WO2024193459A1

WO2024193459A1 - METHODS OF CULTURING Vδ1 T CELLS

Info

Publication number: WO2024193459A1
Application number: PCT/CN2024/081892
Authority: WO
Inventors: Baowei LIU; Qinghe Zhang; Zhongyuan TU; Yafeng Zhang
Original assignee: Nanjing Legend Biotechnology Co Ltd; Legend Biotech Ireland Ltd
Current assignee: Nanjing Legend Biotechnology Co Ltd; Legend Biotech Ireland Ltd
Priority date: 2023-03-17
Filing date: 2024-03-15
Publication date: 2024-09-26
Anticipated expiration: 2025-09-17
Also published as: CN120677232A

Abstract

Provided are methods of preparing engineered and non-engineered Vδ1 T cells and uses thereof. The Vδ1 T cells are useful in the treatment of various cancers, infectious diseases, and immune disorders. Also provided are methods for expanding engineered and non-engineered Vδ1 T cell populations to therapeutically useful quantities. An engineered Vδ1 T cell can be a universal donor, and can be administered to a subject with any MHC haplotype.

Description

METHODS OF CULTURING Vδ1 T CELLS

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority benefits of International Application No. PCT/CN2023/082247, filed on March 17, 2023, the contents of which are incorporated herein by reference in its entirety.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on XML file is incorporated herein by reference in its entirety: a computer readable form (CRF) of the Sequence Listing (file name: IEC240138PCT_Sequence Listing. xml, date recorded: March 14, 2024, size: 3 KB) .

TECHNICAL FIELD

This disclosure relates to methods of culturing Vδ1 T cells and uses thereof.

BACKGROUND

The γδT cells in peripheral lymphocytes exhibits potent cancer antigen recognition independent of classical peptide MHC complexes, making it an attractive candidate for allogeneic cancer adoptive immunotherapy. The Vδ1-T cell receptor (TCR) -expressing subset of peripheral γδT cells has remained enigmatic compared to its more prevalent Vγ9Vδ2-TCR and αβ-TCR-expressing counterparts.

Making cell therapies based on γδT cells is challenging. It took until 2021 before a first patient was dosed with an allogeneic adoptive Vδ1 T cell product. One important factor that limits the clinical progress of the use of the Vδ1 T cells is the lack of robust, consistent, and GMP-compatible expansion protocols. A reliable and GMP-compatible expansion protocol is needed.

SUMMARY

This disclosure relates to methods of preparing engineered or non-engineered Vδ1 T cells and uses thereof. In one aspect, the present disclosure provides a two-step protocol for γδT cell (e.g., Vδ1 T cells) expansion from peripheral blood mononuclear cells (PBMCs) that is further compatible with high-efficiency gene engineering for immunotherapy purposes. The methods as described herein can provide an off-the-shelf CAR-T cell therapy method with a lower cost and more standardized production process, which can be applied to a wider range of hematological tumors or solid tumors disease patients, reducing remission rates and improving drug effect durability.

In one aspect, the disclosure is related to a method for culturing Vδ1 T cells comprising:

(1) culturing γδT 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 comprising IL-15 and IFN-γ.

In some embodiments, the γδT cells comprise Vδ1 T cells.

In some embodiments, the γδT cells is a mixture of Vδ1, Vδ2, or Vδ3 T cells.

In some embodiments, after the two steps culturing, the percentage of Vδ1 T cells that is greater than 60%, 70%, 80%or 90%of the total cells of the culture.

In some embodiments, the disclosure is related to 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

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.

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

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

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.

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

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

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.

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

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

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.

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

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

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.

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

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

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.

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

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

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

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

In some embodiments, step (1) further comprises stimulating the Vδ1 T cells by a Vδ1 T cell-specific antibody.

In some embodiments, the Vδ1 T-specific antibody specifically binds to TCR delta chain.

In some embodiments, the Vδ1 T-specific antibody is TCR delta Monoclonal Antibody TS-1.

In some embodiments, the Vδ1 T-specific antibody is immobilized on the cell culture plate.

In some embodiments, the Vδ1 T-specific antibody is immobilized on the cell culture plate at 0.5 μg/ml per well in a cell culture plate.

In some embodiments, the first culture medium comprises the Vδ1 T-specific antibody.

In some embodiments, the Vδ1 T cell-specific antibody specifically activate Vδ1 T cells and optionally selectively expand Vδ1 T cells.

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 of the culture.

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

In some embodiments, γδT cells are enriched by depleting αβT cells.

In some embodiments, γδT cells are enriched by depleting NK cell.

In some embodiments, γδT cells are enriched by depleting αβT cells and NK cell.

In some embodiments, γδT cells are enriched by isolating γδT cells from the sample.

In some embodiments, αβT cells are depleted prior to step (1) .

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

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

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

In some embodiments, αβT cells are depleted after step (2) .

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

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

In some embodiments, the sample comprises peripheral blood mononuclear cells (PBMCs) . In some embodiments, the cells were cultured for 5-9 days during step (1) .

In some embodiments, the cells were cultured for 7 days during step (1) .

In some embodiments, the cells were cultured for 6-10 days during step (2) .

In some embodiments, the cells were cultured for 8 days during step (2) .

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

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

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

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

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

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

In some embodiments, the serum is FBS.

In some embodiments, the first and/or second culture media further contain 10%human platelet lysate.

In some embodiments, the IL-4 is human IL-4.

In some embodiments, the IL-15 is human IL-15.

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

In some embodiments, the IFN-γ is human IFN-γ.

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

In some embodiments, the cells are transduced with a vector between step (1) and step (2) . In some embodiments, the cells are transduced with a vector after step (2) .

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

In some embodiments, the cells are transduced with a lentiviral vector.

In some embodiments, the cells are transduced with a retroviral vector.

In some embodiments, the resulting Vδ1 T cells have a purity of above 50%, above 60%, above 70%, above 80%, above 90%, above 95%, above 96%, above 97%, above 98%, or above 99%.

In some embodiments, the resulting Vδ1 T cells have a CD27 and CD45RA double-positive rate of above 50%, above 60%, above 70%, above 80%or above 84%.

In some embodiments, the resulting Vδ1 T cells have a NGK2D positive rate of above 50%, above 60%, above 70%, above 80%, above 90%, or above 93%.

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

In some embodiments, the resulting Vδ1 T cells have a PD-1 positive 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%.

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

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

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 GM-CSF, after 48 hours of co-culturing with NCI-H929 target cells.

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-culturing with NCI-H929 target cells.

In one aspect, the disclosure is related to a method for preparing Vδ1 T cells, the method 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 (2) in a second culture medium comprising 60-80 ng/ml (e.g., about 70ng/ml) IL-15 and 20-40 ng/ml (e.g., about 30 ng/ml) IFN-γ.

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

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

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

In some embodiments, the cells are cultured for 5-9 days (e.g., about 7 days) during step (1) .

In some embodiments, the cells are cultured for 6-10 days (e.g., about 8 days) during step (2) .

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

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

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

In some embodiments, the subject has a solid tumor.

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 neoplasms, soft tissue sarcoma, esophageal cancers, or CNS tumors.

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

In some embodiments, the infection is virus infection, bacterial infection, or fungus infection.

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

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

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

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

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

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

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

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

As used herein, the term “purity” refers to the percentage of desired cells (e.g., Vδ1 T cell) out of the total number of culture cells.

As used herein, the term “recombinant protein” is a protein that is derived from the recombinant DNA by expression of the recombinant DNA in a host cell.

As used herein, the term “cancer” refers to cells having the capacity for uncontrolled autonomous growth. Examples of such cells include cells having an abnormal state or condition characterized by rapidly proliferating cell growth. The term is meant to include cancerous growths, e.g., tumors; oncogenic processes, metastatic tissues, and malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. Also included are malignancies of the various organ systems, such as respiratory, cardiovascular, renal, reproductive, hematological, neurological, hepatic, gastrointestinal, and endocrine systems; as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and/or testicular tumors, non-small cell carcinoma of the lung, and cancer of the small intestine. Cancer that is “naturally arising” includes any cancer that is not experimentally induced by implantation of cancer cells into a subject, and includes, for example, spontaneously arising cancer, cancer caused by exposure of a patient to a carcinogen (s) , cancer resulting from insertion of a transgenic oncogene or knockout of a tumor suppressor gene, and cancer caused by infections, e.g., viral infections. The term “carcinoma” is art recognized and refers to malignancies of epithelial or endocrine tissues. The term also includes carcinosarcomas, which include malignant tumors composed of carcinomatous and sarcomatous tissues. An “adenocarcinoma” refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures. The term “sarcoma” is art recognized and refers to malignant tumors of mesenchymal derivation. The term “hematopoietic neoplastic disorders” includes diseases involving hyperplastic/neoplastic cells of hematopoietic origin. A hematopoietic neoplastic disorder can arise from myeloid, lymphoid or erythroid lineages, or precursor cells thereof. A hematologic cancer is a cancer that begins in blood-forming tissue, such as the bone marrow, or in the cells of the immune system. Examples of hematologic cancer include e.g., leukemia, lymphoma, and multiple myeloma etc.

As used herein, the terms “subject” and “patient” are used interchangeably throughout the specification and describe an animal, human or non-human, to whom treatment according to the methods of the present disclosure is provided. Veterinary and non-veterinary applications are contemplated in the present disclosure. Human patients can be adult humans or juvenile humans (e.g., humans below the age of 18 years old) . In addition to humans, patients include but are not limited to mice, rats, hamsters, guinea-pigs, rabbits, ferrets, cats, dogs, and primates. Included are, for example, non-human primates (e.g., monkey, chimpanzee, gorilla, and the like) , rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits) , lagomorphs, swine (e.g., pig, miniature pig) , equine, canine, feline, bovine, and other domestic, farm, and zoo animals.

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

As used herein, the term “chimeric antigen receptor” or “CAR” as used herein refers to genetically engineered receptors, which can be used to graft one or more antigen specificity onto immune effector cells, such as T cells. Some CARs are also known as “artificial T-cell receptors, ” “chimeric T cell receptors, ” or “chimeric immune receptors. ” In some embodiments, the CAR comprises an extracellular antigen binding domain specific for one or more antigens (such as tumor antigens) , a transmembrane domain, and an intracellular signaling domain of a T cell and/or other receptors. “CAR-T cell” refers to a T cell that expresses a CAR.

As used herein, the term “T-cell receptor” or “TCR” as used herein refers to an endogenous or modified T-cell receptor comprising an extracellular antigen binding domain that binds to a specific antigenic peptide bound in an MHC molecule. In some embodiments, the TCR comprises a TCRα polypeptide chain and a TCRβ polypeptide chain. In some embodiments, the TCR comprises a TCRγ polypeptide chain and a TCRδ polypeptide chain. In some embodiments, the TCR specifically binds a tumor antigen. “TCR-T” refers to a T cell that expresses a recombinant TCR. Expression of a heterologous antigen receptor, such as a heterologous TCR or CAR, can alter the immunogenic specificity of the T cells so that they recognize or display improved recognition for one or more tumor antigens that are present on the surface of the cancer cells of an individual with cancer.

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

DESCRIPTION OF DRAWINGS

FIG. 1 shows testing results on Vδ1 T cell expansion under Conditions 1, 2 and 3. Vδ1 T were expanded as described Example 1 and cell expansion were determined on day 15.

