Vδ1 T cells with Vδ1 T cell-specific antibodies.

[0039] In some embodiments, the Vδ1 T-specific antibody specifically binds the TCRδ chain.

[0040] In some embodiments, the Vδ1 T-specific antibody is the TCRδ monoclonal antibody TS-1.

[0041] In some embodiments, Vδ1 T-specific antibodies are immobilized on a cell culture plate.

[0042] In some embodiments, the Vδ1 T-specific antibody is immobilized on the cell culture plate at 0.5 μg / ml / well.

[0043] In some embodiments, the first culture medium comprises Vδ1 T-specific antibodies.

[0044] In some embodiments, the Vδ1 T cell-specific antibody specifically activates Vδ1 T cells and optionally selectively expands Vδ1 T cells.

[0045] In some embodiments, the expanded cell culture comprises a percentage of Vδ1 T cells that is greater than 60%, 70%, 80%, or 90% of the total cells in the culture.

[0046] In some embodiments, prior to step (1), the sample is enriched for γδ T cells.

[0047] In some embodiments, γδ T cells are enriched by depleting αβ T cells.

[0048] In some embodiments, γδ T cells are enriched by depleting NK cells.

[0049] In some embodiments, γδ T cells are enriched by depleting αβ T cells and NK cells.

[0050] In some embodiments, γδ T cells are enriched by isolating γδ T cells from a sample.

[0051] In some embodiments, αβ T cells are depleted prior to step (1).

[0052] In some embodiments, NK cells are depleted prior to step (1).

[0053] In some embodiments, αβ T cells are depleted between step (1) and step (2).

[0054] In some embodiments, NK cells are depleted between steps (1) and (2).

[0055] In some embodiments, αβ T cells are depleted after step (2).

[0056] In some embodiments, NK cells are depleted after step (2).

[0057] In some embodiments, the sample is selected from blood, peripheral blood, umbilical cord blood, lymphoid tissue, bone marrow, or spleen.

[0058] In some embodiments, the sample comprises peripheral blood mononuclear cells (PBMCs).

[0059] In some embodiments, the cells are cultured for 5-9 days during step (1).

[0060] In some embodiments, the cells are cultured for 7 days during step (1).

[0061] In some embodiments, the cells are cultured for 6-10 days during step (2).

[0062] In some embodiments, the cells are cultured for 8 days during step (2).

[0063] In some embodiments, cells are harvested prior to 35 days of culture.

[0064] In some embodiments, cells are harvested prior to 21 days of culture.

[0065] In some embodiments, the first culture medium and / or the second culture medium comprises AIM-V.

[0066] In some embodiments, the first culture medium and / or the second culture medium comprises L-glutamine, streptomycin sulfate, and gentamicin sulfate.

[0067] In some embodiments, the first culture medium and / or the second culture medium further contains serum.

[0068] In some embodiments, serum is present in an amount of about 0.5% to about 25% by volume.

[0069] In some embodiments, the serum is FBS.

[0070] In some embodiments, the first culture medium and / or the second culture medium further contains 10% human platelet lysate.

[0071] In some embodiments, the IL-4 is human IL-4.

[0072] In some embodiments, the IL-15 is human IL-15.

[0073] In some embodiments, the IL-1β is human IL-1β.

[0074] In some embodiments, the IFN-γ is human IFN-γ.

[0075] In some embodiments, prior to step (1), the cells are transduced with a vector.

[0076] In some embodiments, between steps (1) and (2), cells are transduced with a vector.

[0077] In some embodiments, after step (2), the cells are transduced with the vector.

[0078] In some embodiments, the vector comprises a nucleic acid sequence encoding a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

[0079] In some embodiments, cells are transduced with a lentiviral vector.

[0080] In some embodiments, cells are transduced with a retroviral vector.

[0081] In some embodiments, the resulting Vδ1 T cells have a purity greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99%.

[0082] In some embodiments, the resulting Vδ1 T cells have a CD27 and CD45RA double-positive rate higher than 50%, higher than 60%, higher than 70%, higher than 80%, or higher than 84%.

[0083] In some embodiments, the resulting Vδ1 T cells have an NGK2D positivity rate higher than 50%, higher than 60%, higher than 70%, higher than 80%, higher than 90%, or higher than 93%.

[0084] In some embodiments, the resulting Vδ1 T cells have a TIGIT positivity rate of less than 20%, or less than 10%, less than 8%, or less than 7%.

[0085] In some embodiments, the resulting Vδ1 T cells have a PD-1 positivity rate of less than 20%, less than 10%, less than 5%, less than 2%, less than 1%, or less than 0.8%, less than 0.7%, or less than 0.6%.

[0086] In some embodiments, after 9 rounds of tumor stimulation with NCI-H929 target cells, the resulting Vδ1 T cells can kill greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, or greater than 95% of the target cells.

[0087] In some embodiments, after 8 rounds of tumor stimulation with NCI-H929 target cells, the resulting Vδ1 T cells have an expansion fold greater than 1k, greater than 2k, greater than 5k, greater than 10k, greater than 20k, greater than 50k, greater than 100k, greater than 150k, greater than 200k, or greater than 250k.

[0088] In some embodiments, the resulting Vδ1 T cells secrete less than 2000 pg / ml, less than 3000 pg / ml, less than 4000 pg / ml, less than 5000 pg / ml, less than 6000 pg / ml, or less than 7000 pg / ml of GM-CSF after 48 hours of co-culture with NCI-H929 target cells.

[0089] In some embodiments, the resulting Vδ1 T cells secrete less than 2000 pg / ml, less than 3000 pg / ml, less than 4000 pg / ml, less than 5000 pg / ml, less than 6000 pg / ml, or less than 7000 pg / ml INF-γ after 48 hours of co-culture with NCI-H929 target cells.

[0090] In one aspect, the disclosure relates to a method for preparing Vδ1 T cells, the method comprising:

[0091] (1) culturing cells in the sample in a first culture medium comprising 80-120 ng / ml (e.g., about 100 ng / ml) IL-4, 5-15 ng / ml (e.g., about 10 ng / ml) IL-15, 10-20 ng / ml (e.g., about 15 ng / ml) IL-1β, and 60-80 ng / ml (e.g., about 70 ng / ml) IFN-γ; and

[0092] (2) culturing the cells obtained in step (1) in a second culture medium containing 60-80 ng / ml (e.g., about 70 ng / ml) IL-15 and 20-40 ng / ml (e.g., about 30 ng / ml) IFN-γ.

[0093] In some embodiments, prior to step (1), the sample is depleted of αβ T cells and / or NK cells.

[0094] In some embodiments, during step (1), the cells are exposed to a Vδ1 T-specific antibody.

[0095] In some embodiments, the Vδ1 T cell-specific antibody specifically activates Vδ1 T cells and optionally selectively expands Vδ1 T cells.

[0096] In some embodiments, prior to step (2), the cells are transfected with a vector encoding an engineered receptor (e.g., CAR).

[0097] In some embodiments, the cells are cultured for 5-9 days (eg, about 7 days) during step (1).

[0098] In some embodiments, the cells are cultured for 6-10 days (eg, about 8 days) during step (2).

[0099] In one aspect, the disclosure relates to cell preparations prepared using the methods described herein.

[0100] In one aspect, the disclosure relates to a pharmaceutical composition comprising a cell preparation described herein and a pharmaceutically acceptable carrier.

[0101] In one aspect, the disclosure relates to a method of treating a subject having cancer, the method comprising administering to a subject in need thereof a therapeutically effective amount of a cell preparation described herein.

[0102] In some embodiments, the subject has a solid tumor.

[0103] In some embodiments, the cancer is breast cancer, lung cancer, pancreatic cancer, melanoma, oral cancer, mesothelioma, ovarian cancer, colorectal cancer, gastric cancer, cervical cancer, brain cancer, skin cancer, multiple myeloma, lymphoma, epithelial tumor, soft tissue sarcoma, esophageal cancer, or a CNS tumor.

[0104] In one aspect, the disclosure relates to a method of treating a subject having an infection, the method comprising administering to a subject in need thereof a therapeutically effective amount of a cell preparation described herein.

[0105] In some embodiments, the infection is a viral infection, a bacterial infection, or a fungal infection.

[0106] In one aspect, the disclosure relates to a method of treating a subject having an immune disorder, the method comprising administering to a subject in need thereof a therapeutically effective amount of a cell preparation described herein.

[0107] As used herein, when applied to one or more destination values, the terms "about" and "approximately" refer to a value similar to the reference value. In certain embodiments, the terms "about" or "approximately" refer to the value of (greater than or less than) 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less scope falling on either direction of the reference value, unless otherwise stated or in addition it is apparent from the context (unless such a numeral will exceed 100% of a possible value). For example, when used in the context of the amount of a given compound in a composition, "about" can mean the + / - 10% of the listed value. For example, the culture medium comprising the given compound of about 100ng / ml can include the compound of 90-110ng / ml.

[0108] As used herein, the term "IL-15" refers to a polypeptide derived from wild-type IL-15 or a functional variant thereof. In some embodiments, IL-15 is wild-type IL-15 (e.g., human IL-15). In some embodiments, IL-15 may have one or more mutations (e.g., insertions, deletions, or substitutions). In some embodiments, IL-15 is human IL-15. In some embodiments, IL-15 is recombinant IL-15.

[0109] As used herein, the term "IL-4" refers to a polypeptide derived from wild-type IL-4 or a functional variant thereof. In some embodiments, IL-4 is wild-type IL-4 (e.g., human IL-4). In some embodiments, IL-4 may have one or more mutations (e.g., insertions, deletions, or substitutions). In some embodiments, IL-4 is human IL-4. In some embodiments, IL-4 is recombinant IL-4.

[0110] As used herein, the term "IL-1β" refers to a polypeptide derived from wild-type IL-1β or a functional variant thereof. In some embodiments, IL-1β is wild-type IL-1β (e.g., human IL-1β). In some embodiments, IL-1β may have one or more mutations (e.g., insertions, deletions, or substitutions). In some embodiments, IL-1β is human IL-1β. In some embodiments, IL-1β is recombinant IL-1β.

[0111] As used herein, the term "IFN-γ" refers to a polypeptide derived from wild-type IFN-γ or a functional variant thereof. In some embodiments, IFN-γ is wild-type IFN-γ (e.g., human IFN-γ). In some embodiments, IFN-γ may have one or more mutations (e.g., insertions, deletions, or substitutions). In some embodiments, IFN-γ is human IFN-γ. In some embodiments, IFN-γ is recombinant IFN-γ.

[0112] As used herein, the term "IL-21" refers to a polypeptide derived from wild-type IL-21 or a functional variant thereof. In some embodiments, IL-21 is wild-type IL-21 (e.g., human IL-21). In some embodiments, IL-21 may have one or more mutations (e.g., insertions, deletions, or substitutions). In some embodiments, IL-21 is human IL-21. In some embodiments, IL-21 is recombinant IL-21.

[0113] As used herein, the term "IL-2" refers to a polypeptide derived from wild-type IL-2 or a functional variant thereof. In some embodiments, IL-2 is wild-type IL-2 (e.g., human IL-2). In some embodiments, IL-2 may have one or more mutations (e.g., insertions, deletions, or substitutions). In some embodiments, IL-2 is human IL-2. In some embodiments, IL-2 is recombinant IL-2.

[0114] As used herein, the term "purity" refers to the percentage of desired cells (eg, Vδ1 T cells) based on the total number of cells in culture.

[0115] As used herein, the term "recombinant protein" is a protein derived from recombinant DNA by expressing the recombinant DNA in a host cell.

[0116] As used herein, the term "cancer" refers to a cell with an uncontrolled autonomous growth capacity. Examples of such cells include cells with an abnormal state or condition characterized by rapid proliferation of cell growth. The term is meant to include cancerous growths, such as tumors; oncogenic processes, metastatic tissues, and malignantly transformed cells, tissues, or organs, regardless of the histopathological type or invasive stage. Also included are malignant tumors of various organ systems (e.g., respiratory, cardiovascular, renal, reproductive, blood, nervous, liver, gastrointestinal, and endocrine systems); and adenocarcinomas including malignant tumors, such as most colon cancers, renal cell carcinomas, prostate cancer, and / or testicular tumors, non-small cell lung cancer, and small intestinal cancer. "Naturally occurring" cancers include any cancer that is not experimentally induced by implanting cancer cells into a subject, and include, for example, spontaneously occurring cancers, cancers caused by exposure of patients to carcinogens, cancers caused by transgenic oncogene insertion or tumor suppressor gene knockout, and cancers caused by infection (e.g., viral infection). The term "cancer" is generally recognized and refers to a malignant tumor of epithelial or endocrine tissue. The term also includes carcinosarcoma, which includes malignant tumors composed of cancerous and sarcomatous tissues. "Adenocarcinoma" refers to a cancer that is derived from glandular tissue or in which the tumor cells form a recognizable glandular structure. The term "sarcoma" is well-known and refers to a malignant tumor of mesenchymal origin. The term "hematopoietic neoplastic disorder" includes diseases involving hyperplastic / neoplastic cells of hematopoietic origin. Hematopoietic neoplastic disorders can originate from the myeloid, lymphoid, or erythroid lineages or their precursor cells. Hematological cancers are cancers that begin in hematopoietic tissue (such as the bone marrow) or cells of the immune system. Examples of hematological cancers include, for example, leukemias, lymphomas, and multiple myeloma.

[0117] 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 is provided according to the methods of the present disclosure. Veterinary and non-veterinary applications are contemplated in the present disclosure. Human patients can be adults or adolescents (e.g., humans under the age of 18). In addition to humans, patients include, but are not limited to, mice, rats, hamsters, guinea pigs, rabbits, ferrets, cats, dogs, and primates. For example, non-human primates (e.g., monkeys, chimpanzees, gorillas, etc.), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), lagomorphs, pigs (e.g., pigs, miniature pigs), horses, dogs, cats, cattle, and other domestic animals, farm animals, and zoo animals are included.

[0118] 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.

