Methods for gamma delta t cells manufacturing

Abstract

The present disclosure relates to methods of preparing of T cells, specifically γδ T cells.

Description

METHODS FOR GAMMA DELTA T CELLS MANUFACTURING BACKGROUND

[0001] The present disclosure relates to the activation, expansion and re-stimulation of T cells, in particular of Y<5 T cells. The disclosure further provides for uses and methods of using the disclosed y6 T cells.

[0002] Y<5 T cells represent a subset of T cells expressing the ySTCR instead of the a|3TCR. Y<5 T cells can be divided into two primary subsets - the tissue-bound Vy2-negative cells and the peripheral circulating V62 positive cells, more specifically Vy962. Both subsets have been shown to have anti-viral and anti-tumor activities. Unlike the conventional a|3 TCR expressing cells, Y<5 TCR-expressing cells recognize their targets independent of the classical MHC I and II. Similarto natural killer (NK) T cells, Y<5 T cells express NKG2D, which binds to the non-classical MHC molecules, i.e., MHC class I polypeptide-related sequence A (MICA) and MHC class I polypeptide-related sequence B (MICB), present on stressed cells and / or tumor cells. ySTCRs recognize a variety of ligands, e.g., stress and / or tumor-related phosphoantigens. Y<5 T cells can mediate direct cytolysis of their targets via multiple mechanisms, i.e., TRAIL, FasL, perforin and granzyme secretion.

[0003] Engineered Y<5 T cells have shown promising results in anti-cancer immunotherapy. These cells can be genetically modified to express chimeric antigen receptors (CARs) orT cell receptors (TCRs) that enable them to recognize and attack cancer cells. CAR Y<5 T cells have been used in clinical trials to treat solid tumors, such as ovarian and lung cancer, with encouraging results. In addition, TCR-engineered gamma delta T cells have been developed to target specific tumor antigens, such as NY-ESO-1, and have shown promising results in preclinical studies. These approaches have the potential to overcome some of the limitations of conventional Y<5 T cell therapies and offer new opportunities for cancer treatment.

[0004] However, one issue with y3T cells is that they are typically found only in small quantities in the peripheral blood, making it difficult to obtain a sufficient number of pure Y<5 T cells for medical treatment. This challenge is compounded when a small amount of blood is collected and then the cells are activated and / or expanded. Increasing the amount of blood collected from a patient to obtain the necessary number of Y<5 T cells presents a significant burden to the patient. To address this challenge, clinical trials have used an autologous Vy962 T cell expansion protocol, which involves a 14-day treatment of PBMC with bisphosphonate, such as zoledronate and pamidronate, and 100 U / ml IL-2. However, this process may only result in a 100-fold increase intotal Vy952 T cells within 14 days, and thereafter, the expansion rate decreases while cell death increases. As such, the conventional Vy952 expansion protocol may not yield a sufficient number of cells to qualify as a commercially viable allogeneic product.

[0005] U. S. 7,749,760 describes a Vy9V62 T cell proliferation agent containing bisphosphonate, interleukin 2 (IL-2), and interleukin 18 (IL-18).

[0006] U. S. 8,962,313 describes a method for simultaneous proliferation of disease antigen specific cytotoxic T lymphocytes (CTLs) and y6 T cells by adding a disease antigen to isolated peripheral blood; and culturing the resultant combination in a culture media containing an interleukin.

[0007] WO 2014 / 072446 describes a method of inducing IL-2-free proliferation of y6 T cells using a combination of a y6 T cell activator and IL-33 for use in therapy of infection, cancer, autoimmunity as well as other diseases.

[0008] WO 2016 / 166544 describes y5T cells may be expanded in the presence of a phosphoantigen isopentenyl pyrophosphate (IPP) and cytokines may be provided in the step of culturing to encourage proliferation of y5T cells and to maintain cellular phenotype of the peripheral blood mononuclear cells.

[0009] U. S. 2011 / 0158954 describes a method for preparing a y5 T cell population, in which the method includes the step of culturing a cell population containing y5 T cells, in the presence of (a) fibronectin, a fibronectin fragment or a mixture thereof and (b) an activating factor of y5 T cells.