FIGs. 2A-2D show testing results on purity (FIGs. 2A and 2C) and CAR positive rate (FIGs. 2B and 2D) of the Vδ1 T cells prepared under Condition 3. Vδ1 T were expanded with Condition 3 and transduced with retrovirus (FIGs. 2A and 2B) and lentivirus (FIGs. 2C and 2D) encoding anti-BCMA-CAR (SEQ ID NO: 1) as described in Example 1 and cell purity and transduction rate were determined on day 15.

FIGs. 3A-3B show testing results on the phenotype of the Vδ1 T cells prepared under Condition 3. In FIG. 3A, the dotted line shows the results from a negative control. In FIG. 3B, the X-axis measures CD27 and the Y-axis measures CD45RA. Vδ1 T were expanded under Condition 3 and transduced with anti-BCMA-CAR (SEQ ID NO: 1) as described in Example 1 and cell phenotype was determined on day 15.

FIGs. 4A-4B show testing results on in vitro long-term cytotoxicity (FIG. 4A) and persistence (FIG. 4B) of Vδ1 T-cell prepared under Condition 1, 2 and 3. Vδ1 T cells were prepared under different conditions and transduced with anti-BCMA-CAR (SEQ ID NO: 1) as described in Example 1. In vitro anti-tumor cytotoxicity and cell proliferation were determined with a long-term co-culture assay as described in Example 2.

FIGs. 5A-5B show testing results on the cytokine release from Vδ1 T cells prepared under Condition 1, 2 and 3. 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-γwere evaluated with the HTRF assay as described in Example 3.

FIG. 6 shows testing results on the in vivo efficacy of Vδ1 T-cell on multiple myeloma in a NCG mouse model. Vδ1 T cells were expanded with Condition 2 and Condition 3 and transduced with anti-BCMA-CAR (SEQ ID NO: 1) as described in Example 1. In vivo anti-tumor cytotoxicity was determined with multiple myeloma tumor (RPMI-8226) -bearing NOD/SCID IL-2RγCnull (NSG) mice model as described in Example 4.

FIG. 7 lists amino acid sequences discussed in the disclosure.

DETAILED DESCRIPTION

In humans, γδT cells are a subset of T cells that provide a link between the innate and adaptive immune responses. These cells undergo V- (D) -J segment rearrangement to generate antigen-specific γδT cell receptors (γδ TCRs) , and γδT cells and can be directly activated via the recognition of an antigen by either the γδ TCR or other, non-TCR proteins, acting independently or together to activate γδT cell effector functions, γδT cells represent a small fraction of the overall T cell population in mammals, approximately 1-5%of the T cells in peripheral blood and lymphoid organs, and they appear to reside primarily in epithelial cell-rich compartments like skin, liver, digestive, respiratory, and reproductive tracks. Unlike αβ TCRs, which recognize antigens bound to major histocompatibility complex molecules (MHC) , γδ TCRs can directly recognize bacterial antigens, viral antigens, stress antigens expressed by diseased cells, and tumor antigens in the form of intact proteins or non-peptide compounds.

The ability of γδT cells to recognize a broad spectrum of antigens can be enhanced by genetic engineering of the γδT cells. γδT cells can also be engineered to provide a universal allogeneic therapy that recognizes an antigen of choice in vivo.

Particularly, γδT cells are highly cytotoxic against tumor cells. They function through TCR, NCR (natural cytotoxicity receptor) and other mechanisms to recognize and kill tumors. Unlike T cell receptors in αβT cells, γδ TCR recognizes antigens in a MHC-independent manner, thus paving the way for the use of γδT cells as allogeneic, “off-the-shelf” products to treat cancer, since it does not cause GvHD.

Human γδT cells consist of three major populations, according to their Vδ chains. There are Vδ1, Vδ2 and Vδ3 cells. Typically, Vδ2 T cells are paired with Vγ9 chains within γδTCR complex and are primarily distributed in peripheral blood, while Vδ1 T cells are paired with Vγ2/3/4/5/8/9 within γδTCR complex and can be found in peripheral blood, skin, gut, spleen and liver owing to its diversity. Ex vivo culture of Vδ2 T cells is well-developed in the field with phosphoantigens such as isoprene pyrophosphate (IPP) or bromohydrin pyrophosphate (BrHPP) as stimulating agent, starting from PBMC containing 5-10%Vδ2 T cells. Such Vδ2 T cells have been used in adoptive cell therapy in clinical trials for nearly two decades. In contrast, since PBMC contains less than 1%Vδ1 T cells, it remains a major challenge in the field to produce ex vivo culture of Vδ1 T cells with high purity, viability and anti-tumor cytotoxicity in a relatively large scale.

Vδ1-expressing γδT cells constitute typically 10%to 30%of all γδT cells in the peripheral blood, but their major fraction in epithelial tissues. Moreover, Vδ1+ T cells are usually predominant (over Vδ2+) in tumor infiltrates, and Vδ1+ TIL (tumor-infiltrating lymphocyte) -derived lines generally outperformed Vδ2+ TIL lines in in vitro tumor cytotoxicity assays. Leukemia targeting by peripheral blood Vδ1+ (compared with Vδ2+ T cells) can be enhanced through selective induction of natural cytotoxicity receptors (NCRs: NKp30, NKp44, and NKp46) upon stimulation with TCR agonists and cytokines in vitro. This expanded repertoire of activating/cytotoxicity receptors, together with their increased resistance to activation-induced cell death and exhaustion upon continuous stimulation, make Vδ1+ T cells very attractive candidates for adoptive cell therapy (ACT) of cancer. However, difficulties in selectively expanding them to large numbers in good manufacturing practice (GMP) conditions have hindered the clinical application of Vδ1+ T cells. More details regarding Vδ1 cells can be found in detail, e.g., in Almeida et al. “Delta One T Cells for Immunotherapy of Chronic Lymphocytic Leukemia: Clinical-Grade Expansion/Differentiation and Preclinical Proof of 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.

Current protocols to expand Vδ1+ T cells in vitro make use of mitogenic plant lectins (phytohemagglutinin (PHA) or concanavalin-A (ConA) ) and unsafe materials that cannot be directly applied in the clinic. Also, findings on tumor-promoting effects of Vδ1+ T cells producing IL-17 have raised concerns about their application and stressed the need for a detailed characterization of effector Vδ1+ lymphocytes that might be considered for ACT.

The present disclosure provides improved methods for culturing and expanding γδT cells (e.g., Vδ1 T cells) , after testing multiple combinations of cytokines for their capacity to culture and expand peripheral blood γδT cells (e.g., Vδ1 T cells) in culture. γδT cells (e.g., Vδ1 T cells) can be selectively expanded in vitro by culturing these cells in two phases. In the first phase, these cells can be cultured in a first culture medium comprising IL-4, IL-15, IL-1β, and IFN-γ, and stimulated by a γδT-specific antibody (e.g., Vδ1 T-specific antibody) . In the second phase, these cells can be expanded in a second culture medium containing IL-15 and IFN-γ. These cells can also be isolated, cultured and expanded in culture in the absence of feeder cells.

In some embodiments, the γδT cells (e.g., Vδ1 T cells) have a mean 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, the Vδ1 T cells have a mean expansion rate of more than 1 cell division, more than 2 cell divisions, more than 3 cell divisions, more than 4 cell divisions, more than 5 cell divisions, more than 6 cell divisions, more than 7 cell divisions, more than 8 cell divisions, more than 9 cell divisions, or more than 10 cell divisions in less than 24 hours. In some embodiments, the Vδ1 T cells have a mean 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.

In some embodiments, the γδT cells (e.g., Vδ1 T cells) have a mean expansion rate of about 1 division per about 4 hours, 1 division per about 5 hours, 1 division per about 6 hours, 1 division per about 7 hours, 1 division per about 8 hours, 1 division per about 9 hours, 1 division per about 10 hours, 1 division per about 11 hours, 1 division per about 12 hours, 1 division per about 13 hours, 1 division per about 14 hours, 1 division per about 15 hours, 1 division per about 16 hours, 1 division per about 17 hours, 1 division per about 18 hours, 1 division per about 19 hours, 1 division per about 20 hours, 1 division per about 21 hours, 1 division per about 22 hours, 1 division per about 23 hours, 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.

In some embodiments, the γδT cells (e.g., Vδ1 T cells) have a fast expansion rate over a period of time from 1 day to 36 days of culture, resulting in a greater than 10 fold, greater than 100 fold, greater than 200 fold, greater than 300 fold, greater than 400 fold, greater than 500 fold, greater than 1, 000 fold, greater than 2, 000 fold, greater than 5, 000 fold, greater than 10, 000 fold, greater than 20, 000 fold, greater than 50, 000 fold, greater than 100, 000 fold, greater than 200, 000 fold, greater than 500, 000 fold, or greater than 1, 000, 000 fold expansion. In some embodiments, the Vδ1 T cells have an expansion rate over a period of time from 1 day to 36 days of culture, resulting in a less than 10 fold, less than 100 fold, less than 200 fold, less than 300 fold, less than 400 fold, less than 500 fold, less than 1, 000 fold, less than 2, 000 fold, less than 5, 000 fold, less than 10, 000 fold, less than 20, 000 fold, less than 50, 000 fold, less than 100, 000 fold, less than 200, 000 fold, less than 500, 000 fold, or less than 1, 000, 000 fold expansion. In some embodiments, the Vδ1 T cells have a fast expansion rate over 14 days, resulting in a greater than 10 fold, greater than 100 fold, greater than 200 fold, greater than 300 fold, greater than 400 fold, greater than 500 fold, greater than 1, 000 fold, greater than 2, 000 fold, greater than 5, 000 fold, greater than 10, 000 fold, greater than 20, 000 fold, greater than 50, 000 fold, greater than 100, 000 fold, greater than 200, 000 fold, greater than 500, 000 fold, or greater than 1, 000, 000 fold expansion.

In some embodiments, the expanded cell culture comprises a percentage of γδT cells (e.g., Vδ1 T cells) that is greater than 5%, greater than 10%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99%. In some embodiments, the expanded cell culture comprises a percentage of γδT cells (e.g., Vδ1 T cells) that is less than 5%, less than 10%, less than 20%, less than 30%, less than 40%, less than 50%, less than 60%, less than 70%, less than 75%, less than 80%, less than 85%, less than 90%, less than 91%, less than 92%, less than 93%, less than 94%, less than 95%, less than 96%, less than 97%, less than 98%, or less than 99%. In some embodiments, the expanded cell culture comprises a percentage of Vδ1 T cells that is about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%.

In some cases, the cell culture further comprises an 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 moiety (e.g., CAR, TCR) . In some embodiments, the expanded cell culture comprises a percentage of engineered Vδ1 T cells that is greater than 5%, greater than 10%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99%. In some embodiments, the expanded cell culture comprises a percentage of engineered γδT cells (e.g., Vδ1 T cells) that is less than 5%, less than 10%, less than 20%, less than 30%, less than 40%, less than 50%, less than 60%, less than 70%, less than 75%, less than 80%, less than 85%, less than 90%, less than 91%, less than 92%, less than 93%, less than 94%, less than 95%, less than 96%, less than 97%, less than 98%, or less than 99%. In some embodiments, the expanded cell culture comprises a percentage of engineered Vδ1 T cells that is about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%.

In some embodiments, the engineered γδT cells (e.g., Vδ1 T cells) do not express human HLA locus.