[0119] As used herein, the term "chimeric antigen receptor" or "CAR" as used herein refers to a genetically engineered receptor that can be used to specifically transplant one or more antigens to immune effector cells such as T cells. Some CARs are also referred to as "artificial T cell receptors," "chimeric T cell receptors," or "chimeric immune receptors." In some embodiments, CAR comprises an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain of T cells and / or other receptors that are specific for one or more antigens (such as tumor antigens). "CAR-T cells" refer to T cells expressing CAR.

[0120] 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 antigen peptide bound in an MHC molecule. In some embodiments, the TCR comprises a TCR alpha polypeptide chain and a TCR beta polypeptide chain. In some embodiments, the TCR comprises a TCR gamma polypeptide chain and a TCR delta polypeptide chain. In some embodiments, the TCR specifically binds to a tumor antigen. "TCR-T" refers to a T cell expressing a recombinant TCR. The expression of a heterologous antigen receptor (e.g., a heterologous TCR or CAR) can change the immunogenic specificity of the T cell so that they recognize one or more tumor antigens present on the surface of cancer cells of an individual with cancer, or show improved recognition of one or more tumor antigens present on the surface of cancer cells of an individual with cancer.

[0121] 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. The methods and materials described herein are used in this disclosure; other suitable methods and materials known in the art may also be used. The materials, methods, and examples are provided for illustrative purposes only and are 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 the event of a conflict, the present specification, including definitions, will control. BRIEF DESCRIPTION OF THE DRAWINGS

[0122] Figure 1 Shown are the results of testing Vδ1 T cell expansion under conditions 1, 2, and 3. Vδ1 T cells were expanded as described in Example 1, and cell expansion was determined on day 15.

[0123] Figures 2A-2DThe results of the purity test of Vδ1 T cells prepared under condition 3 (Figures 2A and 2C) and the CAR positive rate test results (Figures 2B and 2D) are shown. As described in Example 1, Vδ1 T was amplified using condition 3 and transduced with retrovirus (Figures 2A and 2B) and lentivirus (Figures 2C and 2D) encoding anti-BCMA-CAR (SEQ ID NO: 1), and the cell purity and transduction rate were determined on day 15.

[0124] Figures 3A-3B The results of the phenotype test of Vδ1 T cells prepared under condition 3 are shown. In Figure 3A, the dotted line shows the results of the negative control. In Figure 3B, the X-axis measures CD27 and the Y-axis measures CD45RA. As described in Example 1, Vδ1 T cells were expanded under condition 3 and transduced with anti-BCMA-CAR (SEQ ID NO: 1), and the cell phenotype was determined on day 15.

[0125] Figures 4A-4B The results of the in vitro long-term cytotoxicity test ( FIG. 4A ) and the persistence test ( FIG. 4B ) of Vδ1 T cells prepared under conditions 1, 2, and 3 are shown. As described in Example 1, Vδ1 T cells were prepared under different conditions and transduced with anti-BCMA-CAR (SEQ ID NO: 1). As described in Example 2, in vitro anti-tumor cytotoxicity and cell proliferation were determined using a long-term co-culture assay.

[0126] Figures 5A-5B Results of cytokine release assays of Vδ1 T cells prepared under conditions 1, 2, and 3 are shown. Vδ1 T cells were prepared under different conditions and transduced with anti-BCMA-CAR (SEQ ID NO: 1) as described in Example 1. Cytokine release of GM-CSF and IFN-γ was assessed using HTRF assays as described in Example 3.

[0127] Figure 6 The results of the in vivo efficacy test of Vδ1 T cells against multiple myeloma in the NCG mouse model are shown. As described in Example 1, Vδ1 T cells were expanded using conditions 2 and 3 and transduced with anti-BCMA-CAR (SEQ ID NO: 1). As described in Example 4, in vivo anti-tumor cytotoxicity was determined using a NOD / SCID IL-2RγCnull (NSG) mouse model bearing multiple myeloma tumors (RPMI-8226).

[0128] Figure 7 The amino acid sequences discussed in this disclosure are listed. DETAILED DESCRIPTION

[0129] In humans, γδ T cells are a subpopulation of T cells that provide a link between innate and adaptive immune responses. These cells undergo V-(D)-J segment rearrangement to generate antigen-specific γδ T cell receptors (γδTCRs). γδ T cells can be directly activated by recognizing antigens through the γδTCR or other non-TCR proteins that act independently or in combination to activate the effector functions of γδ T cells. γδ T cells constitute a small fraction of the overall mammalian T cell population, approximately 1%–5% of T cells in peripheral blood and lymphoid organs, and they appear to reside primarily in epithelial cell-rich areas such as the skin, liver, digestive tract, respiratory tract, and reproductive tract. Unlike αβ TCRs, which recognize antigens bound to major histocompatibility complex (MHC) molecules, γδ TCRs can directly recognize bacterial and viral antigens, stress antigens expressed by diseased cells, and tumor antigens in the form of intact proteins or non-peptide compounds.

[0130] The ability of γδ T cells to recognize a broad spectrum of antigens can be enhanced by genetically engineering them. γδ T cells can also be engineered to provide universal allogeneic therapies that recognize selected antigens in vivo.

[0131] In particular, γδ T cells are highly cytotoxic to tumor cells. They work through TCRs, NCRs (natural cytotoxicity receptors), and other mechanisms to recognize and kill tumors. Unlike the T cell receptors of αβ T cells, γδ TCRs recognize antigens in an MHC-independent manner, making it possible for γδ T cells to be used as allogeneic "off-the-shelf" cancer treatments because they do not induce GvHD.

[0132] Human γδT cells are composed of three main populations based on their Vδ chains. There are Vδ1, Vδ2, and Vδ3 cells. Typically, Vδ2T cells pair with the Vγ9 chain in the γδTCR complex and are primarily found in the peripheral blood, while Vδ1 T cells pair with Vγ2 / 3 / 4 / 5 / 8 / 9 in the γδTCR complex and, due to their diversity, can be found in the peripheral blood, skin, intestines, spleen, and liver. In the field, ex vivo cultures of Vδ2T cells have been developed, starting with PBMCs containing 5%-10% Vδ2T cells and using phosphoantigens such as isoprenyl pyrophosphate (IPP) or bromohydrin pyrophosphate (BrHPP) as stimulators. For nearly two decades, such Vδ2T cells have been used in clinical trials for adoptive cell therapy. In contrast, because PBMCs contain less than 1% Vδ1 T cells, generating ex vivo cultures of highly pure, active, and anti-tumor cytotoxic Vδ1 T cells on a large scale remains a major challenge in the field.

[0133] γδT cells expressing Vδ1 typically comprise 10% to 30% of all γδT cells in peripheral blood, but their primary distribution is in epithelial tissues. Furthermore, Vδ1+ T cells often predominate (over Vδ2+) within tumor infiltrates, and Vδ1+ TIL (tumor-infiltrating lymphocyte)-derived lines often outperform Vδ2+ TIL lines in in vitro tumor cytotoxicity assays. The leukemia-targeting capacity of peripheral blood Vδ1+ T cells (compared to Vδ2+ T cells) can be enhanced by selectively inducing natural cytotoxicity receptors (NCRs: NKp30, NKp44, and NKp46) upon stimulation with TCR agonists and cytokines in vitro. This expanded repertoire of activating / cytotoxic receptors, coupled with their enhanced resistance to activation-induced cell death and exhaustion upon sustained stimulation, makes Vδ1+ T cells highly attractive candidates for adoptive cell therapy (ACT) of cancer. However, the difficulty of selectively amplifying them to large quantities under good manufacturing practice (GMP) conditions has hindered the clinical application of Vδ1+ T cells. More details about Vδ1 cells can be found in Almeida et al. "Delta One T Cells for Immunotherapy of Chronic Lymphocytic Leukemia: Clinic-Grade Expansion / Differentiation and Preclinical proof concept Delta One T cells for Adoptive immunotherapy of CLL." Clinical Cancer Research 22.23(2016):5795-5804, which is incorporated herein by reference in its entirety.

[0134] Current protocols for the in vitro expansion of Vδ1+ T cells use mitogenic phytohemagglutinins (phytohemagglutinin PHA or concanavalin A (ConA)) and unsafe materials that are not directly suitable for clinical use. In addition, the discovery of the tumor-promoting effects of IL-17-producing Vδ1+ T cells has raised concerns about their use and emphasized the need for detailed characterization of effector Vδ1+ lymphocytes that may be used for ACT.

[0135] The present disclosure provides an improved method for culturing and amplifying γδT cells (such as Vδ1 T cells) after testing the ability of multiple cytokine combinations to culture and amplify peripheral blood γδT cells (such as Vδ1 T cells) in culture. γδT cells (such as Vδ1 T cells) can be selectively amplified by culturing these cells in vitro in two stages. In the first stage, these cells can be cultured in a first culture medium comprising IL-4, IL-15, IL-1β and IFN-γ and stimulated by γδT-specific antibodies (such as Vδ1 T-specific antibodies). In the second stage, these cells can be amplified in a second culture medium containing IL-15 and IFN-γ. These cells can also be isolated, cultured and amplified in culture in the absence of feeder cells.

[0136] In some embodiments, γδ T cells (e.g., Vδ1 T cells) have an average expansion rate of about 1 cell division, about 2 cell divisions, about 3 cell divisions, about 4 cell divisions, about 5 cell divisions, about 6 cell divisions, about 7 cell divisions, about 8 cell divisions, about 9 cell divisions, or about 10 cell divisions in less than 24 hours. In some embodiments, Vδ1 T cells have an average expansion rate of greater than 1 cell division, greater than 2 cell divisions, greater than 3 cell divisions, greater than 4 cell divisions, greater than 5 cell divisions, greater than 6 cell divisions, greater than 7 cell divisions, greater than 8 cell divisions, greater than 9 cell divisions, or greater than 10 cell divisions in less than 24 hours. In some embodiments, Vδ1 T cells have an average expansion rate of less than 1 cell division, less than 2 cell divisions, less than 3 cell divisions, less than 4 cell divisions, less than 5 cell divisions, less than 6 cell divisions, less than 7 cell divisions, less than 8 cell divisions, less than 9 cell divisions, or less than 10 cell divisions in less than 24 hours.

[0137] In some embodiments, the γδ T cells (e.g., Vδ1 T cells) have a frequency of about 1 division every about 4 hours, 1 division every about 5 hours, 1 division every about 6 hours, 1 division every about 7 hours, 1 division every about 8 hours, 1 division every about 9 hours, 1 division every about 10 hours, 1 division every about 11 hours, 1 division every about 12 hours, 1 division every about 13 hours, 1 division every about 14 hours, 1 division every about 15 hours, 1 division every about 16 hours, 1 division every about 17 hours, 1 division every about 18 hours, 1 division every about 19 hours, 1 division every about 20 hours, The average expansion rate is 1 division per about 24 hours, 1 division per about 25 hours, 1 division per about 26 hours, 1 division per about 27 hours, 1 division per about 28 hours, 1 division per about 29 hours, 1 division per about 30 hours, 1 division per about 31 hours, 1 division per about 32 hours, 1 division per about 33 hours, 1 division per about 34 hours, 1 division per about 35 hours, or 1 division per about 36 hours.

[0138] In some embodiments, the γδ T cells (e.g., Vδ1 T cells) have a rapid expansion rate over a period of 1 to 36 days in culture, resulting in 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. In some embodiments, the Vδ1 T cells have an expansion rate over a period of 1 day to 36 days in culture resulting in 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. In some embodiments, the Vδ1 T cells have a rapid expansion rate within 14 days, resulting in 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.

[0139] In some embodiments, the expanded cell culture comprises 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% of γδ T cells (e.g., Vδ1 T cells). In some embodiments, the expanded cell culture comprises 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% of γδ T cells (e.g., Vδ1 T cells). In some embodiments, the expanded cell culture comprises 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% of Vδ1 T cells.

[0140] In some cases, the cell culture further comprises a certain amount of engineered γδT cells (e.g., Vδ1 T cells), wherein the engineered γδT cells (e.g., Vδ1 T cells) are engineered to express an antigen recognition portion (e.g., CAR, TCR). In some embodiments, the expanded cell culture comprises engineered Vδ1 T cells at a percentage 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%. In some embodiments, the expanded cell culture comprises 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% of engineered γδ T cells (e.g., Vδ1 T cells). In some embodiments, the expanded cell culture comprises 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% engineered Vδ1 T cells.

[0141] In some embodiments, the engineered γδ T cells (eg, Vδ1 T cells) do not express the human HLA locus.

[0142] sample

[0143] The present disclosure provides methods for selectively culturing and expanding γδT cells (e.g., Vδ1 T cells) in culture. The methods described in the present disclosure are performed on a sample, which is also referred to herein as a "starting sample." The methods use an unfractionated sample or a sample that has been enriched for T cells, γδT cells, or Vδ1 T cells. In some embodiments, the sample can be enriched for γδT cells. In some embodiments, the sample can be enriched for Vδ1 T cells.

[0144] The sample can be any sample containing Vδ1 T cells or their precursors, including but not limited to blood, bone marrow, lymphoid tissue, thymus, spleen, lymph node tissue, infected tissue, fetal tissue and parts or enriched parts thereof. The sample is optionally blood, including peripheral blood or umbilical cord blood or parts thereof, which include buffy coat cells, leukocyte apheresis products, peripheral blood mononuclear cells (PBMC) and low-density mononuclear cells (LDMC). In some embodiments, the sample is human blood or a part thereof. Cells can be obtained from a blood sample using techniques known in the art (such as density gradient centrifugation). For example, whole blood can be layered onto equal volumes of Ficoll-Hypaque TM The interphase material will contain low-density mononuclear cells, which can be collected in culture medium, washed, and centrifuged at 200 × g for 10 minutes at room temperature.

[0145] In some embodiments, the αβT cells in the sample can be depleted. In some embodiments, the αβT cells in the sample are depleted using antibodies targeting αβT cells. In some embodiments, the antibodies targeting αβT cells are linked to magnetic beads. In some embodiments, these cells can be removed together with these beads. In some embodiments, the αβT cells in the sample are depleted using a TCRαβ cell depletion kit (Miltenyi, 200-070-407).