[0010] U. S. 2016 / 0175358 describes positive and / or negative selection of cell surface markers expressed on the collected y5 T cells can be used to directly isolate y5 T cells from various sources, e.g., 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. y5 T cells can be isolated from a complex sample based on positive or negative expression of CD4, CD8, TCRa, TCRp, TCR5, and other suitable cell surface markers.

[0011] There remains a need for methods that could prepare sufficient number of y5 T cells as a commercially viable therapeutic product. Specifically, there is a need for large-scale production of y5T cells for allogeneic use as off-the-shelf products. A solution to this technical problem is provided by the embodiments characterized in the claims.BRIEF SUMMARY

[0012] In an aspect, the disclosure provides fora method of preparing Y<5 T cells which includes activatin y6 T cells in the presence of an aminobisphosphonate and a cytokine composition, and expandingthe activated Y<5 T cells in the absence of an aminobisphosphonate and in the presence of the cytokine composition, wherein expanding comprises re-stimulating the Y<5 T cells with a suitable Y<5 T cell restimulation agent. Preferably, the aminobiphosphonate is zoledronate.Preferably, the Y<5 T cell restimulation agents are anti-CD3 antibodies, anti-CD28 antibodies or a combination thereof. In some embodiments, activation and / or expansion are further in the presence of L-ascorbic acid or a derivative thereof, specifically L-ascorbic acid 2-phosphate. In other words, the inventive method comprises preparing (or manufacturing or expanding) Y<5 T cells by activating Y<5 T cells in the presence of an aminobisphosphonate and a cytokine composition, and expanding the activated Y<5 T cells in the absence of an aminobisphosphonate and in the presence of the cytokine composition, a suitable Y<5 T cell restimulation agent such as anti-CD3 antibodies, anti-CD28 antibodies or a combination thereof, and optionally L-ascorbic acid or a derivative thereof, specifically L-ascorbic acid 2-phosphate. The appended Examples show that adding anti-CD3 and / or anti-CD28 antibodies and optionally phospo-Vitamin C (L-ascorbic acid 2-phosphate increases Y<5 T cell expansion and relative cell numbers. It is believed that these effects are, inter alia, based on the re-stimulation of Y<5 T cells during the expansion step. The methods described herein are preferably performed in vitro.

[0013] In an aspect, the disclosure provides fora method of preparing Y<5 T cells including activating Y<5 T cells in the presence of an aminobisphosphonate and a cytokine composition, and expandingthe activated Y<5 T cells in the absence of an aminobisphosphonate and in the presence of the cytokine composition, wherein expanding comprises re-stimulating the Y<5 T cells with a suitable Y<5 T cell restimulation agent. Preferably, the aminobiphosphonate is zoledronate.Preferably, the Y<5 T cell restimulation agents are anti-CD3 antibodies, anti-CD28 antibodies or a combination thereof. In some embodiments, activation and / or expansion are further in the presence of L-ascorbic acid or a derivative thereof, specifically L-ascorbic acid 2-phosphate.

[0014] The inventive manufacturing method can achieve from up to ~5000x up to ~50.000x Y<5 fold expansion of Y<5 T cells and overcomes the hurdle to manufacture Y<5 T cells in sufficient numbers without feeder cells. Overall, the inventive method contributes to the potential advancement of Y<5 T adoptive cell therapy including genetically modified therapy for cancer treatment. This manufacturing process may hold promise for future clinical applications, potentially improving patient outcomes.

[0015] Specifically, the present application relates to methods of manufacturing y5T cells, including activating y5T cells in the presence of an aminobisphosphonate, a cytokine composition comprising or consisting of interleukin 2 (IL-2) and interleukin 15 (IL-15), expanding the activated y5 T cells in the absence of the aminobiphosphonate and in the presence of the cytokine composition comprising or consisting of interleukin 2 (IL-2), and interleukin 15 (IL-15), wherein expanding comprises restimulating the y5 T cells with a combination of anti-CD3 antibodies and anti-CD28 antibodies.