Samples

The present disclosure provides methods for selectively culturing and expanding γδT cells (e.g., Vδ1 T cells) in culture. The methods as described in the present disclosure are carried out on a sample, which is also referred to herein as a “starting sample” . The methods can use either unfractionated samples or samples that have been enriched for T cells, γδT cells, or Vδ1 T cells. In some embodiments, the samples can be enriched for γδT cells. In some embodiments, the samples can be enriched for Vδ1 T cells.

The sample can be any sample that contains Vδ1 T cells or precursors thereof including, but not limited to, blood, bone marrow, lymphoid tissue, thymus, spleen, lymph node tissue, infected tissue, fetal tissue and fractions or enriched portions thereof. The sample is optionally blood including peripheral blood or umbilical cord blood or fractions thereof, including buffy coat cells, leukapheresis products, peripheral blood mononuclear cells (PBMCs) and low density mononuclear cells (LDMCs) . In some embodiments, the sample is human blood or a fraction thereof. The cells can be obtained from a sample of blood using techniques known in the art such as density gradient centrifugation. For example, whole blood can be layered onto an equal volume of Ficoll-Hypaque^TMfollowed by centrifugation at 400×g for 15-30 minutes at room temperature. The interface material will contain low-density mononuclear cells that can be collected and washed in culture medium and centrifuged at 200×g for 10 minutes at room temperature.

In some embodiments, αβT cells in the sample can be depleted. In some embodiments, 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 along with these beads. In some embodiments, αβT cells in the sample are depleted using a TCRαβ cells depletion kit (Miltenyi, 200-070-407) .

In some embodiments, natural killer (NK) cells in the sample can be depleted. In some embodiments, NK cells in the sample are depleted using antibodies targeting NK cells. In some embodiments, the antibodies targeting NK cells are linked to magnetic beads. In some embodiments, the antibodies targeting NK cells specifically bind to CD56. In some embodiments, NK cells in the sample are depleted using a CD56+ cells depletion kit (Miltenyi, 130-050-401) as per the manufacturer’s instructions.

In some embodiments, αβT cells in the sample are depleted using a TCRαβ cells depletion kit (Miltenyi, 200-070-407) and CD56+ cells depletion kit (Miltenyi, 130-050-401) as per the manufacturer’s instructions, which are incorporated herein by reference in the entirety. In some embodiments, αβT cells can be depleted before the first phase, between the first phase and the second phase, or after the second phase. In some embodiments, NK cells can be depleted before the first phase, between the first phase and the second phase, or after the second phase.

Methods of preparing Vδ1 T cells

Certain cytokines (e.g., IL-4, IL-21, IL-1β, and IFN-γ) have strong stimulatory effects on multiple immune cells, including TCR αβT and Vδ2 T cells. Consequently, these cytokines are often not appropriate for expanding Vδ1 T cells in vitro, as they will also expand other cells in culture, compromising Vδ1 T cell purity. There is a need for methods based on more selective reagents for expanding Vδ1 T cells from impure starting samples (e.g., human PBMC) .

The present disclosure provided methods to produce CAR-Vδ1 T cells with high purity, high expansion rate and high transduction rate for clinical use and production. Such cells display high activation, low exhaustion and predominantphenotype. In vitro validation shows superior anti-tumor activity and safer profile than cells obtained from some other existing methods.

In one aspect, the methods for expanding human γδT cells (e.g., Vδ1 T cells) have a culturing phase and an expanding phase. In the culturing phase, these cells can be cultured in a cell culture medium comprising one or more of cytokines selected from e.g., IL-4, IL-15, IL-1β, and IFN-γ. In some embodiments, the cells are stimulated by a Vδ1 T-specific antibody. In the expanding phase, these cells can be expanded in a cell expansion culture medium containing one or more of cytokines selected from IL-15 and IFN-γ. In some embodiments, the cells are cultured and expanded without the need for the use of feeder cells or microbial or viral components. In some embodiments, the cells are cultured and expanded without the need for IL-21. In some embodiments, the cells are cultured and expanded without the need for IL-2.

Accordingly, in a first aspect, the method for culturing and expanding γδT cells (e.g., Vδ1 T cells) in a sample comprising:

(1) culturing cells in the sample in a first culture medium comprising one or more cytokines selected from IL-4, IL-15, IL-1β, IFN-γ; and

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

In some embodiments, during step (1) , the cells are stimulated by a Vδ1 T-specific antibody. In some embodiments, the first culture medium is located in a container (e.g., cell culture plate) that is coated with a Vδ1 T-specific antibody. In some embodiments, the Vδ1 T-specific antibody is a TCR V delta 1 monoclonal antibody. In some embodiments, the Vδ1 T-specific antibody is an anti-Vδ1 TCR TS-1 antibody (e.g., Thermo fisher, TCR1055) . Other Vδ1 T-specific antibodies are known in the art. They are described in detail, e.g., in U. S. patent application publication No. US20230028110A1, PCT patent application publication Nos. WO2019147735A1, WO2017197347A1 WO2021032960A1, WO2022034562A1, and/or WO2022175413A1, each of which is incorporated by reference in its entirety.

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.

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

In some embodiments, IL-15 is present in an amount from about 1 to about 500 ng/ml. Optionally, IL-15 is present in an amount from about 2 to about 200 ng/ml. Optionally, IL-15 is present in an amount from 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 that is greater than 1 ng/ml, greater than 2 ng/ml, greater than 5 ng/ml, greater than 10 ng/ml, greater than 20 ng/ml, greater than 30 ng/ml, greater than 40 ng/ml, greater than 50 ng/ml, greater than 60 ng/ml, greater than 70 ng/ml, greater than 80 ng/ml, greater than 90 ng/ml, greater than 100 ng/ml, greater than 110 ng/ml, or greater than 120 ng/ml. In some embodiments, IL-15 is present in an amount that is less than 1 ng/ml, less than 2 ng/ml, less than 5 ng/ml, less than 10 ng/ml, less than 20 ng/ml, less than 30 ng/ml, less than 40 ng/ml, less than 50 ng/ml, less than 60 ng/ml, less than 70 ng/ml, less than 80 ng/ml, less than 90 ng/ml, less than 100 ng/ml, less than 110 ng/ml, or less than 120 ng/ml. In some embodiments, IL-15 is present in an amount that is about 1 ng/ml, about 2 ng/ml, about 5 ng/ml, about 10 ng/ml, about 20 ng/ml, about 30 ng/ml, about 40 ng/ml, about 50 ng/ml, about 60 ng/ml, about 70 ng/ml, about 80 ng/ml, about 90 ng/ml, about 100 ng/ml, about 110 ng/ml, or about 120 ng/ml. In some embodiments, IL-15 is present in an amount that is from about 1 to about 1000 ng/ml, from about 1 to 100 ng/ml, from about 1 to 75 ng/ml, from about 1 to 50 ng/ml, from about 1 to 25 ng/ml, from about 5 to 20 ng/ml, from about 5 to 15 ng/ml, or from about 7.5 to 12.5 ng/ml.

In some embodiments, IL-1β is present in an amount from about 1 to about 500 ng/ml. Optionally, IL-1β is present in an amount from about 2 to about 200 ng/ml. Optionally, IL-1β is present in an amount from 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 that is greater than 1 ng/ml, greater than 2 ng/ml, greater than 3 ng/ml, greater than 4 ng/ml, greater than 5 ng/ml, greater than 6 ng/ml, greater than 7 ng/ml, greater than 8 ng/ml, greater than 9 ng/ml, greater than 10 ng/ml, greater than 11 ng/ml, greater than 12 ng/ml, greater than 13 ng/ml, greater than 14 ng/ml, greater than 15 ng/ml, greater than 16 ng/ml, greater than 17 ng/ml, greater than 18 ng/ml, greater than 19 ng/ml, greater than 20 ng/ml, greater than 25 ng/ml, or greater than 30 ng/ml. In some embodiments, IL-1β is present in an amount that is less than 1 ng/ml, less than 2 ng/ml, less than 3 ng/ml, less than 4 ng/ml, less than 5 ng/ml, less than 6 ng/ml, less than 7 ng/ml, less than 8 ng/ml, less than 9 ng/ml, less than 10 ng/ml, less than 11 ng/ml, less than 12 ng/ml, less than 13 ng/ml, less than 14 ng/ml, less than 15 ng/ml, less than 16 ng/ml, less than 17 ng/ml, less than 18 ng/ml, less than 19 ng/ml, less than 20 ng/ml, less than 25 ng/ml, or less than 30 ng/ml. In some embodiments, IL-1β is present in an amount that is about 1 ng/ml, about 2 ng/ml, about 3 ng/ml, about 4 ng/ml, about 5 ng/ml, about 6 ng/ml, about 7 ng/ml, about 8 ng/ml, about 9 ng/ml, about 10 ng/ml, about 11 ng/ml, about 12 ng/ml, about 13 ng/ml, about 14 ng/ml, about 15 ng/ml, about 16 ng/ml, about 17 ng/ml, about 18 ng/ml, about 19 ng/ml, about 20 ng/ml, about 25 ng/ml, or about 30 ng/ml. In some embodiments, IL-1β is present in an amount that is from about 1 to about 1000 ng/ml, from about 5 to 100 ng/ml, from about 10 to 75 ng/ml, from about 10 to 50 ng/ml, from about 10 to 25 ng/ml, from about 10 to 20 ng/ml, from about 12.5 to 17.5 ng/ml, or from about 14 to 16 ng/ml.

In some embodiments, IFN-γ is present in an amount from about 1 to about 1000 ng/ml. Optionally, IFN-γ is present in an amount from about 2 to about 500 ng/ml. Optionally, IFN-γ is present in an amount from 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 that is greater than 1 ng/ml, greater than 2 ng/ml, greater than 5 ng/ml, greater than 10 ng/ml, greater than 20 ng/ml, greater than 30 ng/ml, greater than 40 ng/ml, greater than 50 ng/ml, greater than 60 ng/ml, greater than 70 ng/ml, greater than 80 ng/ml, greater than 90 ng/ml, greater than 100 ng/ml, greater than 110 ng/ml, or greater than 120 ng/ml. In some embodiments, IFN-γ is present in an amount that is less than 1 ng/ml, less than 2 ng/ml, less than 5 ng/ml, less than 10 ng/ml, less than 20 ng/ml, less than 30 ng/ml, less than 40 ng/ml, less than 50 ng/ml, less than 60 ng/ml, less than 70 ng/ml, less than 80 ng/ml, less than 90 ng/ml, less than 100 ng/ml, less than 110 ng/ml, or less than 120 ng/ml. In some embodiments, IFN-γ is present in an amount that is about 1 ng/ml, about 2 ng/ml, about 5 ng/ml, about 10 ng/ml, about 20 ng/ml, about 30 ng/ml, about 40 ng/ml, about 50 ng/ml, about 60 ng/ml, about 70 ng/ml, about 80 ng/ml, about 90 ng/ml, about 100 ng/ml, about 110 ng/ml, or about 120 ng/ml. In some embodiments, IFN-γ is present in an amount that is from about 1 to about 1000 ng/ml, from about 10 to 100 ng/ml, from about 20 to 200 ng/ml, from about 30 to 300 ng/ml, from about 40 to 400 ng/ml, from about 50 to 500 ng/ml, from about 50 to 150 ng/ml, from about 50 to 90 ng/ml, or from about 60 to 80 ng/ml.