[0146] In some embodiments, the natural killer (NK) cells in the sample can be depleted. In some embodiments, the NK cells in the sample are depleted using antibodies targeting NK cells. In some embodiments, the antibodies targeting NK cells are connected to magnetic beads. In some embodiments, the antibodies targeting NK cells specifically bind to CD56. In some embodiments, the NK cells in the sample are depleted using a CD56+ cell depletion kit (Miltenyi, 130-050-401) according to the manufacturer's instructions.

[0147] In some embodiments, αβ T cells in the sample are depleted using the TCRαβ Cell Depletion Kit (Miltenyi, 200-070-407) and the CD56+ Cell Depletion Kit (Miltenyi, 130-050-401) according to the manufacturer's instructions, which are incorporated herein by reference in their entirety.

[0148] In some embodiments, αβ T cells may be depleted before the first stage, between the first and second stages, or after the second stage. In some embodiments, NK cells may be depleted before the first stage, between the first and second stages, or after the second stage.

[0149] Method for preparing Vδ1 T cells

[0150] Certain cytokines (e.g., IL-4, IL-21, IL-1β, and IFN-γ) have potent stimulatory effects on a variety of immune cells, including TCRαβT and Vδ2T cells. Therefore, these cytokines are generally not suitable for in vitro expansion of Vδ1 T cells because they also expand other cells in the culture, thereby reducing the purity of Vδ1 T cells. Methods based on more selective reagents are needed for expanding Vδ1 T cells from impure starting samples (e.g., human PBMCs).

[0151] The present disclosure provides methods for producing CAR-Vδ1 T cells with high purity, high expansion rate, and high transduction rate for clinical use and production. Such cells show high activation, low exhaustion, and a predominantly naive phenotype. In vitro validation has shown superior anti-tumor activity and safety compared to cells obtained from some other existing methods.

[0152] On the one hand, the method for expanding human γδT cells (e.g., Vδ1 T cells) has a culture phase and an expansion phase. In the culture phase, these cells can be cultured in a cell culture medium comprising one or more cytokines selected from, for example, IL-4, IL-15, IL-1β and IFN-γ. In some embodiments, cells are stimulated by Vδ1 T-specific antibodies. In the expansion phase, these cells can be expanded in a cell expansion medium containing one or more cytokines selected from IL-15 and IFN-γ. In some embodiments, cells are cultured and expanded without the need for feeder cells or microorganisms or viral components. In some embodiments, cells are cultured and expanded without the need for IL-21. In some embodiments, cells are cultured and expanded without the need for IL-2.

[0153] Thus, in a first aspect, a method for culturing and expanding γδ T cells (e.g., Vδ1 T cells) in a sample comprises:

[0154] (1) culturing cells in a sample in a first culture medium, wherein the first culture medium comprises one or more cytokines selected from the group consisting of IL-4, IL-15, IL-1β, and IFN-γ; and

[0155] (2) culturing the cells obtained in step (1) in a second culture medium containing one or more cytokines selected from the group consisting of IL-15 and IFN-γ.

[0156] In some embodiments, during step (1), the cells are stimulated by Vδ1 T-specific antibodies. In some embodiments, the first culture medium is located in a container (e.g., a cell culture plate) coated with a Vδ1 T-specific antibody. In some embodiments, the Vδ1 T-specific antibody is a TCR Vδ1 monoclonal antibody. In some embodiments, the Vδ1 T-specific antibody is an anti-Vδ1TCR TS-1 antibody (e.g., Thermo Fisher, TCR1055). Other Vδ1 T-specific antibodies are known in the art. They are described in detail in, for example, U.S. Patent Application Publication No. US 20230028110A1, PCT Patent Application Publication No. WO2019147735A1, WO 2017197347A1, WO 2021032960A1, WO 2022034562A1, and / or WO2022175413A1, each of which is incorporated by reference in its entirety.

[0157] In some embodiments, the first culture medium comprises 1, 2, 3, 4, or more than 4 cytokines. In some embodiments, the first culture medium comprises only 1, only 2, only 3, only 4, or only 5 cytokines. In some embodiments, the first culture medium comprises or consists of IL-4. In some embodiments, the first culture medium comprises or consists of IL-15. In some embodiments, the first culture medium comprises or consists of IL-1β. In some embodiments, the first culture medium comprises or consists of IFN-γ. In some embodiments, the first culture medium comprises or consists of IL-4 and IL-15. In some embodiments, the first culture medium comprises or consists of IL-4, IL-15, and IL-1β. In some embodiments, the first culture medium comprises or consists of IL-4, IL-15, IL-1β, and IFN-γ. In some embodiments, the first culture medium comprises or consists of IL-15 and IL-1β. In some embodiments, the first culture medium comprises or consists of IL-15, IL-1β, and IFN-γ. In some embodiments, the first culture medium comprises or consists of IL-1β and IFN-γ.

[0158] In some embodiments, IL-4 is present in an amount of about 1 to about 1000 ng / ml. Optionally, IL-4 is present in an amount of about 2 to about 500 ng / ml. Optionally, IL-4 is present in an amount of about 20 to about 200 ng / ml. Optionally, IL-4 is present in an amount of about 100 ng / ml. In some embodiments, IL-4 is present in an amount of 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. In some embodiments, IL-4 is present in an amount of 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. In some embodiments, IL-4 is present in an amount of 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. In some embodiments, IL-4 is present in an amount of about 1 to about 1000 ng / ml, about 10 to 100 ng / ml, about 20 to 200 ng / ml, about 30 to 300 ng / ml, about 40 to 400 ng / ml, about 50 to 500 ng / ml, about 50 to 150 ng / ml, about 80 to 120 ng / ml, or about 90 to 100 ng / ml.

[0159] In some embodiments, IL-15 is present in an amount of about 1 to about 500 ng / ml. Optionally, IL-15 is present in an amount of about 2 to about 200 ng / ml. Optionally, IL-15 is present in an amount of about 5 to about 100 ng / ml. Optionally, in the first culture medium, IL-15 is present in an amount of about 10 ng / ml. Optionally, in the second culture medium, IL-15 is present in an amount of about 70 ng / ml. In some embodiments, IL-15 is present in an amount 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. In some embodiments, IL-15 is present in an amount of 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. In some embodiments, IL-15 is present in an amount of 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. In some embodiments, IL-15 is present in an amount of about 1 to about 1000 ng / ml, about 1 to 100 ng / ml, about 1 to 75 ng / ml, about 1 to 50 ng / ml, about 1 to 25 ng / ml, about 5 to 20 ng / ml, about 5 to 15 ng / ml, or about 7.5 to 12.5 ng / ml.

[0160] In some embodiments, IL-1β is present in an amount of about 1 to about 500 ng / ml. Optionally, IL-1β is present in an amount of about 2 to about 200 ng / ml. Optionally, IL-1β is present in an amount of about 5 to about 100 ng / ml. Optionally, IL-1β is present in an amount of about 15 ng / ml. In some embodiments, IL-1β is present in an amount of 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. In some embodiments, IL-1β is present in an amount of 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. In some embodiments, IL-1β is present in an amount of 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. In some embodiments, IL-1β is present in an amount of about 1 to about 1000 ng / ml, about 5 to 100 ng / ml, about 10 to 75 ng / ml, about 10 to 50 ng / ml, about 10 to 25 ng / ml, about 10 to 20 ng / ml, about 12.5 to 17.5 ng / ml, or about 14 to 16 ng / ml.

[0161] In some embodiments, IFN-γ is present in an amount of about 1 to about 1000 ng / ml. Optionally, IFN-γ is present in an amount of about 2 to about 500 ng / ml. Optionally, IFN-γ is present in an amount of about 20 to about 200 ng / ml. Optionally, in the first culture medium, IFN-γ is present in an amount of about 70 ng / ml. Optionally, in the second culture medium, IFN-γ is present in an amount of about 30 ng / ml. In some embodiments, IFN-γ is present in an amount 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. In some embodiments, IFN-γ is present in an amount of 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. In some embodiments, IFN-γ is present in an amount of 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. In some embodiments, IFN-γ is present in an amount of about 1 to about 1000 ng / ml, about 10 to 100 ng / ml, about 20 to 200 ng / ml, about 30 to 300 ng / ml, about 40 to 400 ng / ml, about 50 to 500 ng / ml, about 50 to 150 ng / ml, about 50 to 90 ng / ml, or about 60 to 80 ng / ml.

[0162] In some embodiments, the first culture medium further comprises a Vδ1 T-specific antibody. In some embodiments, the Vδ1 T-specific antibody is present in an amount of about 0.1 to about 100 μg / ml. Optionally, the Vδ1 T-specific antibody is present in an amount of about 0.1 to about 10 μg / ml. Optionally, the Vδ1 T-specific antibody is present in an amount of about 0.5 to about 5 μg / ml. Optionally, in the first culture medium, the Vδ1 T-specific antibody is present in an amount of about 1 μg / ml. In some embodiments, the first culture medium is in a container (e.g., a cell culture plate) coated with the Vδ1 T-specific antibody.

[0163] In some embodiments, the Vδ1 T-specific antibody is immobilized on the cell culture plate at about 1 to about 5000 ng / ml. Optionally, the Vδ1 T-specific antibody is immobilized on the cell culture plate at about 50 to about 1000 ng / ml. Optionally, the Vδ1 T-specific antibody is immobilized on the cell culture plate at about 200 to about 1000 ng / ml. Optionally, the Vδ1 T-specific antibody is immobilized on the cell culture plate at about 500 ng / ml. In some embodiments, the Vδ1 T-specific antibody is present in an amount of about 0.1 to about 100 μg / well. Optionally, the Vδ1 T-specific antibody is present in an amount of about 0.1 to about 10 μg / well. Optionally, the Vδ1 T-specific antibody is present in an amount of about 0.1 to about 5 μg / well. Optionally, in the first culture medium, the Vδ1 T-specific antibody is present in an amount of about 0.5 μg / well.

[0164] In some embodiments, the second culture medium comprises 1, 2, 3, 4, or more than 4 cytokines. In some embodiments, the second culture medium comprises only 1, only 2, only 3, only 4, or only 5 cytokines. In some embodiments, the second culture medium comprises or consists of IFN-γ. In some embodiments, the second culture medium comprises or consists of IL-15. In some embodiments, the second culture medium comprises or consists of IFN-γ and IL-15.

[0165] In some embodiments, IFN-γ is present in an amount of about 1 to about 1000 ng / ml. Optionally, IFN-γ is present in an amount of about 2 to about 500 ng / ml. Optionally, IFN-γ is present in an amount of about 20 to about 200 ng / ml. Optionally, in the first culture medium, IFN-γ is present in an amount of about 70 ng / ml. Optionally, in the second culture medium, IFN-γ is present in an amount of about 30 ng / ml. In some embodiments, IFN-γ is present in an amount 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. In some embodiments, IFN-γ is present in an amount of 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. In some embodiments, IFN-γ is present in an amount of 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. In some embodiments, IFN-γ is present in an amount of about 1 to about 1000 ng / ml, about 1 to 100 ng / ml, about 1 to 50 ng / ml, about 10 to 50 ng / ml, or about 20 to 40 ng / ml.

[0166] In some embodiments, IL-15 is present in an amount of about 1 to about 500 ng / ml. Optionally, IL-15 is present in an amount of about 2 to about 200 ng / ml. Optionally, IL-15 is present in an amount of about 5 to about 100 ng / ml. Optionally, in the first culture medium, IL-15 is present in an amount of about 10 ng / ml. Optionally, in the second culture medium, IL-15 is present in an amount of about 70 ng / ml. In some embodiments, IL-15 is present in an amount 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. In some embodiments, IL-15 is present in an amount of 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. In some embodiments, IL-15 is present in an amount of 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. In some embodiments, IL-15 is present in an amount of about 1 to about 1000 ng / ml, about 10 to 100 ng / ml, about 20 to 200 ng / ml, about 30 to 300 ng / ml, about 40 to 400 ng / ml, about 50 to 500 ng / ml, about 50 to 150 ng / ml, about 50 to 90 ng / ml, or about 60 to 80 ng / ml.

[0167] In some embodiments, the concentration of IL-15 in the second culture medium is at least 1, 2, 3, 4, or 5 times greater 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 greater than the concentration of IFN-γ in the second culture medium.

[0168] In some embodiments, the cells are cultured in the first culture medium for a period of time 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 are cultured in the first culture medium for 5 days. In some embodiments, the cells are cultured in the first culture medium for greater than 1 day, greater than 2 days, greater than 3 days, greater than 4 days, greater than 5 days, greater than 6 days, greater than 7 days, greater than 8 days, greater than 9 days, greater than 10 days, greater than 11 days, greater than 12 days, greater than 13 days, or greater than 14 days. In some embodiments, the cells are 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. In some embodiments, the cells are 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. In some embodiments, the cells are cultured in the first culture medium for a period of time ranging from about 2 days to about 21 days, about 3 days to about 20 days, about 3 days to about 10 days, about 3 days to about 7 days, about 2 days to about 7 days, about 3 days to about 6 days, or about 4 days to about 6 days.

[0169] The cells are optionally 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 7 days to 10 days. Optionally, the cells are cultured in the second culture medium for 9 days. In some embodiments, the cells are cultured in the second culture medium for greater than 1 day, greater than 2 days, greater than 3 days, greater than 4 days, greater than 5 days, greater than 6 days, greater than 7 days, greater than 8 days, greater than 9 days, greater than 10 days, greater than 11 days, greater than 12 days, greater than 13 days, or greater than 14 days. In some embodiments, the cells are 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. In some embodiments, the cells are 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. In some embodiments, the cells are cultured in the second culture medium for a period ranging from about 2 days to about 21 days, about 3 days to about 20 days, about 3 days to about 15 days, about 5 days to about 15 days, about 5 days to about 12 days, about 7 days to about 10 days, or about 8 days to about 10 days.