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 that is from about 0.1 to about 100 μg/ml. Optionally, the Vδ1 T-specific antibody is present in an amount from about 0.1 to about 10 μg/ml. Optionally, the Vδ1 T-specific antibody is present in an amount from 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., cell culture plate) that is coated with a Vδ1 T-specific antibody.

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

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.

In some embodiments, IFN-γ is present in an amount from about 1 to about 1000 ng/ml. Optionally, IFN-γ is present in an amount from about 2 to about 500 ng/ml. Optionally, IFN-γ is present in an amount from 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 that is greater than 1 ng/ml, greater than 2 ng/ml, greater than 5 ng/ml, greater than 10 ng/ml, greater than 20 ng/ml, greater than 30 ng/ml, greater than 40 ng/ml, greater than 50 ng/ml, greater than 60 ng/ml, greater than 70 ng/ml, greater than 80 ng/ml, greater than 90 ng/ml, greater than 100 ng/ml, greater than 110 ng/ml, or greater than 120 ng/ml. In some embodiments, IFN-γ is present in an amount that is less than 1 ng/ml, less than 2 ng/ml, less than 5 ng/ml, less than 10 ng/ml, less than 20 ng/ml, less than 30 ng/ml, less than 40 ng/ml, less than 50 ng/ml, less than 60 ng/ml, less than 70 ng/ml, less than 80 ng/ml, less than 90 ng/ml, less than 100 ng/ml, less than 110 ng/ml, or less than 120 ng/ml. In some embodiments, IFN-γ is present in an amount that is about 1 ng/ml, about 2 ng/ml, about 5 ng/ml, about 10 ng/ml, about 20 ng/ml, about 30 ng/ml, about 40 ng/ml, about 50 ng/ml, about 60 ng/ml, about 70 ng/ml, about 80 ng/ml, about 90 ng/ml, about 100 ng/ml, about 110 ng/ml, or about 120 ng/ml. In some embodiments, IFN-γ is present in an amount that is from about 1 to about 1000 ng/ml, from about 1 to 100 ng/ml, from about 1 to 50 ng/ml, from about 10 to 50 ng/ml, or from about 20 to 40 ng/ml.

In some embodiments, IL-15 is present in an amount from about 1 to about 500 ng/ml. Optionally, IL-15 is present in an amount from about 2 to about 200 ng/ml. Optionally, IL-15 is present in an amount from 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 that is greater than 1 ng/ml, greater than 2 ng/ml, greater than 5 ng/ml, greater than 10 ng/ml, greater than 20 ng/ml, greater than 30 ng/ml, greater than 40 ng/ml, greater than 50 ng/ml, greater than 60 ng/ml, greater than 70 ng/ml, greater than 80 ng/ml, greater than 90 ng/ml, greater than 100 ng/ml, greater than 110 ng/ml, or greater than 120 ng/ml. In some embodiments, IL-15 is present in an amount that is less than 1 ng/ml, less than 2 ng/ml, less than 5 ng/ml, less than 10 ng/ml, less than 20 ng/ml, less than 30 ng/ml, less than 40 ng/ml, less than 50 ng/ml, less than 60 ng/ml, less than 70 ng/ml, less than 80 ng/ml, less than 90 ng/ml, less than 100 ng/ml, less than 110 ng/ml, or less than 120 ng/ml. In some embodiments, IL-15 is present in an amount that is about 1 ng/ml, about 2 ng/ml, about 5 ng/ml, about 10 ng/ml, about 20 ng/ml, about 30 ng/ml, about 40 ng/ml, about 50 ng/ml, about 60 ng/ml, about 70 ng/ml, about 80 ng/ml, about 90 ng/ml, about 100 ng/ml, about 110 ng/ml, or about 120 ng/ml. In some embodiments, IL-15 is present in an amount that is from about 1 to about 1000 ng/ml, from about 10 to 100 ng/ml, from about 20 to 200 ng/ml, from about 30 to 300 ng/ml, from about 40 to 400 ng/ml, from about 50 to 500 ng/ml, from about 50 to 150 ng/ml, from about 50 to 90 ng/ml, or from about 60 to 80 ng/ml.

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

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

The cells 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 more than 1 day, more than 2 days, more than 3 days, more than 4 days, more than 5 days, more than 6 days, more than 7 days, more than 8 days, more than 9 days, more than 10 days, more than 11 days, more than 12 days, more than 13 days, or more than 14 days. 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 of time ranging from about 2 days to about 21 days, from about 3 days to about 20 days, from about 3 days to about 15 days, from about 5 days to about 15 days, from about 5 days to about 12 days, from about 7 days to about 10 days, or from about 8 days to about 10 days.

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

Any suitable mammalian cell culture medium 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) can be used. 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.

In some embodiments, both the first and second culture media are supplemented with serum or plasma. The amount of plasma in the first and second culture media is optionally from about 0.5%to about 25%by volume, for example from about 2%to about 20%by volume or from about 2.5%to about 10%by volume, for example is about 10%by volume. The serum or plasma can be obtained from any source including, but not limited to, human peripheral blood, umbilical cord blood, or blood derived from another mammalian species. The plasma may be from a single donor or may be pooled from several donors. In some embodiments, if autologous Vδ1 T cells are to be used clinically, i.e. reinfused into the same patient from whom the original sample was obtained, then autologous plasma (i.e. from the same patient) can be used to avoid the introduction of hazardous products (e.g. viruses) into that patient. In some embodiments, both the first and second culture media supplemented with FBS. In some embodiments, the FBS is present in an amount that is greater than 0.5%, greater than 1%, greater than 2%, greater than 3%, greater than 4%, greater than 5%, greater than 6%, greater than 7%, greater than 8%, greater than 9%, greater than 10%, greater than 11%, greater than 12%, greater than 13%, greater than 14%, greater than 15%, greater than 20%, greater than 25%, or greater than 30%by volume. In some embodiments, the FBS is present in an amount that is less than 0.5%, less than 1%, less than 2%, greater than 3%, less than 4%, less than 5%, less than 6%, less than 7%, less than 8%, less than 9%, less than 10%, less than 11%, less than 12%, less than 13%, less than 14%, less than 15%, less than 20%, less than 25%, or less than 30%by volume. In some embodiments, the FBS is present in an amount that is about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, les about 15%, about 20%, about 25%, or about 30%by volume. In some embodiments, the FBS is present in an amount that is from about 0.5%to about 30%, from about 1%to about 25%, from about 2%to about 20%, from about 5%to about 20%, from about 5%to about 15%, from about 8%to about 12%, or from about 9%to about 11%by volume.

In some embodiments, both the first and second culture media supplemented with human platelet lysate. In some embodiments, the human platelet lysate is present in an amount that is greater than 0.5%, greater than 1%, greater than 2%, greater than 3%, greater than 4%, greater than 5%, greater than 6%, greater than 7%, greater than 8%, greater than 9%, greater than 10%, greater than 11%, greater than 12%, greater than 13%, greater than 14%, greater than 15%, greater than 20%, greater than 25%, or greater than 30%by volume. In some embodiments, the human platelet lysate is present in an amount that is less than 0.5%, less than 1%, less than 2%, greater than 3%, less than 4%, less than 5%, less than 6%, less than 7%, less than 8%, less than 9%, less than 10%, less than 11%, less than 12%, less than 13%, less than 14%, less than 15%, less than 20%, less than 25%, or less than 30%by volume. In some embodiments, the human platelet lysate is present in an amount that is about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, les about 15%, about 20%, about 25%, or about 30%by volume. In some embodiments, the human platelet lysate is present in an amount that is from about 0.5%to about 30%, from about 1%to about 25%, from about 2%to about 20%, from about 5%to about 20%, from about 5%to about 15%, from about 8%to about 12%, or from about 9%to about 11%by volume.

Prior to culturing the sample or fraction thereof (such as PBMCs) in the first culture medium, the sample or fraction thereof may be enriched for certain cell types and/or depleted for other cell types. In particular, the sample or fraction thereof may be enriched for T cells, or enriched for γδT cells, or enriched for Vδ1 T cells, or depleted of aβT cells or depleted of non-Vδ1 T cells. In some embodiments, prior to culturing the sample in the first culture medium, the sample is depleted of aβT cells.

The first or second culture medium, or both culture media, can additionally include other ingredients that can assist in the growth and expansion of the Vδ1 T cells. Examples of other ingredients that can be added, include, but are not limited to, plasma or serum, purified proteins such as albumin, a lipid source such as low density lipoprotein (LDL) , vitamins, amino acids, steroids and any other supplements supporting or promoting cell growth and/or survival.

The first or second culture medium, or both culture media, may also contain other growth factors, including cytokines that can further enhance the expansion of Vδ1 T cells. Examples of other growth factors that can be added include co-stimulatory molecules such as an IL-21, human anti-SLAM antibody, any soluble ligand of CD27, or any soluble ligand of CD7. Any combination of these growth factors can be included in the first or second culture medium, or in both media.

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.

In some embodiments, the method of preparing Vδ1 cells comprises

(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

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

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

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) .

The president disclosure also provides some alternative methods of culturing and expanding Vδ1 T cells.

In some embodiments, the method of preparing Vδ1 cells comprises (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) , 5-9 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 (2) in a second culture medium comprising 60-80 ng/ml (e.g., about 70ng/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 were 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) .

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

Depletion of αβT cells and enrichment of γδT cells

In some embodiments, αβT cells are depleted at various stages of the method described herein. In some embodiments, αβT cells are depleted prior to the first culture step. In some embodiments, αβT cells are depleted prior to the second culture step. In some embodiments, αβT cells are depleted after the second culture step.

The γδT cells can be enriched by various means. γδT cells can be directly enriched from a sample, for example, by sorting γδT cells that express one or more cell surface markers with flow cytometry techniques. Wild-type γδT cells exhibit numerous antigen recognition, antigen-presentation, co-stimulation, and adhesion molecules that can be associated with a γδT cells. One or more cell surface markers such as specific γδ TCRs, antigen recognition, antigen-presentation, ligands, adhesion molecules, or co-stimulatory molecules may be used to isolate a wild-type γδT cell from a sample. Various molecules associated with, or expressed by, a γδT cell may be used to isolate a γδT cell from a sample. In some cases, the present disclosure provides methods for enrichment of mixed population of Vδ1+, Vδ2+, Vδ3+ cells or any combination thereof.

Peripheral blood mononuclear cells can be collected from a subject, for example, with an apheresis machine, including the Ficoll-Paque^TM PLUS (GE Healthcare) system, or another suitable device/system, γδT cells, or a desired subpopulation of γδT cells, can be purified from the collected sample with, for example, with flow cytometry techniques. Cord blood cells can also be obtained from cord blood during the birth of a subject.

Positive and/or negative selection of cell surface markers expressed on the collected γδT cells can be used to directly isolate a γδT cell, or a population of γδT cells expressing similar cell surface markers from a peripheral blood sample, a cord blood sample, a tumor, a tumor biopsy, a tissue, a lymph, or from an epithelial sample of a subject. For instance, a γδT cell can be isolated from a complex sample based on positive or negative expression of CD2, CD3, CD4, CD8, CD24, CD25, CD44, Kit, TCR a, TCR β, TCR γ, TCR δ, NKG2D, CD70, CD27, CD30, CD 16, CD337 (NKp30) , CD336 (NKp46) , OX40, CD46, CCR7, and other suitable cell surface markers.