[0170] The culture medium can be supplemented as needed. This can be achieved by adding fresh culture medium to the first culture medium, optionally after removing a portion of the first culture medium. This can be accomplished by centrifuging and / or decanting the cells, removing a portion of the first culture medium, and resuspending the cells in a second culture medium. In some embodiments, supplementation involves removing at least ¾ of the previous culture medium.

[0171] Any suitable mammalian cell culture medium may be used, such as AIM-V TM , TexMACS, RPMI 1640, OPTMIZER CTS TM (Gibco, Life Technologies), X-VIVO 10, X-VIVO 15, or X-VIVO 20 (Lonza). In some embodiments, the mammalian cell culture medium comprises L-glutamine, streptomycin sulfate, and gentamicin sulfate. In some embodiments, the mammalian cell culture medium comprises L-glutamine, 50 μg / mL streptomycin sulfate, and 10 μg / mL gentamicin sulfate. In some embodiments, the mammalian cell culture medium comprises serum or plasma.

[0172] In certain embodiments, the first culture medium and the second culture medium are both supplemented with serum or plasma. The amount of plasma in the first culture medium and the second culture medium is optionally about 0.5% to about 25% by volume, for example, about 2% to about 20% by volume or about 2.5% to about 10% by volume, for example, about 10% by volume. 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. Plasma can be from a single donor or can be collected from several donors. In certain embodiments, if autologous Vδ1 T cells are to be used clinically, i.e., re-infused into the same patient from which the original sample is obtained, autologous plasma (i.e., from the same patient) can be used to avoid introducing dangerous products (e.g., viruses) into the patient. In certain embodiments, the first culture medium and the second culture medium are both supplemented with FBS. In some embodiments, FBS is present in an amount 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. In some embodiments, FBS is present in an amount 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. In some embodiments, FBS is present in an amount of 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. In some embodiments, FBS is present in an amount of about 0.5% to about 30%, about 1% to about 25%, about 2% to about 20%, about 5% to about 20%, about 5% to about 15%, about 8% to about 12%, or about 9% to about 11% by volume.

[0173] In some embodiments, both the first culture medium and the second culture medium are supplemented with human platelet lysate. In some embodiments, the human platelet lysate is present in an amount 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. In some embodiments, the human platelet lysate is present in an amount 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. In some embodiments, the human platelet lysate is present in an amount of 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. In some embodiments, the human platelet lysate is present in an amount of about 0.5% to about 30%, about 1% to about 25%, about 2% to about 20%, about 5% to about 20%, about 5% to about 15%, about 8% to about 12%, or about 9% to about 11% by volume.

[0174] Before culturing the sample or a portion thereof (e.g., PBMC) in the first culture medium, the sample or a portion thereof can be enriched for certain cell types and / or depleted for other cell types. In particular, the sample or a portion thereof can be enriched for T cells, or enriched for γδ T cells, or enriched for Vδ1 T cells, or depleted for aβ T cells, or depleted for non-Vδ1 T cells. In some embodiments, the sample is depleted for aβ T cells before culturing the sample in the first culture medium.

[0175] The first culture medium or the second culture medium, or both culture media, may further contain other components that can assist in the growth and expansion of Vδ1 T cells. Examples of other components that can be added include, but are not limited to, plasma or serum, purified proteins (such as albumin), lipid sources (such as low-density lipoprotein (LDL)), vitamins, amino acids, steroids, and any other supplements that support or promote cell growth and / or survival.

[0176] The first culture medium or the second culture medium, or both culture mediums, may also contain other growth factors, including cytokines that can further enhance Vδ1 T cell expansion. Examples of other growth factors that can be added include costimulatory molecules such as IL-21, human anti-SLAM antibodies, any soluble ligands of CD27, or any soluble ligands of CD7. Any combination of these growth factors may be included in the first culture medium or the second culture medium, or in both culture mediums simultaneously.

[0177] In one aspect, the first culture medium comprises IL-4, IL-15, IL-1β, and IFN-γ. In some embodiments, the second culture medium comprises IFN-γ and IL-15.

[0178] In some embodiments, the method of preparing Vδ1 T cells comprises:

[0179] (1) culturing cells in the sample in a first culture medium comprising 80-120 ng / ml (e.g., about 100 ng / ml) interleukin-4 (IL-4), 5-15 ng / ml (e.g., about 10 ng / ml) interleukin-15 (IL-15), 10-20 ng / ml (e.g., about 15 ng / ml) interleukin-1β (IL-1β), and 60-80 ng / ml (e.g., about 70 ng / ml) interferon-γ (IFN-γ); and

[0180] (2) culturing the cells obtained in step (1) in a second culture medium containing 60-80 ng / ml (e.g., about 70 ng / ml) IL-15 and 20-40 ng / ml (e.g., about 30 ng / ml) IFN-γ.

[0181] In some embodiments, prior to step (1), the sample is depleted of αβ T cells.

[0182] In some embodiments, during step (1), the cells are exposed to a Vδ1 T-specific antibody.

[0183] In some embodiments, prior to step (2), the cells are transfected with a vector encoding an engineered receptor (e.g., CAR).

[0184] In some embodiments, the cells are cultured for about 7 days during step (1).

[0185] In some embodiments, the cells are cultured for about 8 days during step (2).

[0186] The present disclosure also provides some alternative methods for culturing and expanding Vδ1 T cells.

[0187] In some embodiments, a method for preparing Vδ1 T cells comprises (1) culturing cells in a sample in a first culture medium comprising 80-120 ng / ml (e.g., about 100 ng / ml) interleukin-4 (IL-4), 5-9 ng / ml (e.g., about 7 ng / ml) interleukin-21 (IL-21), 10-20 ng / ml (e.g., about 15 ng / ml) interleukin-1β (IL-1β), and 60-80 ng / ml (e.g., about 70 ng / ml) interferon-γ (IFN-γ); and (2) culturing the cells obtained in step (1) in a second culture medium comprising 60-80 ng / ml (e.g., about 70 ng / ml) IL-15 and 20-40 ng / ml (e.g., about 30 ng / ml) IFN-γ. 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 about 7 days during step (1). In some embodiments, the cells are cultured for about 8 days during step (2).

[0188] In some embodiments, the method of preparing Vδ1 T cells comprises culturing cells in a sample in a medium comprising IL-2. In some embodiments, the cells are exposed to a Vδ1 T-specific antibody. In some embodiments, after culturing the cells in the sample in a medium comprising IL-2, αβ T cells are depleted.

[0189] Depletion of αβ T cells and enrichment of γδ T cells

[0190] In some embodiments, αβ T cells are depleted at various stages of the methods described herein. In some embodiments, αβ T cells are depleted prior to the first culturing step. In some embodiments, αβ T cells are depleted prior to the second culturing step. In some embodiments, αβ T cells are depleted after the second culturing step.

[0191] γδT cells can be enriched by various means. γδT cells can be directly enriched from a sample, for example, by sorting γδT cells expressing one or more cell surface markers using flow cytometry techniques. Wild-type γδT cells exhibit many antigen recognition, antigen presentation, co-stimulation and adhesion molecules that can associate with γδT cells. One or more cell surface markers, such as specific γδTCR, antigen recognition, antigen presentation, ligands, adhesion molecules, or co-stimulatory molecules can be used to separate wild-type γδT cells from a sample. Various molecules associated with γδT cells or expressed by γδT cells can be used to separate γδT cells from a sample. In some cases, the present disclosure provides a method for enriching a mixed population of Vδ1+, Vδ2+, Vδ3+ cells or any combination thereof.

[0192] Peripheral blood mononuclear cells can be collected using apheresis machines (including Ficoll-Paque TM PLUS (GE Healthcare) system) or another suitable device / system is collected from the subject, and γδ T cells or desired γδ T cell subsets can be purified from the collected sample using, for example, flow cytometry techniques. Umbilical cord blood cells can also be obtained from umbilical cord blood during the subject's birth.

[0193] Positive and / or negative selection of cell surface markers expressed by the collected γδ T cells can be used to isolate γδ T cells, or a population of γδ T cells expressing similar cell surface markers, directly from a subject's peripheral blood sample, umbilical cord blood sample, tumor, tumor biopsy, tissue, lymph fluid, or epithelial sample. For example, γδ T cells can be isolated from a complex sample based on the positive or negative expression of CD2, CD3, CD4, CD8, CD24, CD25, CD44, Kit, TCRa, TCRβ, TCRγ, TCRδ, NKG2D, CD70, CD27, CD30, CD16, CD337 (NKp30), CD336 (NKp46), OX40, CD46, CCR7, and other suitable cell surface markers.

[0194] γδT cells can be separated from complex samples cultured in vitro. In some embodiments, complete PBMC groups can be activated and amplified without previously depleting specific cell populations (such as monocytes, αβT cells, B cells, and NK cells). In some embodiments, the enriched γδT cell population can be produced before its specific activation and amplification. In some aspects, the activation and amplification of γδT cells are carried out in the absence of natural or engineered APCs. In some aspects, immobilized γδT cell mitogens (including antibodies specific for γδTCR) and other γδTCR activators (including lectins) can be used to separate and amplify γδT cells in tumor specimens.

[0195] In some embodiments, Vδ1 T cells or γδ T cells can be isolated using techniques known in the art, including fluorescence-activated cell sorting, immunomagnetic separation, affinity column chromatography, density gradient centrifugation, and cell panning. In some embodiments, γδ T cells can be isolated using a TCRγ / δ+ T cell isolation kit (Miltenyi Biotec, 130-092-892) according to the manufacturer's instructions.

[0196] In some embodiments, αβ T cells in the sample are depleted using antibodies targeting αβ T cells. In some embodiments, antibodies targeting αβ T cells are linked to magnetic beads. In some embodiments, αβ T cells in the sample are depleted using a TCR αβ cell depletion kit (Miltenyi, 200-070-407).

[0197] In some embodiments, the natural killer (NK) cells in the sample can be depleted. In some embodiments, the NK cells in the sample are depleted using antibodies targeting NK cells. In some embodiments, the antibodies targeting NK cells are connected to magnetic beads. In some embodiments, the antibodies targeting NK cells specifically bind to CD56. In some embodiments, the NK cells in the sample are depleted using a CD56+ cell depletion kit (Miltenyi, 130-050-401) according to the manufacturer's instructions.

[0198] In some embodiments, αβ T cells in a sample can be depleted using a TCRαβ Cell Depletion Kit (Miltenyi, 200-070-407) and a CD56+ Cell Depletion Kit (Miltenyi, 130-050-401) according to the manufacturer's instructions.

[0199] Characteristics of the resulting Vδ1 T cells

[0200] In another aspect, the present disclosure provides a cell preparation prepared according to the methods described herein. In some embodiments, the Vδ1 T cell preparation has a purity greater than 80%. Optionally, the resulting Vδ1 T cell preparation has a purity greater than 80%, optionally greater than 90%, and optionally greater than 95%. In some embodiments, the Vδ1 T cell preparation has a purity 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%. In some embodiments, the Vδ1 T cell preparation has a purity of 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%. In some embodiments, the Vδ1 T cell preparation has a purity of 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%.

[0201] In some embodiments, Vδ1 T cells can be transfected with a vector encoding an engineered receptor (e.g., CAR or TCR). In some embodiments, prior to the first culture step, Vδ1 T cells are transfected with a vector encoding an engineered receptor (e.g., CAR or TCR). In some embodiments, after the first culture step, Vδ1 T cells are transfected with a vector encoding an engineered receptor (e.g., CAR or TCR). In some embodiments, after the second culture step, Vδ1 T cells are transfected with a vector encoding an engineered receptor (e.g., CAR or TCR).

[0202] The Vδ1 T cells obtained by the methods described herein can be used in cell therapy. Vδ1 T cells are considered to be the first line of defense against infectious pathogens. In addition, Vδ1 T cells have inherent cytolytic activity against transformed cells of various sources (including B-cell lymphomas, sarcomas, and carcinomas). Therefore, Vδ1 T cells obtained and cultured in vitro according to the methods disclosed herein can be infused into patients to treat or prevent infections, cancers, or diseases caused by immunosuppression.

[0203] The cell culture method described here is robust, highly reproducible, and fully compatible with large-scale clinical applications. It generates sufficient numbers of differentiated Vδ1 T cells for adoptive immunotherapy of cancer and a variety of other therapeutic applications.

[0204] In some embodiments, the Vδ1 T cells prepared according to the methods described herein have higher cytotoxicity. In some embodiments, the Vδ1 T cells prepared according to the methods described herein have a higher expansion rate than Vδ1 T cells prepared according to other methods. In some embodiments, the Vδ1 T cells prepared according to the methods described herein have an expansion fold greater than 1k, greater than 2k, greater than 5k, greater than 10k, greater than 20k, greater than 50k, greater than 100k, greater than 150k, greater than 200k, or greater than 250k as determined by a cell expansion assay.

[0205] In some embodiments, the Vδ1 T cells prepared according to the methods described herein have a higher purity. In some embodiments, the Vδ1 T cells prepared according to the methods described herein have a purity greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, greater than 95%, greater than 96%, greater than 97%, or greater than 97.4%.

[0206] In some embodiments, the Vδ1 T cells prepared according to the methods described herein have a higher CAR positive rate. In some embodiments, the Vδ1 T cells prepared according to the methods described herein have a CAR positive rate higher than 20%, higher than 30%, higher than 40%, higher than 50%, higher than 60%, higher than 70%, or higher than 80%.

[0207] In some embodiments, the Vδ1 T cells prepared according to the methods described herein exhibit high expression of activation markers, such as NGK2D expression (e.g., 93.8%), and low expression of T cell exhaustion markers, such as PD-1 (e.g., 0.519%) and TIGIT (e.g., 6.5%). In some embodiments, the Vδ1 T cells prepared according to the methods described herein have an NGK2D positivity rate of greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, greater than 92.5%, greater than 93%, or greater than 93.5%. In some embodiments, the Vδ1 T cells prepared according to the methods described herein have a PD-1 positivity rate of less than 0.5%, less than 0.7%, less than 0.7%, less than 0.8%, less than 0.9%, less than 1%, less than 2%, less than 5%, or less than 10%. In some embodiments, the Vδ1 T cells prepared according to the methods described herein have a TIGIT positivity rate of less than 0.5%, less than 1%, less than 2%, less than 5%, less than 6%, less than 7%, less than 8%, less than 9%, less than 10%, or less than 20%.