A γδT cell can be isolated from a complex sample that is cultured in vitro. In some embodiments, whole PBMC population, without prior depletion of specific cell populations such as monocytes, αβT cells, B-cells, and NK cells, can be activated and expanded. In other embodiments, enriched γδT cell populations can be generated prior to their specific activation and expansion. In some aspects, activation and expansion of γδT cell are performed without the presence of native or engineered APCs. In some aspects, isolation and expansion of γδT cells from tumor specimens can be performed using immobilized γδT cell mitogens, including antibodies specific to γδ TCR, and other γδ TCR activating agents, including lectins.

In some embodiments, the Vδ1 T or γδT cells can be separated using techniques known in the art including fluorescence activated cell sorting, immunomagnetic separation, affinity column chromatography, density gradient centrifugation and cellular panning. In some embodiments, γδT cells can be isolated by TCRγ/δ+ T Cell Isolation Kit (Miltenyi Biotec, 130-092-892) as per the manufacturer’s instructions.

In some embodiments, 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, αβT cells in the sample are depleted using a TCRαβ cells depletion kit (Miltenyi, 200-070-407) .

In some embodiments, αβT cells in the sample are depleted using a TCRαβ cells depletion kit (Miltenyi, 200-070-407) and CD56+ cells depletion kit (Miltenyi, 130-050-401) as per the manufacturer’s instructions.

Characteristics of the resulting Vδ1 T cells

In another aspect, the present disclosure provides a cell preparation prepared according the method described herein. In some embodiments, the Vδ1 T cell preparation has a purity that is greater than 80%. Optionally, the resulting Vδ1 T cell preparation has a purity that is greater than 80%, Optionally greater than 90%, and optionally greater than 95%. In some embodiments, the Vδ1 T cell preparation has a purity that is greater than 5%, greater than 10%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99%. In some embodiments, Vδ1 T cell preparation has a purity that is less than 5%, less than 10%, less than 20%, less than 30%, less than 40%, less than 50%, less than 60%, less than 70%, less than 75%, less than 80%, less than 85%, less than 90%, less than 91%, less than 92%, less than 93%, less than 94%, less than 95%, less than 96%, less than 97%, less than 98%, or less than 99%. In some embodiments, Vδ1 T cell preparation has a purity that is about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%

In some embodiments, the Vδ1 T cells can be transfected with a vector encoding an engineered receptor (e.g., CAR or TCR) . In some embodiments, the Vδ1 T cells are transfected with a vector encoding an engineered receptor (e.g., CAR or TCR) before the first culturing step. In some embodiments, the Vδ1 T cells are transfected with a vector encoding an engineered receptor (e.g., CAR or TCR) after the first culturing step. In some embodiments, the Vδ1 T cells are transfected with a vector encoding an engineered receptor (e.g., CAR or TCR) after the second culturing step.

The Vδ1 T cells obtained by the method described herein can be used in cell therapies. Vδ1 T cells are considered as a first line of defense against infectious pathogens. In addition, Vδ1 T cells possess intrinsic cytolytic activity against transformed cells of various origins including B-cell lymphomas, sarcomas and carcinomas. As a result, the Vδ1 T cells obtained and cultured ex vivo according to the methods of the disclosure can be transfused into a patient for the treatment or prevention of infections, cancer or diseases resulting from immunosuppression.

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

In some embodiments, the Vδ1 T cells prepared according to the method described herein have a higher cytotoxicity. In some embodiments, the expansion rate of Vδ1 T cells prepared under the methods described herein is higher comparing to the Vδ1 T cells prepared according to other methods. In some embodiments, the Vδ1 T cells prepared according to the method described herein has an expansion fold of more than 1k, more than 2k, more than 5k, more than 10k, more than 20k, more than 50k, more than 100k, more than 150k, more than 200k, or more than 250k, as determined by a cell expansion assay.

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

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

In some embodiments, the Vδ1 T cells prepared according to the methods described herein showed 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 has NGK2D positive rate that is higher than 20%, higher than 30%, higher than 40%, higher than 50%, higher than 60%, higher than 70%, higher than 80%, higher than 90%, higher than 92.5%, higher than 93%, or higher than 93.5%. In some embodiments, the Vδ1 T cells prepared according to the methods described herein has PD-1 positive rate that is lower than 0.5%, lower than 0.7%, lower than 0.7%, lower than 0.8%, lower than 0.9%, lower than 1%, lower than 2%, lower than 5%, or lower than 10%. In some embodiments, the Vδ1 T cells prepared according to the methods described herein has TIGIT positive rate that is lower than 0.5%, lower than 1%, lower than 2%, lower than 5%, lower than 6%, lower than 7%, lower than 8%, lower than 9%, lower than 10%, or lower than 20%.

In some embodiments, the Vδ1 T cells prepared according to the method described herein have a phenotype that is close tophenotype.

In some embodiments, the Vδ1 T cells prepared according to the method described herein have a higher CD27 positive rate. In some embodiments, the Vδ1 T cells prepared according to the method described herein has a CD27 positive rate of higher than 20%, higher than 30%, higher than 40%, higher than 50%, higher than 60%, higher than 70%, higher than 80%, or higher than 90%. In some embodiments, the Vδ1 T cells prepared according to the method described herein have a higher CD45RA positive rate. In some embodiments, the Vδ1 T cells prepared according to the method described herein has a CD45RA positive rate of higher than 20%, higher than 30%, higher than 40%, higher than 50%, higher than 60%, higher than 70%, higher than 80%, higher than 90%, higher than 95%, or higher than 96%.

In some embodiments, the Vδ1 T cells prepared according to the method described herein have a higher cytotoxicity. In some embodiments, the cytotoxicity of Vδ1 T cells prepared according to the methods described herein is stronger comparing to Vδ1 T cells prepared according to other methods. In some embodiments, the Vδ1 T cells prepared according to the method described herein can kill target cells by more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, or more than 95%, as determined by a CAR-T cytotoxicity assay. In some embodiments, the Vδ1 T cells prepared according to the method described herein can kill target cells by more than 80%after 9 days.

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

In some embodiments, the Vδ1 T cells prepared according to the method described herein can secrete less cytokines. In some embodiments, the Vδ1 T cells prepared according to the methods described herein secrete less cytokines comparing to the Vδ1 T cells prepared according to other methods. 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 GM-CSF. 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.

In some embodiments, the Vδ1 T cells prepared according to the method described herein can suppress tumor growth. In some embodiments, the Vδ1 T cells prepared according to the methods described herein have a tumor growth inhibition percentage (TGI%) that is greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. In some embodiments, the Vδ1 T cells prepared according to the method described herein have a tumor growth inhibition percentage that is less than 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. The TGI%can be determined, e.g., at 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days after the treatment starts, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after the treatment starts. As used herein, the tumor growth inhibition percentage (TGI%) is calculated using the following formula:
TGI (%) = [1- (Ti-T0) / (Vi-V0) ] ×100

Ti is the average tumor volume in the treatment group on day i. T0 is the average tumor volume in the treatment group on day zero. Vi is the average tumor volume in the control group on day i. V0 is the average tumor volume in the control group on day zero.

In some embodiments, the tumor suppression effect of Vδ1 T cells prepared according to the methods described herein is stronger comparing to Vδ1 T cells prepared according to other methods. In some embodiments, the Vδ1 T cells prepared according to the methods described herein can inhibit tumor growth by more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, or more than 95%.

Contrasting with Vδ1 T cells prepared according to other methods, Vδ1 T cells prepared according to the methods described herein displayed higher purity, yield and persistence with better anti-tumor cytotoxicity.

Contrasting with Vδ1 T cells prepared according to other methods, Vδ1 T cells prepared according to the methods described herein displayed better cell fitness judged by exhaustion markers and persistence with better anti-tumor cytotoxicity.

In some embodiments, upon infusion in mice, the differentiated Vδ1 T cells maintained their cytotoxic phenotype and inhibited tumor growth in vivo.

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

Any of the T cells (e.g., γδT cells, Vδ1 T cells) described above may further express an engineered receptor. Exemplary engineered receptor include, but are not limited to, CAR, engineered TCR, and TAC receptors. In some embodiments, the engineered receptor comprises an extracellular domain that specifically binds to an antigen (e.g., a tumor antigen) , a transmembrane domain, and an intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain and/or a co-stimulatory 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 (such as a constitutive promoter or an inducible promoter) . In some embodiments, the engineered receptor is introduced to the Vδ1 T cells by inserting proteins into the cell membrane while passing cells through a microfluidic system, such as CELL (see, for example, U. S. Patent Application Publication No. 20140287509) . The engineered receptor may 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 cytotoxicity of the modified Vδ1 T cells. In some embodiments, the modified Vδ1 T cells do not express an engineered receptor, such as CAR, TCR, or TAC receptor.

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.

In some embodiments, the engineered receptor is a chimeric antigen receptor (CAR) . Many chimeric antigen receptors are known in the art and may be suitable for the modified Vδ1 T cells of the present disclosure. CARs can also be constructed with a specificity for any cell surface marker by utilizing antigen binding fragments or antibody variable domains of, for example, antibody molecules.

CARs of the present disclosure comprise an extracellular domain comprising at least one targeting domain that specifically binds at least one tumor antigen, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the intracellular signaling domain generates a signal that promotes an immune effector function of the CAR-containing cell, e.g., a CAR-T cell. “Immune effector function or immune effector response” refers to function or response, e.g., of an immune effector cell, that enhances or promotes an immune attack of a target cell. For example, an immune effector function or response may refer to a property of a T or NK cell that promotes killing or the inhibition of growth or proliferation, of a target cell. Examples of immune effector function, e.g., in a CAR-T cell, include cytolytic activity (such as antibody-dependent cellular toxicity, or ADCC) and helper activity (such as the secretion of cytokines) . In some embodiments, the CAR has an intracellular signaling domain with an attenuated immune effector function. In some embodiments, the CAR has an intracellular signaling domain having no more than about any of 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%or less of an immune effector function (such as cytolytic function against target cells) compared to a CAR having a full-length and wildtype CD3ζand optionally one or more co-stimulatory domains. In some embodiments, the intracellular signaling domain generates a signal that promotes proliferation and/or survival of the CAR containing cell. In some embodiments, the 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 can comprise the entire intracellular (i.e., cytoplasmic) portion, or the entire native intracellular signaling domain, of the molecule, or a fragment or derivative thereof.

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

In some embodiments, the intracellular signaling domain of a CAR comprises one or more (such as any of 1, 2, 3, or more) co-stimulatory domains. “Co-stimulatory domain” can be the intracellular portion of a co-stimulatory molecule. The term “co-stimulatory molecule” refers to a cognate binding partner on an immune cell (such as T cell) that specifically binds with a co-stimulatory ligand, thereby mediating a co-stimulatory response by the immune cell, such as, but not limited to, proliferation and survival. Co-stimulatory molecules are cell surface molecules other than antigen receptors or their ligands that contribute to an efficient immune response. A co-stimulatory molecule can be represented in the following protein families: TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins) , and activating NK cell receptors. Co-stimulatory molecules include, but are not limited to an MHC class I molecule, BTLA and a Toll ligand receptor, as well as OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18) , ICOS (CD278) , and 4-1BB (CD137) . Further examples of such co-stimulatory molecules include CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR) , SLAMF7, NKp80 (KLRF1) , NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8alpha, CD8beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226) , SLAMF4 (CD244, 2B4) , CD84, CD96 (Tactile) , CEACAM1, CRTAM, Ly9 (CD229) , CD160 (BY55) , PSGL1, 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 a ligand that specifically binds with CD83.

In some embodiments, the CAR comprises a single co-stimulatory domain. In some embodiments, the CAR comprises two or more co-stimulatory domains. In some embodiments, the intracellular signaling domain comprises a functional primary intracellular signaling domain and one or more co-stimulatory domains. In some embodiments, the CAR does not comprise a functional primary intracellular signaling domain (such as CD3ζ) . In some embodiments, the CAR comprises an intracellular signaling domain consisting of or consisting essentially of one or more co-stimulatory domains. In some embodiments, the CAR comprises an intracellular signaling domain consisting of or consisting essentially of a nonfunctional or attenuated primary intracellular signaling domain (such as a mutant CD3ζ) and one or more co-stimulatory domains. Upon binding of the targeting domain to tumor antigen, the co-stimulatory domains of the CAR may transduce signals for enhanced proliferation, survival and differentiation of the engineered immune cells having the CAR (such as T cells) , and inhibit activation induced cell death. In some embodiments, the one or more co-stimulatory 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 ligands that specially bind to CD83.

In some embodiments, the intracellular signaling domain of the CAR comprises a co-stimulatory signaling domain derived from CD28. In some embodiments, the intracellular signaling domain comprises a cytoplasmic signaling domain of CD3ζ and a co-stimulatory signaling domain of CD28. In some embodiments, the intracellular signaling domain in the chimeric receptor of the present application comprises a co-stimulatory signaling domain derived from 4-1BB (i.e., CD137) . In some embodiments, the intracellular signaling domain comprises a cytoplasmic signaling domain of CD3ζ and a co-stimulatory signaling domain of 4-1BB.

In some embodiments, the intracellular signaling domain of the CAR comprises a co-stimulatory signaling domain of CD28 and a co-stimulatory signaling domain of 4-1BB. In some embodiments, the intracellular signaling domain comprises a cytoplasmic signaling domain of CD3ζ, a co-stimulatory signaling domain of CD28, and a co-stimulatory signaling domain of 4-1BB. In some embodiments, the intracellular signaling domain comprises a polypeptide comprising from the N-terminus to the C-terminus: a co-stimulatory signaling domain of CD28, a co-stimulatory signaling domain of 4-1BB, and a cytoplasmic signaling domain of CD3ζ.

In some embodiments, the targeting domain of the CAR is an antibody or an antibody fragment, such as an scFv, a Fv, a Fab, a (Fab′) ₂, a single domain antibody (sdAb) , or a V_HH domain. In some embodiments, the targeting domain of the CAR is a ligand or an extracellular portion of a receptor that specifically binds to a tumor antigen. In some embodiments, the one or more targeting domains of the CAR specifically bind to a single tumor antigen. In some embodiments, the CAR is a bispecific or multispecific CAR with targeting domains that bind 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 (such as EGFRvIII) , GD2, HER2, IGF1R, mesothelin, PSMA, ROR1, WT1, and other tumor antigens with 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 (such as EGFRvIII) , GD2, HER2, IGF1R, mesothelin, PSMA, ROR1, WT1, and other tumor antigens with clinical significance. In some embodiments, the tumor antigen is derived from an intracellular protein of tumor cells. Many TCRs specific for tumor antigens (including tumor-associated antigens) have been described, including, for example, NY-ESO-1 cancer-testis antigen, the p53 tumor suppressor antigens, TCRs for tumor antigens in melanoma (e.g., MARTI, gp 100) , leukemia (e.g., WT1, minor histocompatibility antigens) , and breast cancer (e.g., HER2, NY-BR1) .

In some embodiments, the CAR is an anti-BCMA CAR. A wide variety of antigen binding domain sequences can be used as the targeting domains of the CAR. In some embodiments, the anti-BCMA CAR comprises from the N-terminus to the C-terminus: a CD8 leader, an anti-BCMA sdAb, a CD8 hinge, a CD8 transmembrane, a 4-1BB intracellular co-stimulatory domain, and a CD3ζintracellular signaling domain.

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 (such as EGFRvIII) , GD2, HER2, IGF1R, mesothelin, PSMA, ROR1, WT1, and other tumor antigens with clinical significance. In some embodiments, the tumor antigen is derived from an intracellular protein of tumor cells. Many TCRs specific for tumor antigens (including tumor-associated antigens) have been described, including, for example, NY-ESO-1 cancer-testis antigen, the p53 tumor suppressor antigens, TCRs for tumor antigens in melanoma (e.g., MARTI, gp 100) , leukemia (e.g., WT1, minor histocompatibility antigens) , and breast cancer (e.g., HER2, NY-BR1) . Any of the TCRs known in the art may be used in the present application. In some embodiments, the TCR has an enhanced affinity to the tumor antigen. Exemplary TCRs and methods for introducing the TCRs to immune cells have been described, for example, in U. S. Pat. No. 5, 830, 755, and Kessels et al.

Immunotherapy through TCR gene transfer. Nat. Immunol. 2, 957-961 (2001) . In some embodiments, the modified Vδ1 T cell is a TCR-T cell.

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

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

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

In some embodiments, the TFP comprises a TCR subunit comprising at least a portion of a TCR extracellular domain, and a TCR intracellular domain comprising a stimulatory domain from an intracellular signaling domain of CD3 gamma; and an antigen binding domain wherein the TCR subunit and the antigen binding domain are operatively linked, and wherein the TFP incorporates into a TCR when expressed in a T cell.

In some embodiments, the TFP comprises a TCR subunit comprising at least a portion of a TCR extracellular domain, and a TCR intracellular domain comprising a stimulatory domain from an intracellular signaling domain of CD3 delta; and an antigen binding domain, wherein the TCR subunit and the antigen binding domain are operatively linked, and wherein the TFP incorporates into a TCR when expressed in a T cell.

In some embodiments, the TFP comprises a TCR subunit comprising at least a portion of a TCR extracellular domain, and a TCR intracellular domain comprising a stimulatory domain from an intracellular signaling domain of TCR alpha; and an antigen binding domain wherein the TCR subunit and the antigen binding domain are operatively linked, and wherein the TFP incorporates into a TCR when expressed in a T cell.

In some embodiments, the TFP comprises a TCR subunit comprising at least a portion of a TCR extracellular domain, and a TCR intracellular domain comprising a stimulatory domain from an intracellular signaling domain of TCR beta; and an antigen binding domain wherein the TCR subunit and the antigen binding domain are operatively linked, and wherein the TFP incorporates into a TCR when expressed in a T cell.

In some embodiments, the engineered receptor is a T-cell antigen coupler (TAC) receptor. Exemplary TAC receptors have been described, for example, in US20160368964A1, which is incorporated herein by reference. In some embodiments, the TAC comprises a targeting domain, a TCR-binding domain that specifically binds a protein associated with the TCR complex, and a T-cell receptor signaling domain. In some embodiments, the targeting domain is an antibody fragment, such as scFv or V_HH, which specifically binds to a tumor antigen. 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 (such as EGFRvIII) , GD2, HER2, IGF1R, mesothelin, PSMA, ROR1, WT1, and other tumor antigens with 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 a V_HH. In some embodiments, the TCR-binding domain is derived from UCHT1. In some embodiments, the TAC receptor comprises a cytosolic domain and a transmembrane domain. In some embodiments, the T-cell receptor signaling domain comprises a cytosolic domain derived from a TCR co-receptor. Exemplary TCR co-receptors include, but are not limited to, CD4, CD8, CD28, CD45, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD 154. In some embodiments, the TAC receptor comprises a transmembrane domain and a cytosolic domain derived from CD4. In some embodiments, the TAC receptor comprises a transmembrane domain and a cytosolic domain derived from CD8 (such as CD8a) .

T cell co-receptors are expressed as membrane protein on T cells. They can provide stabilization of the TCR: peptide: MEC complex and facilitate signal transduction. The two subtypes of T cell co-receptor, CD4 and CD8, display strong specificity for particular MEC classes. The CD4 co-receptor can only stabilize TCR: MEC II complexes while the CD8 co-receptor can only stabilize the TCR: MEC I complex. The differential expression of CD4 and CD8 on different T cell types results in distinct T cell functional subpopulations. CD8+ T cells are cytotoxic T cells.

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

In some embodiments, the engineered receptor (such as CAR, TCR, or TAC) expressed by the modified Vδ1 T cells targets one or more tumor antigens. Tumor antigens are proteins that are produced by tumor cells that can elicit an immune response, particularly T-cell mediated immune responses. The selection of the targeted antigen will depend on the particular type of cancer to be treated. Exemplary tumor antigens include, for example, a glioma-associated antigen, carcinoembryonic antigen (CEA) , β-human chorionic gonadotropin, alphafetoprotein (AFP) , lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CAIX, human telomerase reverse transcriptase, RU1, RU2 (AS) , intestinal carboxyl esterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA) , PAP, NY-ESO-1, LAGE-la, p53, prostein, PSMA, HER2/neu, survivin and telomerase, prostate-carcinoma tumor antigen-1 (PCTA-1) , MAGE, ELF2M, neutrophil elastase, ephrinB2, CD22, insulin growth factor (IGF) -I, IGF-II, IGF-I receptor and mesothelin.

In some embodiments, the tumor antigen comprises one or more antigenic cancer epitopes associated with a malignant tumor. Malignant tumors express a number of proteins that can serve as target antigens for an immune attack. These molecules include but are not limited to tissue-specific antigens such as MART-1, tyrosinase and gp100 in melanoma and prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to the group of transformation-related molecules such as the oncogene HER2/Neu/ErbB-2. Yet another group of target antigens are onco-fetal antigens such as carcinoembryonic antigen (CEA) . In B-cell lymphoma the tumor-specific idiotype immunoglobulin constitutes a truly tumor-specific immunoglobulin antigen that is unique to the individual tumor. B cell differentiation antigens such as CD19, CD20 and CD37 are other candidates for target antigens in B-cell lymphoma.

In some embodiments, the tumor antigen is a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA) . A TSA is unique to tumor cells and does not occur on other cells in the body. A TAA associated antigen is not unique to a tumor cell, and instead is also expressed on a normal cell under conditions that fail to induce a state of immunologic tolerance to the antigen. The expression of the antigen on the tumor may occur under conditions that enable the immune system to respond to the antigen. TAAs may be antigens that are expressed on normal cells during fetal development, when the immune system is immature, and unable to respond or they may be antigens that are normally present at extremely low levels on normal cells, but which are expressed at much higher levels on tumor cells.

Non-limiting examples of TSA or TAA antigens include the following: Differentiation antigens such as MART-1/MelanA (MART-I) , gp 100 (Pmel 17) , tyrosinase, TRP-1, TRP-2 and tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor-suppressor genes such as p53, Ras, HER2/neu; unique tumor antigens resulting from chromosomal translocations; such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such as the Epstein Barr virus antigens EBVA and the human papillomavirus (HPV) antigens E6 and E7. Other large, protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23HI, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-Catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB/70K, NY-CO-1, RCAS 1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-associated protein, TAAL6, TAG72, TLP, and TPS.

Engineered Vδ1 T cells of the disclosure can be designed to home to a specific physical location in the body of a subject and hence target an antigen at a particular tissue, organ or body site. Endogenous T-cells have distinct repertoires of trafficking ligands and receptors that influence their patterns of migration. An engineered Vδ1 T cell of the disclosure can be designed to express, from the expression cassette comprising the tumor recognition moiety or from a separate expression cassette, one or more trafficking ligand (s) , or receptor (s) that guide the migration of the engineered Vδ1 T cell to a particular tissue, organ, or body site.