[0208] In some embodiments, the Vδ1 T cells prepared according to the methods described herein have a phenotype that is close to the naive phenotype.

[0209] In some embodiments, the Vδ1 T cells prepared according to the methods described herein have a higher CD27 positivity rate. In some embodiments, the Vδ1 T cells prepared according to the methods described herein have a CD27 positivity rate of greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, or greater than 90%. In some embodiments, the Vδ1 T cells prepared according to the methods described herein have a higher CD45RA positivity rate. In some embodiments, the Vδ1 T cells prepared according to the methods described herein have a CD45RA positivity rate of greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, greater than 95%, or greater than 96%.

[0210] In some embodiments, the Vδ1 T cells prepared according to the methods described herein have higher cytotoxicity. In some embodiments, the Vδ1 T cells prepared according to the methods described herein are more cytotoxic than Vδ1 T cells prepared according to other methods. In some embodiments, as determined by a CAR-T cell toxicity assay, the Vδ1 T cells prepared according to the methods described herein can kill greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, or greater than 95% of target cells. In some embodiments, the Vδ1 T cells prepared according to the methods described herein can kill greater than 80% of target cells after 9 days.

[0211] In some embodiments, the Vδ1 T cells prepared according to the methods described herein have longer persistence. In some embodiments, the Vδ1 T cells prepared according to the methods described herein have higher persistence than Vδ1 T cells prepared according to other methods. In some embodiments, the Vδ1 T cells prepared according to the methods described herein have an expansion fold greater than 1k, greater than 2k, greater than 5k, greater than 10k, greater than 20k, greater than 50k, greater than 100k, greater than 150k, greater than 200k, or greater than 250k as determined by a CAR-T cell expansion assay. In some embodiments, the Vδ1 T cells prepared according to the methods described herein have an expansion fold greater than 200k after 8 days.

[0212] In some embodiments, the Vδ1 T cells prepared according to the methods described herein can secrete less cytokines. In some embodiments, the Vδ1 T cells prepared according to the methods described herein secrete less cytokines than Vδ1 T cells prepared according to other methods. In some embodiments, the Vδ1 T cells prepared according to the methods described herein secrete less GM-CSF than 2000 pg / ml, less than 3000 pg / ml, less than 4000 pg / ml, less than 5000 pg / ml, less than 6000 pg / ml, or less than 7000 pg / ml. In some embodiments, the Vδ1 T cells prepared according to the methods described herein secrete less than 2000 pg / ml, less than 3000 pg / ml, less than 4000 pg / ml, less than 5000 pg / ml, less than 6000 pg / ml, or less than 7000 pg / ml INF-γ.

[0213] In some embodiments, the Vδ1 T cells prepared according to the methods described herein can inhibit tumor growth. In some embodiments, the Vδ1 T cells prepared according to the methods described herein have a tumor growth inhibition percentage (TGI%) greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. In some embodiments, the Vδ1 T cells prepared according to the methods described herein have a tumor growth inhibition percentage of less than 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. TGI% can be measured, for example, 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 start of treatment, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after the start of treatment. As used herein, percent tumor growth inhibition (TGI%) is calculated using the formula:

[0214] TGI(%)=[1-(Ti-T0) / (Vi-V0)]×100

[0215] Ti is the mean tumor volume of the treatment group on day i. T0 is the mean tumor volume of the treatment group on day 0. Vi is the mean tumor volume of the control group on day i. V0 is the mean tumor volume of the control group on day 0.

[0216] In some embodiments, the Vδ1 T cells prepared according to the methods described herein have a stronger tumor suppression effect than Vδ1 T cells prepared according to other methods. In some embodiments, the Vδ1 T cells prepared according to the methods described herein can suppress tumor growth by greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, or greater than 95%.

[0217] Compared with Vδ1 T cells prepared according to other methods, Vδ1 T cells prepared according to the method described herein showed higher purity, yield and persistence and better anti-tumor cytotoxicity.

[0218] Compared with Vδ1 T cells prepared according to other methods, Vδ1 T cells prepared according to the method described herein showed better cell fitness and persistence judged by exhaustion markers and better anti-tumor cytotoxicity.

[0219] In some embodiments, differentiated Vδ1 T cells maintain their cytotoxic phenotype and inhibit tumor growth in vivo following infusion in mice.

[0220] Engineered receptors (e.g., CARs and TCRs)

[0221] Any of the above-mentioned T cells (e.g., γδT cells, Vδ1 T cells) may further express an engineered receptor. Exemplary engineered receptors include, but are not limited to, CAR, engineered TCR, and TAC receptors. In some embodiments, the engineered receptor comprises an extracellular domain, a transmembrane domain, and an intracellular signaling domain that specifically binds to an antigen (e.g., a tumor antigen). In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain and / or a costimulatory domain. In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain of a TCR co-receptor. In some embodiments, the engineered receptor is encoded by a heterologous nucleic acid operably linked to a promoter (e.g., a constitutive promoter or an inducible promoter). In some embodiments, the cell is expressed by inserting a protein into the cell membrane while passing the cell through a microfluidic system (e.g., CELL OUT). ), introducing an engineered receptor into Vδ1 T cells (see, for example, U.S. Patent Application Publication No. 20140287509). The engineered receptor can enhance the function of the modified Vδ1 T cells, such as by targeting the modified Vδ1 T cells, by transducing signals, and / or by enhancing the cytotoxicity of the modified Vδ1 T cells. In some embodiments, the modified Vδ1 T cells do not express an engineered receptor, such as a CAR, TCR, or TAC receptor.

[0222] In some embodiments, the engineered receptor comprises 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 domains.

[0223] In some embodiments, the engineered receptor is a chimeric antigen receptor (CAR). Many chimeric antigen receptors are known in the art and can be applied to the modified Vδ1 T cells of the present disclosure. CAR can also be constructed to be specific for any cell surface marker by using, for example, an antigen binding fragment of an antibody molecule or an antibody variable domain.

[0224] The CAR of the present disclosure includes an extracellular domain, a transmembrane domain and an intracellular signaling domain, which includes at least one targeting domain that specifically binds to at least one tumor antigen. In some embodiments, the intracellular signaling domain produces a signal that promotes the immune effector function of CAR cells (e.g., CAR-T cells). "Immune effector function or immune effector response" refers to a function or response of, for example, immune effector cells that enhance or promote the immune attack of target cells. For example, immune effector function or response can refer to T or NK cells that promote killing or inhibiting target cell growth or proliferation. Examples of immune effector functions, for example, in CAR-T cells, include cytolytic activity (such as antibody-dependent cellular toxicity, or ADCC) and auxiliary activity (such as secretion of cytokines). In some embodiments, CAR has an intracellular signaling domain that has weakened immune effector functions. In some embodiments, the CAR has an intracellular signaling domain of no more than about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10% or less of any one of the immune effector functions (such as cytolytic function for target cells) compared to a CAR with full-length and wild-type CD3ζ and optionally one or more costimulatory domains. In some embodiments, the intracellular signaling domain produces a signal that promotes the proliferation and / or survival of CAR-containing cells. In some embodiments, CAR comprises 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 may comprise the entire intracellular (intracellular) (i.e., intracellular (cytoplasmic)) portion of a molecule or its fragment or derivative or the entire native intracellular signaling domain.

[0225] In some embodiments, the intracellular signaling domain of CAR includes a primary intracellular signaling domain. "Primary intracellular signaling domain" refers to an intracellular signaling sequence that acts in a stimulating manner to induce immune effector function. In some embodiments, the primary intracellular signaling domain contains a signaling motif called an immunoreceptor tyrosine-based activation motif or ITAM. In some embodiments, the primary intracellular signaling domain includes a functional signaling domain of a protein selected from the group consisting of CD3ζ, CD3γ, CD3δ, CD3ε, common FcRγ (FCER1G), FcRβ (FcεRib), CD79a, CD79b, FcγR IIa, DAP10, and DAP12. In some embodiments, the primary intracellular signaling domain comprises a non-functional or weakened signaling domain of a protein selected from the group consisting of CD3ζ, CD3γ, CD3δ, CD3ε, common FcRγ (FCER1G), FcRβ (FcεRib), CD79a, CD79b, FcγRIIa, DAP10 and DAP 12. A non-functional or weakened signaling domain can be a mutant signaling domain having a point mutation, insertion or deletion that weakens or eliminates one or more immune effector functions, such as cytolytic activity or auxiliary activity, including antibody-dependent cellular toxicity (ADCC). In some embodiments, the CAR comprises a non-functional or weakened CD3ζ (i.e., CD3ζ or CD3z) signaling domain. In some embodiments, the intracellular signaling domain does not comprise a primary intracellular signaling domain. The attenuated primary intracellular signaling domain may induce no more than about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10% or less of any of the immune effector functions (e.g., cytolytic function against target cells) compared to a CAR having the same construct but having a wild-type primary intracellular signaling domain.

[0226] In some embodiments, the intracellular signal transduction domain of CAR includes one or more (such as any one of 1, 2, 3 or more) costimulatory domains." Costimulatory domain" can be the intracellular part of a costimulatory molecule. The term "costimulatory molecule" refers to a cognate binding partner on an immune cell (such as a T cell), which specifically binds to a costimulatory ligand, thereby mediating the costimulatory response of the immune cell, such as but not limited to proliferation and survival. Costimulatory molecules are cell surface molecules that contribute to an efficient immune response other than an antigen receptor or its ligand. Costimulatory molecules can be represented by the following protein family: TNF receptor protein, immunoglobulin-like protein, cytokine receptor, integrin, signaling lymphocyte activation molecule (SLAM protein) and activating NK cell receptor. Costimulatory molecules include but are not limited to MHC class I molecules, BTLA and Toll ligand receptors, and OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278) and 4-1BB (CD137). Additional examples of such costimulatory molecules include CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8α, CD8β, IL-2Rβ, IL-2Rγ, IL-7Rα, 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, L FA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CDIOO (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a and ligands that specifically bind to CD83.

[0227] In some embodiments, CAR comprises a single costimulatory domain. In some embodiments, CAR comprises two or more costimulatory domains. In some embodiments, the intracellular signaling domain comprises a functional primary intracellular signaling domain and one or more costimulatory domains. In some embodiments, CAR does not comprise a functional primary intracellular signaling domain (such as CD3 ζ). In some embodiments, CAR comprises an intracellular signaling domain consisting of one or more costimulatory domains or consisting essentially of one or more costimulatory domains. In some embodiments, CAR comprises an intracellular signaling domain consisting of a non-functional or weakened primary intracellular signaling domain (such as a mutation CD3 ζ) and one or more costimulatory domains or consisting essentially of a non-functional or weakened primary intracellular signaling domain (such as a mutation CD3 ζ) and one or more costimulatory domains. After the targeting domain binds to the tumor antigen, the costimulatory domain of CAR can transduce signals to enhance the proliferation, survival and differentiation of engineered immune cells (such as T cells) with CAR, and inhibit activation-induced cell death. In some embodiments, the one or more costimulatory signaling domains are 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 a ligand that specifically binds to CD83.

[0228] In some embodiments, the intracellular signaling domain of CAR includes a costimulatory signaling domain derived from CD28. In some embodiments, the intracellular signaling domain includes an intracellular signaling domain of CD3 ζ and a costimulatory signaling domain of CD28. In some embodiments, the intracellular signaling domain in the chimeric receptor of the present application includes a costimulatory signaling domain derived from 4-1BB (i.e., CD137). In some embodiments, the intracellular signaling domain includes an intracellular signaling domain of CD3 ζ and a costimulatory signaling domain of 4-1BB.

[0229] In some embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling domain of CD28 and a costimulatory signaling domain of 4-1BB. In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain of CD3 zeta, a costimulatory signaling domain of CD28, and a costimulatory signaling domain of 4-1BB. In some embodiments, the intracellular signaling domain comprises a polypeptide comprising, from N-terminus to C-terminus, a costimulatory signaling domain of CD28, a costimulatory signaling domain of 4-1BB, and an intracellular signaling domain of CD3 zeta.

[0230] In some embodiments, the targeting domain of the CAR is an antibody or antibody fragment, such as scFv, Fv, Fab, (Fab')2, single domain antibody (sdAb), or V H H domain. In some embodiments, the targeting domain of CAR is a ligand or extracellular portion of a receptor that specifically binds to a tumor antigen. In some embodiments, one or more targeting domains of CAR specifically bind to a single tumor antigen. In some embodiments, CAR is a bispecific or multispecific CAR with a targeting domain that binds to two or more tumor antigens. In some embodiments, the tumor antigen is selected from the group consisting of: CD19, BCMA, NY-ESO-1, VEGFR2, MAGE-A3, CD20, CD22, CD33, CD38, CEA, EGFR (eg, EGFRvIII), GD2, HER2, IGF1R, mesothelin, PSMA, ROR1, WT1, and other tumor antigens of clinical significance and combinations thereof. In some embodiments, the tumor antigen is selected from the group consisting of CD19, BCMA, NY-ESO-1, VEGFR2, MAGE-A3, VEGFR2, MAGE-A3, CD20, CD22, CD33, CD38, CEA, EGFR (e.g., EGFRvIII), GD2, HER2, IGF1R, mesothelin, PSMA, ROR1, WT1, and other tumor antigens of clinical significance. In some embodiments, the tumor antigen is derived from an intracellular protein of a tumor cell. Many TCRs specific for tumor antigens (including tumor-associated antigens) have been described, including, for example, NY-ESO-1 cancer-testis antigen, p53 tumor suppressor antigen, 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).