An engineered Vδ1 T cell of the disclosure can be a tumor-specific allogeneic cell. For instance, an engineered Vδ1 T cell may be derived from a non-engineered Vδ1 T cell that is a tumor infiltrating lymphocyte (TIL) isolated from a tumor. Different TILs that can be isolated from different tumor types. An expression cassette encoding a tumor recognition moiety, and activation domain, or another engineered featured can be inserted into the genome of a TIL isolated from various tumors. Such Vδ1 T cell can infiltrate solid tumors, weaken and kill tumors cells expressing one or more target antigens, and they can provide an effective treatment for various malignancies. A tumor specific allogeneic Vδ1 T cell can be engineered to express at least one tumor recognition moiety that recognizes an epitope of choice. In some cases, a tumor specific allogeneic Vδ1 T cell is designed to express at least two different tumor recognition moieties, and each different tumor recognition moiety is designed to recognize a different epitope of the same antigen, distinct antigens, an antigen and an activating or inactivating co-stimulatory/immune modulation receptor (s) , an antigen in complex with an MHC molecule, or a homing receptor.

Therapeutic use

The Vδ1 T cells obtained by the method as described herein can be used in 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 with the objective of improving their therapeutic potential. For example, with the objective of redirecting the specificity of the Vδ1 T cells through the expression of a chimeric antigen receptor (CAR) or TCR on these cells. CAR expression can be induced through electroporation of Vδ1 T cells for the insertion of genetic material, or by infecting these cells with viral vectors, such as lentiviruses or retroviruses containing the desired genetic material. Such genetic editing can improve the potency of the Vδ1 T cells by improving homing, cytokine production, recycle killing, and/or improved engraftment.

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

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

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

Examples of infections that may be treated include, but are not limited to, bacterial infections such as those caused by Mycobacteria (e.g. tuberculosis) , viral infections such as those caused by herpes simplex virus (HSV) , human immunodeficiency virus (HIV) or the hepatitis viruses, and parasitic infections such as those caused by Plasmodium (e.g. malaria) .

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

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

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

The Vδ1 T cells obtained according to the present disclosure can also be used in experimental models, for example, to further study and elucidate the function of the cells. Additionally, these cells may be used for studies directed towards the identification of the antigens/epitopes recognized by Vδ1 T cells and for the design and development of vaccines.

Accordingly, in another aspect, the present disclosure provides a method for vaccinating a subject comprising administering an effective amount of Vδ1 T cells obtained by a method described herein to a subject in need thereof. Such vaccine can be given to immunocompromised patients or individuals with elevated risk of developing an infectious disease or cancer.

The obtained Vδ1 T cells, according to the disclosure may be immediately used in the above therapeutic, experimental or commercial applications or the cells may be cryopreserved for use at a later date.

The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration. The disclosure also provides methods of manufacturing the antibodies or antigen binding fragments thereof for various uses as described herein.

One or more of the Vδ1 T cells described herein can be administered to a subject in a single, unified form, such as an intravenous injection, or in multiple forms, for example, as multiple intravenous infusions or injections, or subcutaneous injections. In some cases, the Vδ1 T cells can expand within a subject's body, in vivo, after administration to a 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 disclosure, and pharmaceutical compositions comprising the same, can be packaged as a kit. A kit may include instructions (e.g., written instructions) on the use of the Vδ1 T cells and compositions comprising the same.

In some embodiments, a method of treatment comprises administering to a subject a therapeutically-effective amount of the Vδ1 T cells. In some embodiments, the therapeutically-effective amount of the 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 the Vδ1 T cells is administered for at least one week. In some embodiments, the therapeutically-effective amount of the Vδ1 T cells is administered for at least two weeks.

The Vδ1 T cells described herein can be administered before, during, or after the occurrence of a disease or condition, and the timing of administering a pharmaceutical composition containing the Vδ1 T cells can vary. For example, the Vδ1 T cells can be used as a prophylactic and can be administered continuously to subjects with a propensity to conditions or diseases in order to lessen a likelihood of the occurrence of the disease or condition. The Vδ1 T cells can be administered to a subject during or as soon as possible after the onset of the symptoms. The administration of the Vδ1 T cells can be initiated immediately within the onset of symptoms, within the first 3 hours of the onset of the symptoms, within the first 6 hours of the onset of the symptoms, within the first 24 hours of the onset of the symptoms, within 48 hours of the onset of the symptoms, or within any period of time from the onset of symptoms. The initial administration can be via any route practical (e.g., intravenous infusions or injections) , such as by any route described herein using any formulation described herein. In some examples, the administration of the Vδ1 T cells of the disclosure is an intravenous administration. One or multiple dosages of the Vδ1 T cells can be administered as soon as is practicable after the onset of a cancer or an infectious disease, and for a length of time necessary for the treatment of the disease, such as, for example, from about 24 hours to about 48 hours, from about 48 hours to about 1 week, from about 1 week to about 2 weeks, from about 2 weeks to about 1 month, from about 1 month to about 3 months. For the treatment of cancer, one or multiple dosages of the Vδ1 T cells can be administered years after onset of the cancer and before or after other treatments. In some examples, the 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 vary for each subject.

The Vδ1 T cells disclosed herein may be formulated in unit dosage forms suitable for single administration of precise dosages. In some cases, the unit dosage forms comprise additional lymphocytes. In unit dosage form, the formulation is divided into unit doses containing appropriate quantities of one or more compounds. The unit dosage can be in the form of a package containing discrete quantities of the formulation. Non-limiting examples are packaged tablets or capsules, and powders in vials or ampoules. Aqueous suspension compositions can be packaged in single-dose non-reclosable containers. Multiple-dose reclosable containers can be used, for example, in combination with a preservative or without a preservative. In some examples, the pharmaceutical composition does not comprise a preservative. Formulations for parenteral injection can be presented in unit dosage form, for example, in ampoules, or in multi-dose containers with a preservative.

The Vδ1 T cells described herein may be present in a composition in an amount 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 × 10³ cells, at least 2 × 10³ cells, at least 3 × 10³ cells, at least 4 × 10³ cells, at least 5 × 10³ cells, at least 6 × 10³ cells, at least 7 × 10³ cells, at least 8 ×10³ cells, at least 9 ×10³ cells, at least 1 ×10⁴ cells, at least 2 × 10⁴ cells, at least 3 × 10⁴ cells, at least 4 × 10⁴ cells, at least 5 × 10⁴ cells, at least 6 × 10⁴ cells, at least 7 × 10⁴ cells, at least 8 × 10⁴ cells, at least 9 × 10⁴ cells, at least 1 × 10⁵ cells, at least 2 × 10⁵ cells, at least 3 × 10⁵ cells, at least 4 × 10⁵ cells, at least 5 × 10⁵ cells, at least 6 × 10⁵ cells, at least 7 × 10⁵ cells, at least 8 × 10⁵ cells, at least 9 × 10⁵ cells, at least 1 × 10⁶ cells, at least 2 × 10⁶ cells, at least 3 × 10⁶ cells, at least 4 × 10⁶ cells, at least 5 × 10⁶ cells, at least 6 × 10⁶ cells, at least 7 × 10⁶ cells, at least 8 × 10⁶ cells, at least 9 × 10⁶ cells, at least 1 × 10⁷ cells, at least 2 × 10⁷ cells, at least 3 × 10⁷ cells, at least 4 × 10⁷ cells, at least 5 × 10⁷ cells, at least 6 × 10⁷ cells, at least 7 × 10⁷ cells, at least 8 × 10⁷ cells, at least 9 × 10⁷ cells, at least 1 × 10⁸ cells, at least 2 × 10⁸ cells, at least 3 × 10⁸ cells, at least 4 × 10⁸ cells, at least 5 × 10⁸ cells, at least 6 × 10⁸ cells, at least 7 × 10⁸ cells, at least 8 × 10⁸ cells, at least 9 × 10⁸ cells, at least 1 × 10⁹ cells, or more.

EXAMPLES

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

Example 1: Preparing Vδ1 T cells

Vδ1 T cells were prepared under Conditions 1-3 as shown in Table 1 and explained in detail below.

Table 1 Conditions 1-3

Condition 1

Vδ1 T cells were expanded from peripheral blood mononuclear cells (PBMC) obtained from a healthy human subject.

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 media in the cultures were replenished once on Day 3.

On Day 5, cells were passaged in fresh media and transduced with the γ-retroviral or lentiviral anti-BCMA CAR construct (SEQ ID NO: 1) in combination with RetroNectin (Takara, T100A) .

Following transduction, cells were expanded in AIM-V with 10%FBS containing 70 ng/ml rIL-15 and 30 ng/ml rIFN-γ in 24-well plates. 50%of the total volume of the cell culture media in each well was replaced every other day. If viable cell culture density increased to 2×10⁶ cells/ml or above, the cell culture is diluted to 1×10⁶ cells /ml using fresh medium. The details of Condition 1 is described in detail in WO 2016/198480 A1, which is incorporated herein by reference in its entirety.

Condition 2

Vδ1 T cells were expanded from PBMC obtained from a healthy donor.

On Day 0, one million PBMC cells were stimulated with anti-Vδ1 TCR TS-1 antibodies (Thermo fisher, TCR1055) immobilized at 0.5 μg/ml per well in non-treated 24-well plates. Cells were activated and expanded in AIM-V with 10%FBS containing 100 IU/ml IL-2. The media in the cultures were replenished once on Day 3.

On Day 5, cells transduced with the γ-retroviral or lentiviral anti-BCMA CAR construct (SEQ ID NO: 1) in combination with RetroNectin (Takara, T100A) .

Following transduction, cells were expanded in the same medium. 50%of the total volume of the cell culture media in each well was replaced every other day. If viable cell culture density increased to 2×10⁶ cells/ml or above, the cell culture is diluted to 1×10⁶ cells /ml using fresh medium.

For αβT cell depletion at harvest, TCRαβ+ and CD56+ cells were depleted with a TCRαβcells depletion kit (Miltenyi, 200-070-407) and CD56+ cells depletion kit (Miltenyi, 130-050-401) as per the manufacturer’s instructions. Such method can significantly reduce the residual αβT cell and NK at harvest, therefore increasing Vδ1 T cell purity. The details of Condition 2 is described in detail in WO 2016/081518 A2, which is incorporated herein by reference in its entirety.

Condition 3

Vδ1 T cells were expanded from PBMC obtained from a healthy donor.

For αβT cell depletion, TCRαβ+ and CD56+ cells were depleted from PBMC with a TCRαβcells depletion kit (Miltenyi, 200-070-407) and CD56+ cells depletion kit (Miltenyi, 130-050-401) as per the manufacturer’s instructions. Such method can significantly reduce the residual αβT cell and NK at harvest, therefore increasing Vδ1 T cell purity.

On Day 0, one million cells (PBMC after TCRαβ depletion) were stimulated with anti-Vδ1 TCR TS-1 antibodies (Thermo fisher, TCR1055) immobilized at 0.5 μg/ml per well in non-treated 24-well plates. Cells were activated and expanded in AIM-V with 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 media in the cultures were replenished once on Day 3.