[0231] In some embodiments, the CAR is an anti-BCMA CAR. A variety of antigen binding domain sequences can be used as the targeting domain of the CAR. In some embodiments, the anti-BCMA CAR comprises, from N-terminus to C-terminus: a CD8 leader sequence, an anti-BCMAsdAb, a CD8 hinge, a CD8 transmembrane, a 4-1BB intracellular costimulatory domain, and a CD3ζ intracellular signaling domain.

[0232] In some embodiments, the engineered receptor is a modified T cell receptor. In some embodiments, the engineered TCR is specific for a tumor antigen. In some embodiments, the tumor antigen is selected from the group consisting of: CD19, BCMA, NY-ESO-1, VEGFR2, MAGE-A3, VEGFR2, MAGE-A3, CD20, CD22, CD33, CD38, CEA, EGFR (e.g., EGFRvIII), GD2, HER2, IGF1R, mesothelin, PSMA, ROR1, WT1, and other tumor antigens of clinical significance. In some embodiments, the tumor antigen is derived from an intracellular protein of a tumor cell. Many TCRs specific for tumor antigens (including tumor-associated antigens) have been described, including, for example, NY-ESO-1 cancer-testis antigen, p53 tumor suppressor antigen, 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 TCR known in the art can be used in this application. In some embodiments, the TCR has an enhanced affinity for tumor antigens. Exemplary TCRs and methods for introducing these TCRs into immune cells are described, for example, in U.S. Patent No. 5,830,755 and Kessels et al. Immunotherapy through TCR gene transfer. Nat. Immunol. 2, 957-961 (2001). In some embodiments, the modified Vδ1 T cell is a TCR-T cell.

[0233] The TCR receptor complex is an octameric complex composed of the variable TCR receptor α and β chains (γ and δ chains in the case of γδ T cells) and three dimeric signaling modules: CD3δ / ε, CD3γ / ε, and CD247 (the T cell surface glycoprotein CD3ζ chain)ζ / ζ or ζ / η. The ionizable residues in the transmembrane domain of each subunit form a polar network of interactions that holds the complex together. The TCR complex has the function of activating the signaling cascade in T cells.

[0234] In some embodiments, the engineered receptor is an engineered TCR comprising one or more T cell receptor (TCR) fusion proteins (TFP). For example, exemplary TFPs have been described in US20170166622A1, which is incorporated herein by reference. In some embodiments, TFP comprises the extracellular domain of a TCR subunit, which comprises an extracellular domain or a portion thereof of a protein selected from the group consisting of: TCR α chain, TCR β chain, CD3εTCR subunit, CD3γTCR subunit, CD3δTCR subunit, its functional fragment and its amino acid sequence with at least one but no more than 20 modifications. In some embodiments, TFP comprises a transmembrane domain, which comprises a transmembrane domain of a protein selected from the group consisting of: TCR α chain, TCR β chain, CD3εTCR subunit, CD3γTCR subunit, CD3δTCR subunit, its functional fragment and its amino acid sequence with at least one but no more than 20 modifications. In some embodiments, the TFP comprises a transmembrane domain comprising a transmembrane domain of a protein selected from the group consisting of: TCRα chain, TCRβ chain, TCRζ chain, CD3εTCR subunit, CD3γTCR subunit, CD3δTCR 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.

[0235] In some embodiments, the TFP comprises a TCR subunit and an antigen binding domain, the TCR subunit comprising at least a portion of a TCR extracellular domain and a TCR intracellular domain comprising a stimulatory domain from the intracellular signaling domain of CD3ε; wherein the TCR subunit is operably linked to the antigen binding domain, and wherein when expressed in a T cell, the TFP is incorporated into the TCR.

[0236] In some embodiments, the TFP comprises a TCR subunit and an antigen binding domain, the TCR subunit comprising at least a portion of a TCR extracellular domain and a TCR intracellular domain comprising a stimulatory domain from the intracellular signaling domain of CD3γ; wherein the TCR subunit is operably linked to the antigen binding domain, and wherein when expressed in a T cell, the TFP is incorporated into the TCR.

[0237] In some embodiments, the TFP comprises a TCR subunit and an antigen binding domain, the TCR subunit comprising at least a portion of a TCR extracellular domain and a TCR intracellular domain comprising a stimulatory domain from the intracellular signaling domain of CD3δ; wherein the TCR subunit is operably linked to the antigen binding domain, and wherein when expressed in a T cell, the TFP is incorporated into the TCR.

[0238] In some embodiments, the TFP comprises a TCR subunit and an antigen binding domain, wherein the TCR subunit comprises at least a portion of a TCR extracellular domain and a TCR intracellular domain comprising a stimulatory domain from the intracellular signaling domain of TCRα; wherein the TCR subunit is operably linked to the antigen binding domain, and wherein when expressed in a T cell, the TFP is incorporated into the TCR.

[0239] In some embodiments, the TFP comprises a TCR subunit and an antigen binding domain, wherein the TCR subunit comprises at least a portion of a TCR extracellular domain and a TCR intracellular domain comprising a stimulatory domain from the intracellular signaling domain of TCRβ; wherein the TCR subunit is operably linked to the antigen binding domain, and wherein when expressed in a T cell, the TFP is incorporated into the TCR.

[0240] In some embodiments, the engineered receptor is a T cell antigen conjugate (TAC) receptor. For example, exemplary TAC receptors have been described in US20160368964 A1, which is incorporated herein by reference. In some embodiments, the TAC comprises a targeting domain, a TCR binding domain that specifically binds to a protein associated with the TCR complex, and a T cell receptor signaling domain. In some embodiments, the targeting domain is an antibody fragment that specifically binds to a tumor antigen, such as an scFv or V H H. In some embodiments, the targeting domain is a designed ankyrin repeat (DARPin) polypeptide. In some embodiments, the tumor antigen is selected from the group consisting of: CD19, BCMA, NY-ESO-1, VEGFR2, MAGE-A3, VEGFR2, MAGE-A3, CD20, CD22, CD33, CD38, CEA, EGFR (e.g., EGFRvIII), GD2, HER2, IGF1R, mesothelin, PSMA, ROR1, WT1, and other tumor antigens of clinical significance. In some embodiments, the protein associated with the TCR complex is CD3, such as CD3E. In some embodiments, the TCR binding domain is a single-chain antibody, such as scFv or V HH. In some embodiments, the TCR binding domain is derived from UCHT1. In some embodiments, the TAC receptor comprises a cytoplasmic domain and a transmembrane domain. In some embodiments, the T cell receptor signaling domain comprises a cytoplasmic 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 CD 154. In some embodiments, the TAC receptor comprises a transmembrane domain and a cytoplasmic domain derived from CD4. In some embodiments, the TAC receptor comprises a transmembrane domain and a cytoplasmic domain derived from CD8 (such as CD8a).

[0241] T cell co-receptors are expressed as membrane proteins on T cells. They provide stability to the TCR:peptide:MEC complex and facilitate signal transduction. Two subtypes of T cell co-receptors, CD4 and CD8, exhibit strong specificity for specific MEC types. The CD4 co-receptor can only stabilize the TCR:MEC II complex, while the CD8 co-receptor can only stabilize the TCR:MEC I complex. Differential expression of CD4 and CD8 on different T cell types results in distinct functional T cell subsets. CD8+ T cells are cytotoxic T cells.

[0242] In some embodiments, the modified Vδ1 T cells express more than one engineered receptor, such as any combination of CAR, TCR, or TAC receptors.

[0243] In some embodiments, the engineered receptors (such as CARs, TCRs, or TACs) expressed by the modified Vδ1 T cells target one or more tumor antigens. Tumor antigens are proteins produced by tumor cells that can trigger an immune response, particularly a T cell-mediated immune response. The choice of target antigen will depend on the specific type of cancer to be treated. Exemplary tumor antigens include, e.g., glioma-associated antigen, carcinoembryonic antigen (CEA), beta-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CAIX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostate enzyme, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostaglandins, PSMA, HER2 / neu, survivin and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, and mesothelin.

[0244] In certain embodiments, tumor antigens comprise one or more antigen cancer epitopes associated with malignant tumors. Malignant tumors express many proteins that can be used as target antigens for immune attack. These molecules include but are not limited to tissue-specific antigens, such as MART-1, tyrosinase and gp100 in melanoma and prostate acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to the transformation-related molecule group, such as oncogene HER2 / Neu / ErbB-2. Another group of target antigens are carcinoembryonic antigens, such as carcinoembryonic antigen (CEA). In B-cell lymphoma, tumor-specific idiotypic immunoglobulins constitute the true tumor-specific immunoglobulin antigens unique to individual tumors. B-cell differentiation antigens (such as CD19, CD20 and CD37) are other candidates for target antigens in B-cell lymphoma.

[0245] In some embodiments, the tumor antigen is a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA). TSA is unique to tumor cells and is not present on other cells in the body. TAA-related antigens are not unique to tumor cells, on the contrary, they can also be expressed on normal cells under conditions that cannot induce an immune tolerance state to the antigen. The expression of antigens on tumors can occur under conditions that enable the immune system to respond to the antigen. TAA may be antigens expressed on normal cells during embryonic development, when the immune system is immature and unable to respond, or they may be antigens that are usually present at very low levels on normal cells but expressed at much higher levels on tumor cells.

[0246] Non-limiting examples of TSA or TAA antigens include the following: differentiation antigens, such as MART-1 / MelanA (MART-I), gp 100 (Pmel 17), tyrosinase, TRP-1, TRP-2, and tumor-specific multi-lineage 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 generated by chromosomal translocations, such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such as Epstein-Barr virus antigen EBVA and 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, p180erbB-3, c-met, nm-23HI, PSA, TAG-72, CA 19-9, CA 72-4, CAM17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, β-HCG, BCA225, BTAA, CA 125, CA 15-3\CA27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-related protein, TAAL6, TAG72, TLP and TPS.

[0247] The engineered Vδ1 T cells of the present disclosure can be designed to home to specific physical locations in the subject and thus target antigens at specific tissues, organs or body parts. Endogenous T cells have different repertoires of transport ligands and receptors that influence their migration patterns. The engineered Vδ1 T cells of the present disclosure can be designed to express one or more transport ligands or receptors from an expression cassette containing a tumor recognition portion, or from a separate expression cassette, that direct the engineered Vδ1 T cells to migrate to specific tissues, organs or body parts.

[0248] The engineered Vδ1 T cells of the present disclosure can be tumor-specific allogeneic cells. For example, engineered Vδ1 T cells can be derived from non-engineered Vδ1 T cells, which are tumor-infiltrating lymphocytes (TILs) isolated from tumors. Different TILs that can be isolated from different tumor types. An expression cassette encoding a tumor recognition portion and an activation domain or another engineered feature can be inserted into the genome of TILs isolated from various tumors. Such Vδ1 T cells can infiltrate solid tumors, weaken and kill tumor cells expressing one or more target antigens, and they can provide effective treatment for various malignancies. Tumor-specific allogeneic Vδ1 T cells can be engineered to express at least one tumor recognition portion that recognizes a selected epitope. In some cases, tumor-specific allogeneic Vδ1 T cells are designed to express at least two different tumor recognition portions, and each different tumor recognition portion is designed to recognize different epitopes of the same antigen, different antigens, antigens and one or more activation or inactivation co-stimulatory / immunomodulatory receptors, antigens complexed with MHC molecules, or homing receptors.

[0249] Therapeutic uses

[0250] The Vδ1 T cells obtained as described herein can be used for a variety of experimental, therapeutic and commercial applications. This includes but is not limited to genetic modification or genetic editing of such cells, for example, for the purpose of improving their therapeutic potential. For example, the purpose is to redirect the specificity of Vδ1 T cells by expressing chimeric antigen receptors (CAR) or TCR on these cells. Vδ1 T cells are electroporated to insert genetic material or by infecting these cells with viral vectors (such as lentivirus or retrovirus containing the desired genetic material) to induce CAR expression. Such gene editing can improve the effectiveness of Vδ1 T cells by improving homing, cytokine production, recirculation killing and / or improving implantation.

[0251] Another aspect of the present disclosure provides a method of modulating an immune response, comprising administering to a subject in need thereof an effective amount of Vδ1 T cells prepared according to the methods described herein.

[0252] As used herein, the term "effective amount" means an amount that works at dosages and for periods of time necessary to achieve the desired result.

[0253] In another aspect, the present disclosure provides a method of treating an infection, comprising administering to a subject in need thereof an effective amount of Vδ1 T cells prepared according to the methods described herein.

[0254] Examples of infections that can be treated include, but are not limited to, bacterial infections (e.g., infections caused by mycobacteria (e.g., tuberculosis)), viral infections (e.g., infections caused by herpes simplex virus (HSV), human immunodeficiency virus (HIV), or hepatitis viruses), and parasitic infections (e.g., infections caused by Plasmodium (e.g., malaria)).

[0255] In another aspect, the present disclosure provides a method for treating cancer, comprising administering to a subject in need thereof an effective amount of Vδ1 T cells prepared according to the method described herein.

[0256] Examples of cancers that can be treated include, but are not limited to, leukemias (including chronic lymphocytic leukemia, chronic myeloid leukemia, acute myeloid leukemia, acute lymphoblastic leukemia, and T-cell and B-cell leukemias), lymphomas (Hodgkin's and non-Hodgkin's), lymphoproliferative disorders, plasmacytomas, histiocytomas, melanomas, adenomas, sarcomas, solid tissue cancers, hypoxic tumors, squamous cell carcinomas, genitourinary cancers (such as cervical and bladder cancer), hematopoietic cancers, head and neck cancers, and cancers of the nervous system.

[0257] These aspects of the disclosure also extend to Vδ1 T cells obtained by the methods described herein for use in methods for modulating an immune response, treating an infection, or treating a cancer as described above. The disclosure further includes the use of Vδ1 T cells obtained according to the methods described herein in the manufacture of a medicament or pharmaceutical composition for modulating an immune response, treating an infection, or treating a cancer as described above.

[0258] For example, Vδ1 T cells obtained according to the present disclosure can also be used in experimental models to further study and elucidate the functions of the cells. In addition, these cells can be used for research aimed at identifying antigens / epitopes recognized by Vδ1 T cells and for the design and development of vaccines.

[0259] In another aspect, the present disclosure provides a method for vaccinating a subject, comprising administering to a subject in need thereof an effective amount of Vδ1 T cells obtained by the methods described herein. Such a vaccine can be administered to immunocompromised patients or individuals at increased risk of developing infectious diseases or cancer.

[0260] The Vδ1 T cells obtained according to the present disclosure can be used immediately for the above-mentioned therapeutic, experimental or commercial applications, or the cells can be cryopreserved for later use.

[0261] The pharmaceutical composition can be included in a container, pack, or dispenser together with instructions for administration.The present disclosure also provides methods of making antibodies or antigen-binding fragments thereof for the various uses as described herein.

[0262] One or more of the Vδ1 T cells described herein can be administered to a subject in a single, unified form (such as intravenous injection) or in multiple forms (e.g., as multiple intravenous infusions or injections, or subcutaneous injections). In some cases, the Vδ1 T cells can be expanded in the subject after administration to the subject. The Vδ1 T cells can be frozen to provide cells for multiple treatments with the same cell preparation. The Vδ1 T cells of the present disclosure and pharmaceutical compositions comprising the same can be packaged as a kit (test kit). The kit can include instructions (e.g., written instructions) for using the Vδ1 T cells and compositions comprising the same.

[0263] In some embodiments, the method of treatment comprises administering a therapeutically effective amount of Vδ1 T cells to the subject. In some embodiments, the therapeutically effective amount of Vδ1 T cells is administered for at least about 10 seconds, 30 seconds, 1 minute, 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or 1 year. In some embodiments, the therapeutically effective amount of Vδ1 T cells is administered for at least one week. In some embodiments, the therapeutically effective amount of Vδ1 T cells is administered for at least two weeks.

[0264] The Vδ1 T cells described herein can be administered before, during, or after the onset of a disease or condition, and the time of administering a pharmaceutical composition containing Vδ1 T cells can vary. For example, Vδ1 T cells can be used as a prophylactic and can be continuously administered to a subject with a condition or disease tendency to reduce the likelihood of the disease or condition. Vδ1 T cells can be administered to a subject during the onset of symptoms or as soon as possible after the onset of symptoms. The administration of Vδ1 T cells can begin immediately upon the onset of symptoms, within 3 hours before the onset of symptoms, within 6 hours before the onset of symptoms, within 24 hours before the onset of symptoms, within 48 hours before the onset of symptoms, or at any time period after the onset of symptoms. The initial administration can be via any practical route (e.g., intravenous infusion or injection), such as by any route described herein using any formulation described herein. In some instances, the administration of the Vδ1 T cells of the present disclosure is intravenous administration. After the onset of cancer or infectious disease, one or more doses of Vδ1 T cells can be administered as soon as feasible and for as long as necessary to treat 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 more doses of Vδ1 T cells can be administered years after the onset of cancer and before or after other treatments. In some examples, Vδ1 T cells can be administered for at least about 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 1 year, at least 2 years, at least 3 years, at least 4 years, or at least 5 years. The length of treatment can be different for each subject.

[0265] The Vδ1 T cells disclosed herein can be formulated into unit dosage forms suitable for single administration of precise doses. In some cases, the unit dosage form contains additional lymphocytes. In the unit dosage form, the preparation is divided into unit doses containing an appropriate amount of one or more compounds. The unit dose can be in the form of a package containing a discrete amount of the preparation. Non-limiting examples are packaged tablets or capsules, and powders in vials or ampoules. The aqueous suspension composition can be packaged in a single-dose non-reclosable container. Multi-dose reclosable containers can be used, for example, in combination with a preservative or not. In some instances, the pharmaceutical composition does not contain a preservative. Preparations for parenteral injection can be in unit dosage form, for example, in ampoules or in multi-dose containers with a preservative.

[0266] The Vδ1 T cells described herein can be expressed in a number of at least 5 cells, at least 10 cells, at least 20 cells, at least 30 cells, at least 40 cells, at least 50 cells, at least 60 cells, at least 70 cells, at least 80 cells, at least 90 cells, at least 100 cells, at least 200 cells, at least 300 cells, at least 400 cells, at least 500 cells, at least 600 cells, at least 700 cells, at least 800 cells, at least 900 cells, at least 1×10 3 cells, at least 2×10 3 cells, at least 3×10 3 cells, at least 4×10 3 cells, at least 5×10 3 cells, at least 6×10 3 cells, at least 7×10 3 cells, at least 8×10 3 cells, at least 9×10 3 cells, at least 1×10 4 cells, at least 2×10 4 cells, at least 3×10 4 cells, at least 4×10 4 cells, at least 5×10 4 cells, at least 6×10 4 cells, at least 7×10 4 cells, at least 8×10 4 cells, at least 9×10 4 cells, at least 1×10 5 cells, at least 2×10 5 cells, at least 3×10 5 cells, at least 4×10 5 cells, at least 5×10 5 cells, at least 6×10 5 cells, at least 7×10 5 cells, at least 8×10 5 cells, at least 9×10 5 cells, at least 1×10 6 cells, at least 2×10 6 cells, at least 3×10 6 cells, at least 4×10 6 cells, at least 5×10 6 cells, at least 6×10 6 cells, at least 7×10 6 cells, at least 8×10 6cells, at least 9×10 6 cells, at least 1×10 7 cells, at least 2×10 7 cells, at least 3×10 7 cells, at least 4×10 7 cells, at least 5×10 7 cells, at least 6×10 7 cells, at least 7×10 7 cells, at least 8×10 7 cells, at least 9×10 7 cells, at least 1×10 8 cells, at least 2×10 8 cells, at least 3×10 8 cells, at least 4×10 8 cells, at least 5×10 8 cells, at least 6×10 8 cells, at least 7×10 8 cells, at least 8×10 8 cells, at least 9×10 8 cells, at least 1×10 9 cells or more are present in the composition.

[0267] Examples

[0268] The present disclosure is further described in the following examples, which do not limit the scope of the present disclosure described in the claims.

[0269] Example 1: Preparation of Vδ1 T cells

[0270] Vδ1 T cells were prepared under conditions 1-3, as shown in Table 1 and explained in detail below.

[0271] Table 1: Conditions 1-3

[0272]

[0273]

[0274] Condition 1

[0275] Vδ1 T cells were expanded from peripheral blood mononuclear cells (PBMCs) obtained from healthy human subjects.

[0276] On day 0, one million PBMC cells were activated and expanded in 24-well plates in AIM-V with 10% fetal bovine serum (FBS) containing 100 ng / ml rIL-4, 70 ng / ml rIFN-γ, 7 ng / ml rIL-21, and 15 ng / ml rIL-1β. The culture medium was replenished once on day 3.

[0277] On day 5, cells were passaged in fresh medium and transduced with a combination of a γ-retroviral or lentiviral anti-BCMACAR construct (SEQ ID NO: 1) and RetroNectin (Takara, T100A).

[0278] After transduction, cells were expanded in 24-well plates in AIM-V containing 10% FBS containing 70 ng / ml rIL-15 and 30 ng / ml rIFN-γ. 50% of the total volume of cell culture medium in each well was replaced every other day. If the viable cell culture density was increased to 2 × 10 6 If the cell culture volume is higher than 1 × 10 cells / ml, dilute the cell culture to 1 × 10 cells / ml using fresh medium. 6 The details of Condition 1 are described in WO 2016 / 198480 A1, which is incorporated herein by reference in its entirety.

[0279] Condition 2

[0280] Vδ1 T cells were expanded from PBMCs obtained from healthy donors.

[0281] On day 0, one million PBMC cells were stimulated with anti-Vδ1 TCR TS-1 antibody (Thermo Fisher, TCR1055) immobilized at 0.5 μg / ml / well in untreated 24-well plates. The cells were activated and expanded in AIM-V with 10% FBS containing 100 IU / ml IL-2. The culture medium was replenished once on day 3.

[0282] On day 5, cells were transduced with a γ-retroviral or lentiviral anti-BCMACAR construct (SEQ ID NO: 1) in combination with RetroNectin (Takara, T100A).

[0283] After transduction, cells were expanded in the same culture medium. 50% of the total volume of cell culture medium in each well was replaced every other day. If the viable cell culture density was increased to 2 × 10 6 If the cell culture volume is higher than 1 × 10 cells / ml, dilute the cell culture to 1 × 10 cells / ml using fresh medium. 6 cells / ml.

[0284] For αβT cell depletion at harvest, TCRαβ+ and CD56+ cells were depleted with a TCRαβ cell depletion kit (Miltenyi, 200-070-407) and a CD56+ cell depletion kit (Miltenyi, 130-050-401) according to the manufacturer's instructions. This method significantly reduces the residual αβT cells and NK cells at harvest, thereby increasing the purity of Vδ1 T cells. The details of condition 2 are described in detail in WO 2016 / 081518 A2, which is incorporated herein by reference in its entirety.

[0285] Condition 3

[0286] Vδ1 T cells were expanded from PBMCs obtained from healthy donors.

[0287] For αβ T cell depletion, TCRαβ+ and CD56+ cells from PBMC were depleted using the TCRαβ Cell Depletion Kit (Miltenyi, 200-070-407) and CD56+ Cell Depletion Kit (Miltenyi, 130-050-401) according to the manufacturer's instructions. This method significantly reduces residual αβ T cells and NK cells at harvest, thereby increasing Vδ1 T cell purity.

[0288] At day 0, one million cells (PBMC after TCRαβ depletion) were stimulated with 0.5 μg / ml / well fixed anti-Vδ1 TCR TS-1 antibody (Thermo fisher, TCR1055) in untreated 24-well plates. Cells were activated and expanded in AIM-V, which had 10% human platelet lysate containing 100 ng / ml rIL-4 (recombinant IL-4), 70 ng / ml rIFN-γ (recombinant IFN-γ), 10 ng / ml rIL-15 (recombinant IL-15) and 15 ng / ml rIL-1β (recombinant IL-1β). The culture medium was supplemented once on day 3.

[0289] A lentiviral packaging plasmid mixture including pCMV-ΔR-8.47 and pMD2.G was purchased from Addgene and mixed with the appropriate CAR encoding plasmid at a pre-optimized ratio with polyethyleneimine. HEK293 cells were transfected with a mixture of lentivirus and CAR construct and cultured overnight. After overnight culture, the supernatant was collected. The supernatant was centrifuged to further remove cell debris and filtered through a 0.45 μm PES filter. The viral particles were precipitated and rinsed with pre-chilled DPBS. The virus was aliquoted and immediately stored at -80 ° C, and the viral titer was determined by measuring the transduction efficiency of the supT1 cell line using flow cytometry.

[0290] On day 5, cells were passaged in fresh medium and transduced with a combination of a γ-retroviral or lentiviral anti-BCMACAR construct (SEQ ID NO: 1) and RetroNectin (Takara, T100A).

[0291] After transduction, cells were expanded in 24-well plates in AIM-V containing 10% human platelet lysate containing 70 ng / ml rIL-15 and 30 ng / ml rIFN-γ. 50% of the total volume of cell culture medium in each well was replaced every other day. If the viable cell culture density was increased to 2 × 10 6 If the cell culture volume is higher than 1 × 10 cells / ml, dilute the cell culture to 1 × 10 cells / ml using fresh medium. 6 Vδ1 T cells can be cultured in plates or culture dishes, such as 12-well plates, 6-well plates, 6-cm culture dishes, and 10-cm culture dishes.

[0292] Characterization of Vδ1 T cells

[0293] On day 14, the phenotype of Vδ1 T cells was assessed by: APC-conjugated anti-human Vδ1 (TS8.2, ThermoFisher, catalog number: 17-5679-42), Brilliant Violet 421 TM Conjugated anti-human TCR Vδ2 antibody (B6, Biolegend, catalog number: 331428), rabbit anti-camelid sdAb pAb Alexa Fluor 488 (GenScript, catalog number: C9042GH240), PE-conjugated anti-CD27 (MT271, Biolegend, catalog number: 356406), Brilliant Violet 421 TMConjugated anti-CD45RA (HI100, Biolegend, catalog number: 304130), PE-conjugated anti-PD1 (A17188A, Biolegend, catalog number: 379210), and Brilliant Violet 786 TM Conjugated anti-TIM3 (F38-2E2, Biolegend, catalog number: 345032).

[0294] Amplification rate data showed that condition 3 resulted in significantly better amplification than condition 1 (17275-fold on day 15) ( Figure 1 ). For retrovirus, the purity and transduction efficiency were determined to be 97.4% and 46.0%, respectively (Figures 2A-2B). For lentivirus, the purity and transduction efficiency were determined to be 98% and 94%, respectively (Figures 2C-2D). In addition, Vδ1 T cells prepared under condition 3 showed high expression of activation markers (such as NGK2D expression (93.8%)) and low expression of T cell exhaustion markers (such as PD-1 (0.519%) and TIGIT (6.5%)) (Figure 3A). In addition, 84.4% of the expanded CAR-Vδ1 T cells prepared under condition 3 were of the initial phenotype (CD27 positive and CD45RA positive), indicating excellent cell fitness (Figure 3B)

[0295] Example 2. Long-term killing efficacy and persistence of Vδ1 T cells

[0296] To evaluate the long-term killing efficacy and persistence of Vδ1 T cells, a long-term co-culture assay was performed to simulate the dynamic killing process in vivo. In the absence of exogenous cytokines (e.g., IL-2), transduced T cells (1 × 10 5 / well) and NCI-H929 target cells (4×10 5 / well) were co-cultured in 24-well plates at an E:T ratio of 1:3. After 2 or 3 days of co-culture, a portion of these cells were harvested and stained for CD3. Vδ1 T cells were identified by CD3 and CAR signals. For serial co-culture assays, the remaining Vδ1 T cells were then attacked again with fresh NCI-H929 target cells at the same E:T ratio. Co-culture was performed until the tumor cells grew beyond the pore volume. The Vδ1 T cell proliferation rate at each time point was calculated by dividing the number of Vδ1 T cells at that time point by the initial number of Vδ1 T cells.