Lentivirus packing plasmid mixture including pCMV-ΔR-8.47 and pMD2. G was purchased from Addgene, and admixed with the appropriate CAR-encoding plasmid at a pre-optimized ratio with polyethylenimine. HEK293 cells were transfected with the mixture of lentivirus and CAR-constructs, and were cultured overnight. Following overnight culture the supernatant was collected. The supernatant was centrifuged to further remove cellular debris, and filtered through a 0.45μm PES filter. The virus particles were pelleted, and rinsed with pre-chilled DPBS. The virus was aliquoted and stored at -80 ℃ immediately, and the virus titer was determined by measuring supT1 cell line transduction efficiency by flow cytometric assay.

Following transduction, cells were expanded in AIM-V with 10%human platelet lysate containing 70 ng/ml rIL-15 and 30 ng/ml rIFN-γ in 24-well plates. 50%of the total volume of the cell culture media in each well was replaced every other day. If viable cell culture density increased to 2×10⁶ cells/ml or above, the cell culture is diluted to 1×10⁶ cells /ml using fresh medium. Vδ1 T cells can be cultivated in plates or dishes, such as 12-well plate, 6-well plate, 6cm dish and 10cm dish.

Characterization of the Vδ1 T cells

On Day 14, the phenotype of Vδ1 T cells was assessed by APC conjugated anti-human Vδ1 (TS8.2, Thermo Fisher, Cat#: 17-5679-42) , Brilliant Violet 421^TM conjugated anti-human TCR Vδ2 Antibody (B6, Biolegend, Cat#: 331428) , Rabbit anti-camel sdAb pAb Alexa Fluor 488 (GenScript, Cat#: C9042GH240) , PE conjugated anti-CD27 (MT271, Biolegend, Cat#: 356406) , Brilliant Violet 421^TM conjugated anti-CD45RA (HI100, Biolegend, Cat#: 304130) , PE conjugated anti-PD1 (A17188A, Biolegend, Cat#: 379210) , and Brilliant Violet 786^TM conjugated anti-TIM3 (F38-2E2, Biolegend, Cat#: 345032) .

The expansion rate data showed Condition 3 resulted in significantly better expansion (17275 fold at day 15) than Condition 1 (FIG. 1) . Purity and transduction efficiency was determined to be 97.4%and 46.0%, respectively, for retrovirus (FIGS. 2A-2B) . Purity and transduction efficiency was determined to be 98 %and 94%, respectively, for lentivirus (FIGS. 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%) (FIG. 3A) . Moreover, 84.4%of expanded CAR-Vδ1 T cells prepared under Condition 3 werephenotype (CD27 positive and CD45RA positive) , suggesting superior cell fitness (FIG. 3B)

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

To evaluate the long-term killing efficacy and persistence of Vδ1 T cells, long-term co-culture assays were performed to mimic the dynamic killing process in vivo. Transduced T cells (1×10⁵/well) were co-cultured with NCI-H929 target cells (4×10⁵ well) at an E: T ratio of 1: 3 in 24-well plates, in the absence of exogenous cytokines (e.g., IL-2) . Part of the cells were harvested and stained for CD3 after 2 or 3 days of co-culture. Vδ1 T cells were identified by CD3 and CAR signals. For serial co-culture assays, the remaining Vδ1 T cells were then re-challenged with fresh NCI-H929 target cells at the same E: T ratio. Co-cultures were carried on until the tumor cells outgrew the well volume. The Vδ1 T cell proliferation rate at each time point was calculated by dividing the number of Vδ1 T cells at the time point by the initial number of Vδ1 T cells.

The killing efficacy of Vδ1 T cells from specified conditions in the repeated tumor stimulation assay is shown in FIGS. 4A-4B. Vδ1 T cells prepared under Conditions 1 and 2 both became exhausted after 7 rounds of tumor stimulation (e.g., could not kill additional target tumor cells, as measured by CD3%) , whereas Vδ1 T cells prepared under Condition 3 persisted killing target tumor cells after 9 rounds of tumor stimulation. In addition, it has been found that the Vδ1 T cells prepared under Condition 3 proliferated and exhibited better long-term persistence than Vδ1 T cells prepared under Conditions 1 and 2.

Example 3. Evaluation of cytokine release of Vδ1 T cells

A measure of effector γδT-cell safety is the production of effector cytokines such as IFN-γand GM-CSF. Supernatants from the in vitro cytotoxicity assay were collected to assess CAR-induced cytokine release. Homogeneous Time Resolved Fluorescence (HTRF) assays for IFN-γ and GM-CSF (Cisbio) were performed according to the manufacturer’s manual.

Vδ1 T cells from specified conditions were co-cultured with NCI-H929 target cells. The culture supernatants were collected after 20h to assess IFN-γ and GM-CSF release as a measure of γδT cell safety. As shown in FIGS. 5A-5B, CAR-Vδ1 T cells prepared under Condition 3 co-cultured with target tumor cells secreted significant less amount of IFN-γ and GM-CSF, in comparison to that of Vδ1 T cells from Conditions 1 and 2. This suggested that Vδ1 T cells prepared under Condition 3 is safe for clinical use.

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

Anti-tumor activity of anti-BCMA CAR-Vδ1 T cells (prepared under Condition 3) was assessed in vivo in an RPMI-8226 xenograft model. Briefly, one million (1×10⁶) RPMI-8226 cells stably expressing the firefly luciferase reporter were implanted subcutaneously/intravenously on day 0 in NOD/SCID IL-2RγCnull (NSG) mice. Fourteen days after tumor inoculation, mice were treated with intravenous injection of 3×10⁶ CAR-Vδ1 T or mock T cells or phosphate-buffered saline (PBS) . Tumor progression was monitored by bioluminescent imaging (BLI) once a week. In addition, T cell proliferation was monitored via FACS analysis from plasma drawn from blood.

The data showed that the CAR-Vδ1 T displayed anti-tumor activity compared to vehicle control (FIG. 6) . Moreover, it was evident that anti-tumor cytotoxicity from CAR-Vδ1 T cells prepared under Condition 3 was much more profound than that of CAR-Vδ1 T cells prepared under Condition 2.

Taken together, the present disclosure provided a method to produce CAR-Vδ1 T cells with high purity, expansion and CAR transduction rate for clinical use and production. Such cells displayed high activation, low exhaustion and predominantphenotype. In vitro validation showed superior anti-tumor activity and safer profile than cells prepared under Condition 1 and 2. Further, the anti-tumor effect was further confirmed in vivo to be far superior than current art.

OTHER EMBODIMENTS

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

Claims

A method for culturing 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 comprising IL-15 and IFN-γ.
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.
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.
The method of any one of claims 1-3, wherein the IL-4 in the first culture medium has a concentration of about 100 ng/ml.
The method of any one of claims 1-4, 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.
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.
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.
The method of any one of claims 1-7, 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.
The method of any one of claims 1-8, wherein the IL-1β in the first culture medium has a concentration of about 10 to about 20 ng/ml.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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 higher than the concentration of IL-15 in the first culture medium.
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 higher than the concentration of IFN-γ in the second culture medium.
The method of any one of claims 1-21, wherein step (1) further comprises stimulating the Vδ1 T cells by a Vδ1 T cell-specific antibody.
The method of claim 22, wherein the Vδ1 T-specific antibody specifically binds to TCR delta chain.
The method of claim 22 or claim 23, wherein the Vδ1 T-specific antibody is TCR delta Monoclonal Antibody TS-1.
The method of any one of claims 22-24, wherein the Vδ1 T-specific antibody is immobilized on the cell culture plate.
The method of any one of claims 22-25, wherein the Vδ1 T-specific antibody is immobilized on the cell culture plate at 0.5 μg/ml per well in a cell culture plate.
The method of any one of claims 22-24, wherein the first culture medium comprises the Vδ1 T-specific antibody.
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 of the culture.
The method of any one of claims 1-28, wherein prior to step (1) , the sample is enriched for γδT cells.
The method of claim 29, wherein γδ T cells are enriched by (1) depleting αβT cells and optionally depleting NK cells or (2) isolating γδT cells from the sample.
The method of any one of claims 1-30, wherein αβT cells are depleted prior to step (1) .
The method of any one of claims 1-30, wherein NK cells are depleted prior to step (1) .
The method of any one of claims 1-30, wherein αβT cells are depleted between step (1) and step (2) .
The method of any one of claims 1-30, wherein NK cells are depleted between step (1) and step (2) .
The method of any one of claims 1-30, wherein αβT cells are depleted after step (2) .
The method of any one of claims 1-30, wherein NK cells are depleted after step (2) .
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.
The method of any one of claims 1-37, wherein the sample comprises peripheral blood mononuclear cells (PBMCs) .
The method of any one of claims 1-38, wherein the cells were cultured for 5-9 days during step (1) .
The method of any one of claim 39, wherein the cells were cultured for 7 days during step (1) .
The method of any one of claims 1-40, wherein the cells were cultured for 6-10 days during step (2) .
The method of claim 41, wherein the cells were cultured for 8 days during step (2) .
The method of any one of claims 1-42, wherein the cells were collected prior to 35 days of culturing.
The method of claim 43, wherein the cells were collected prior to 21 days of culturing.
The method of any one of claims 1-44, wherein the first and/or the second culture medium comprises AIM-V.
The method of any one of claims 1-45, wherein the first and/or the second culture medium comprises L-glutamine, streptomycin sulfate, and gentamicin sulfate.
The method of any one of claims 1-46, wherein the first and/or second culture media further contain serum.
The method of claim 47, wherein the serum is present in an amount from about 0.5 to about 25%by volume.
The method of claim 47 or claim 48, wherein the serum is FBS.
The method of any one of claims 1-46, wherein the first and/or second culture media further contain 10%human platelet lysate.
The method of any one of claims 1-50, wherein the IL-4 is human IL-4.
The method of any one of claims 1-51, wherein the IL-15 is human IL-15.
The method of any one of claims 1-52, wherein the IL-1β is human IL-1β.
The method of any one of claims 1-53, wherein the IFN-γ is human IFN-γ.
The method of any one of claims 1-54, wherein the cells are transduced with a vector prior to step (1) .
The method of any one of claims 1-54, wherein the cells are transduced with a vector between step (1) and step (2) .
The method of any one of claims 1-54, wherein the cells are transduced with a vector after step (2) .
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) .
The method of any one of claims 55-58, wherein the cells are transduced with a lentiviral vector or retroviral vector.
A method for preparing Vδ1 T cells, the method 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 (2) 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.
The method of claim 60, where prior to step (1) , the sample is depleted of αβT cells and/or NK cells.
The method of claim 60 or claim 61, where during step (1) the cells are exposed to a Vδ1 T-specific antibody.
The method of any one of claims 60-62, wherein prior to step (2) , the cells were transfected with a vector encoding an engineered receptor (e. g., CAR) .
The method of any one of claims 60-63, wherein the cells are cultured for 5-9 days (e.g., about 7 days) during step (1) .
The method of any one of claims 60-64, wherein the cells are cultured for 6-10 days (e.g., about 8 days) during step (2) .
A cell preparation prepared using the method of any one of the preceding claims.
A pharmaceutical composition comprising the cell preparation of claim 66, and a pharmaceutically acceptable carrier.
A method of treating a subject having cancer, the method comprising administering to the subject in need thereof a therapeutically effective amount of the cell preparation of claim 66.
The method of claim 68, wherein the subject has a solid tumor.
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 neoplasms, soft tissue sarcoma, esophageal cancers, or CNS tumors.