[0297] In repeated tumor stimulation assays, the killing efficacy of Vδ1 T cells from the indicated conditions was shown to be Figures 4A-4BVδ1 T cells prepared under conditions 1 and 2 became exhausted (e.g., unable to kill additional target tumor cells, as measured by CD3%) after 7 rounds of tumor stimulation, whereas Vδ1 T cells prepared under condition 3 continued to kill target tumor cells after 9 rounds of tumor stimulation. In addition, it was found that Vδ1 T cells prepared under condition 3 proliferated and exhibited better long-term persistence than Vδ1 T cells prepared under conditions 1 and 2.

[0298] Example 3. Assessment of cytokine release by Vδ1 T cells

[0299] The measure of effector γδT cell activation is the production of effector cytokines such as IFN-γ and GM-CSF. Supernatants from in vitro cytotoxicity assays were collected to assess CAR-induced cytokine release. According to the manufacturer's manual, homogeneous time-resolved fluorescence (HTRF) assays of IFN-γ and GM-CSF were performed (Cisbio).

[0300] Vδ1 T cells from the indicated conditions were co-cultured with NCI-H929 target cells. Culture supernatants were collected 20 h later to assess IFN-γ and GM-CSF release as a measure of γδ T cell safety. Figures 5A-5B As shown, CAR-Vδ1 T cells co-cultured with target tumor cells prepared under condition 3 secreted significantly less IFN- and GM-CSF than Vδ1 T cells from conditions 1 and 2. This suggests that Vδ1 T cells prepared under condition 3 are safe for clinical use.

[0301] Example 4. Anti-tumor activity of CAR-Vδ1 T cells

[0302] The anti-tumor activity of anti-BCMACAR-Vδ1 T cells (prepared under condition 3) was evaluated in vivo in an RPMI-8226 xenograft model. Briefly, on day 0, one million (1×10 6 ) RPMI-8226 cells stably expressing the firefly luciferase reporter gene were implanted subcutaneously / intravenously into NOD / SCID IL-2RγCnull (NSG) mice. Fourteen days after tumor inoculation, 3×10 6 Mice were treated with either CAR-Vδ1 T cells, mock T cells, or phosphate-buffered saline (PBS). Tumor progression was monitored weekly by bioluminescence imaging (BLI). In addition, T cell proliferation was monitored by FACS analysis of plasma drawn from the blood.

[0303] The data showed that CAR-Vδ1 T exhibited antitumor activity compared with vehicle control ( Figure 6). In addition, it is obvious that the anti-tumor cytotoxicity of CAR-Vδ1 T cells prepared under condition 3 is much more significant than that of CAR-Vδ1 T cells prepared under condition 2.

[0304] In general, the present disclosure provides a method for producing CAR-Vδ1 T cells with high purity, expansion and CAR transduction rate for clinical use and production. Such cells show high activation, low exhaustion and a predominantly initial phenotype. In vitro validation showed superior anti-tumor activity and safer characteristics than cells prepared under conditions 1 and 2. In addition, in vivo further confirmed that the anti-tumor effect is far superior to the existing technology.

[0305] Other embodiments

[0306] It should be understood that although the disclosure has been described in conjunction with its detailed description, 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

1. A method for culturing Vδ1 T cells, comprising: (1) culturing Vδ1 T cells from a sample in a first culture medium comprising interleukin-4 (IL-4), interleukin-15 (IL-15), interleukin-1β (IL-1β), and interferon-γ (IFN-γ); and (2) Expanding the cells obtained in step (1) in a second culture medium containing IL-15 and IFN-γ.

2. The method of claim 1 , wherein the IL-4 in the first culture medium has a concentration of about 1 to about 1000 ng / ml, about 10 to about 500 ng / ml, about 10 to about 300 ng / ml, about 20 to about 200 ng / ml, about 30 to 180 ng / ml, about 50 to about 150 ng / ml, about 60 to about 140 ng / ml, about 70 to about 130 ng / ml, about 80 to about 120 ng / ml, or about 90 to about 110 ng / ml.

3. The method of any one of claims 1-2, wherein the IL-4 in the first culture medium has a concentration of about 80 to about 120 ng / ml.

4. The method of any one of claims 1 to 3, wherein the IL-4 in the first culture medium has a concentration of about 100 ng / ml.

5. The method of any one of claims 1 to 4, wherein the IL-15 in the first culture medium has a concentration of about 1 to about 1000 ng / ml, about 1 to about 500 ng / ml, about 1 to about 300 ng / ml, about 5 to about 200 ng / ml, about 5 to 150 ng / ml, about 5 to about 100 ng / ml, about 5 to about 50 ng / ml, about 5 to about 25 ng / ml, or about 5 to about 15 ng / ml.

6. The method of any one of claims 1-5, wherein the IL-15 in the first culture medium has a concentration of about 5 to about 15 ng / ml.

7. The method of any one of claims 1-6, wherein the IL-15 in the first culture medium has a concentration of about 10 ng / ml.

8. The method of any one of claims 1 to 7, wherein the IL-1β in the first culture medium has a concentration of about 1 to about 1000 ng / ml, about 5 to about 500 ng / ml, about 5 to about 300 ng / ml, about 5 to about 200 ng / ml, about 5 to 180 ng / ml, about 5 to about 150 ng / ml, about 5 to about 140 ng / ml, about 5 to about 100 ng / ml, about 10 to about 50 ng / ml, or about 10 to about 20 ng / ml.

9. The method of any one of claims 1 to 8, wherein the IL-1β in the first culture medium has a concentration of about 10 to about 20 ng / ml.

10. The method of any one of claims 1-9, wherein the IL-1β in the first culture medium has a concentration of about 15 ng / ml.

11. The method of any one of claims 1-10, wherein the IFN-γ in the first culture medium has a concentration of about 1 to about 1000 ng / ml, about 10 to about 500 ng / ml, about 10 to about 300 ng / ml, about 20 to about 200 ng / ml, about 30 to 150 ng / ml, about 50 to about 150 ng / ml, about 50 to about 140 ng / ml, about 60 to about 120 ng / ml, about 60 to about 100 ng / ml, or about 60 to about 80 ng / ml.

12. The method of any one of claims 1-11, wherein the IFN-γ in the first culture medium has a concentration of about 60 to about 80 ng / ml.

13. The method of any one of claims 1-12, wherein the IFN-γ in the first culture medium has a concentration of about 70 ng / ml.

14. The method of any one of claims 1-13, wherein the IL-15 in the second culture medium has a concentration of about 1 to about 1000 ng / ml, about 10 to about 500 ng / ml, about 10 to about 300 ng / ml, about 20 to about 200 ng / ml, about 30 to 180 ng / ml, about 50 to about 150 ng / ml, about 60 to about 140 ng / ml, about 60 to about 120 ng / ml, about 60 to about 100 ng / ml, or about 60 to about 80 ng / ml.

15. The method of any one of claims 1-14, wherein the IL-15 in the second culture medium has a concentration of about 60 to about 80 ng / ml.

16. The method of any one of claims 1-15, wherein the IL-15 in the second culture medium has a concentration of about 70 ng / ml.

17. The method of any one of claims 1-16, wherein the IFN-γ in the second culture medium has a concentration of about 1 to about 1000 ng / ml, about 5 to about 500 ng / ml, about 5 to about 300 ng / ml, about 10 to about 200 ng / ml, about 10 to 150 ng / ml, about 15 to about 120 ng / ml, about 15 to about 100 ng / ml, about 15 to about 50 ng / ml, or about 20 to about 40 ng / ml.

18. The method of any one of claims 1-17, wherein the IFN-γ in the second culture medium has a concentration of about 20 to about 40 ng / ml.

19. The method of any one of claims 1-18, wherein the IFN-γ in the second culture medium has a concentration of about 30 ng / ml.

20. The method of any one of claims 1-19, wherein the concentration of IL-15 in the second culture medium is at least 1, 2, 3, 4, or 5 times greater than the concentration of IL-15 in the first culture medium.

21. The method of any one of claims 1-20, wherein the concentration of IFN-γ in the first culture medium is at least 1 or 2 times the concentration of IFN-γ in the second culture medium.

22. The method according to any one of claims 1 to 21, wherein step (1) further comprises stimulating the Vδ1 T cells with Vδ1 T cell-specific antibodies. The method of claim 22 , wherein the Vδ1T-specific antibody specifically binds to the TCRδ chain.

24. The method of claim 22 or claim 23, wherein the Vδ1T-specific antibody is the TCRδ monoclonal antibody TS-1. 25 . The method of claim 22 , wherein the Vδ1T-specific antibody is immobilized on a cell culture plate. The method according to any one of claims 22 to 25 , wherein the Vδ1T-specific antibody is immobilized on a cell culture plate at 0.5 μg / ml / well.

27. The method of any one of claims 22-24, wherein the first culture medium comprises the Vδ1T-specific antibody.

28. The method of any one of claims 1-27, wherein the expanded cell culture comprises a percentage of Vδ1 T cells that is greater than 60%, 70%, 80% or 90% of the total cells in the culture.

29. The method of any one of claims 1 to 28, wherein prior to step (1), the sample is enriched for γδ T cells.

30. The method of claim 29, wherein γδ T cells are enriched by (1) depleting αβ T cells and optionally depleting NK cells or (2) isolating γδ T cells from the sample.

31. The method of any one of claims 1-30, wherein αβ T cells are depleted prior to step (1).

32. The method of any one of claims 1-30, wherein NK cells are depleted prior to step (1).

33. The method of any one of claims 1-30, wherein αβ T cells are depleted between steps (1) and (2).

34. The method of any one of claims 1-30, wherein NK cells are depleted between steps (1) and (2).

35. The method of any one of claims 1-30, wherein αβ T cells are depleted after step (2).

36. The method of any one of claims 1-30, wherein NK cells are depleted after step (2).

37. The method of any one of claims 1-36, wherein the sample is selected from blood, peripheral blood, umbilical cord blood, lymphoid tissue, bone marrow, or spleen.

38. The method of any one of claims 1-37, wherein the sample comprises peripheral blood mononuclear cells (PBMCs).

39. The method of any one of claims 1-38, wherein during step (1), the cells are cultured for 5-9 days.

40. The method of claim 39, wherein during step (1), the cells are cultured for 7 days.

41. The method of any one of claims 1-40, wherein during step (2), the cells are cultured for 6-10 days.

42. The method of claim 41, wherein during step (2), the cells are cultured for 8 days.

43. The method of any one of claims 1-42, wherein the cells are harvested prior to 35 days in culture.

44. The method of claim 43, wherein the cells are harvested prior to 21 days in culture.

45. The method of any one of claims 1-44, wherein the first culture medium and / or the second culture medium comprises AIM-V.

46. ​​The method of any one of claims 1 to 45, wherein the first culture medium and / or the second culture medium comprises L-glutamine, streptomycin sulfate, and gentamicin sulfate.

47. The method of any one of claims 1 to 46, wherein the first culture medium and / or the second culture medium further contains serum.

48. The method of claim 47, wherein the serum is present in an amount of about 0.5% to about 25% by volume.

49. The method of claim 47 or claim 48, wherein the serum is FBS.

50. The method of any one of claims 1-46, wherein the first culture medium and / or the second culture medium further contains 10% human platelet lysate.

51. The method of any one of claims 1-50, wherein the IL-4 is human IL-4.

52. The method of any one of claims 1-51, wherein the IL-15 is human IL-15.

53. The method of any one of claims 1-52, wherein the IL-1 β is human IL-1 β.

54. The method of any one of claims 1-53, wherein the IFN-γ is human IFN-γ.

55. The method of any one of claims 1-54, wherein prior to step (1), the cells are transduced with a vector.

56. The method of any one of claims 1-54, wherein the cells are transduced with a vector between steps (1) and (2).

57. The method of any one of claims 1-54, wherein after step (2), the cells are transduced with a vector.

58. The method of any one of claims 55-57, wherein the vector comprises a nucleic acid sequence encoding a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

59. The method of any one of claims 55-58, wherein the cells are transduced with a lentiviral vector or a retroviral vector.

60. A method for preparing Vδ1 T cells, comprising: (1) culturing cells in the sample in a first culture medium comprising 80-120 ng / ml (e.g., about 100 ng / ml) IL-4, 5-15 ng / ml (e.g., about 10 ng / ml) IL-15, 10-20 ng / ml (e.g., about 15 ng / ml) IL-1β, and 60-80 ng / ml (e.g., about 70 ng / ml) IFN-γ; and (2) culturing the cells obtained in step (1) in a second culture medium containing 60-80 ng / ml (e.g., about 70 ng / ml) IL-15 and 20-40 ng / ml (e.g., about 30 ng / ml) IFN-γ.

61. The method of claim 60, wherein prior to step (1), the sample is depleted of αβ T cells and / or NK cells.

62. The method of claim 60 or claim 61, wherein during step (1), the cells are exposed to Vδ1T-specific antibodies.

63. The method of any one of claims 60-62, wherein prior to step (2), the cells are transfected with a vector encoding an engineered receptor (e.g., a CAR).

64. The method of any one of claims 60-63, wherein during step (1), the cells are cultured for 5-9 days (e.g., about 7 days).

65. The method of any one of claims 60-64, wherein during step (2), the cells are cultured for 6-10 days (e.g., about 8 days).

66. A cell preparation prepared using the method of any preceding claim.

67. A pharmaceutical composition comprising the cell preparation according to claim 66, and a pharmaceutically acceptable carrier.

68. A method of treating a subject having cancer, comprising administering to the subject in need thereof a therapeutically effective amount of the cell preparation of claim 66.

69. The method of claim 68, wherein the subject has a solid tumor.

70. The method of claim 68, wherein the cancer is breast cancer, lung cancer, pancreatic cancer, melanoma, oral cancer, mesothelioma, ovarian cancer, colorectal cancer, gastric cancer, cervical cancer, brain cancer, skin cancer, multiple myeloma, lymphoma, epithelial tumor, soft tissue sarcoma, esophageal cancer, or a CNS tumor.

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