[0016] In some embodiments, restimulating is on day 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 after activation. The anti-CD3 antibody and anti-CD28 antibody may be added to the cell culture medium or may be immobilized (i.e. bound to the cultivation plate). The anti-CD3 antibody concentration and / or said an anti-CD28 antibody concentration during restimulation may be between about 0.1 pg / ml and about 2 pg / ml, such as about 0.25, 0.5.,0.75, 1.0, 1.25, 1.5, 1.75 or 2 pg / ml.

[0017] In some embodiments, activation and / or expansion (incl. restimulation) is further in the presence of L-ascorbic acid or a derivative thereof, specifically L-ascorbic acid 2-phosphate (also referred to as phospho-Vitamin C or pVC). In some embodiments, L-ascorbic acid 2-phosphate is used during activation and / or expansion ata concentration of 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975 or 1000 pg / mL pg / mL. Typically, L-ascorbic acid 2-phosphate is added to the cell culture medium during activation and / or expansion (incl. restimulation).

[0018] In some embodiments, the blood sample comprises or consists of peripheral blood mononuclear cells (PBMC) and / or a leukapheresis product, e.g., LeukoPak®, of a subject, for example, a human subject. In some embodiments, the methods comprise obtaining a blood sample from a subject, such as a human subject. In some embodiments, the methods do not comprise a step of treatment or interference of / with the (human) body.

[0019] In some embodiments, enriching includes contacting the leukapheresis product or PBMC with anti-a and / or anti-p T cell receptor (TCR) antibodies, for example, biotin- or magnetic bead-conjugated anti-ap TCR antibodies, for depleting a- and / or p-TCR positive cells from the PBMC, for example, by using streptavidin-microbeads or magnet. In some embodiments, a- and / or p-TCR positive cells include ap T cells and natural killer T (NKT) cells.

[0020] In some embodiments, the aminobisphosphonate may be zoledronate, pamidronate, alendronate, risedronate, ibandronate, incadronate, clodronate, etidronate, or neridronate, a salt thereof and / or a hydrate thereof. Preferably, the only aminobisphosphonate utilized in methods described herein is zoledronate.

[0021] In some embodiments, the activation and / or expansion (specifically restimulation) is in the absence of a phosphoantigen, in particular (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), bromohydrin pyrophosphate (BrHPP) isoprenoid pyrophosphates (farnesyl pyrophosphate (FPP), geranylgeranyl pyrophosphate (GGPP), isopentenyl pyrophosphate (IPP), or dimethylallyl diphosphate (DMAPP).

[0022] The method includes activating y6 T cells in the presence of an aminobisphosphonate, in particular zoledronate, and a cytokine composition comprising or consisting of IL-2 and IL-15. Preferably, the cytokine composition consists of IL-2 and IL-15, meaning that no other interleukins are added as the y6 T cells are activated.

[0023] The aminobisphosphonate, in particular zoledronate, may be at a concentration from aboutO.1 pM to about 500 pM, from about 0.1 pM to about 400 pM, from about 0.1 pM toabout300 pM, from about 0.1 pM to about 200 pM, from about 0.1 pM to about 100 pM, from about 0.5 pM to about 100 pM, from about 1 pM to about 500 pM, from about 1 pM to about 400 pM, from about 1 pM to about 300 pM, from about 1 pM to about 200 pM, from about 1 pM to about 100 pM, from about 1 pM to about 90 pM, from about 1 pM to about 80 pM, from about 1 pM to about 70 pM, from about 1 pM to about 60 pM, from about 1 pM to about 50 pM, from about 1 pM to about 40 pM, from about 1 pM to about 30 pM, from about 1 pM to about 20 pM, from about 1 pM to about 15 pM, from about l pM to about 10 pM, from about 1 pM to about 9 pM, from about 1 pM to about 8 pM, from about 1 pM to about 7 pM, from about 1 pM to about 6 pM, from about 1 pM to about 5 pM, from about 2 pM to about 5 pM, from about 3 pM to about 5 pM, from about 2 pM to about 5 pM, from about 2 pM to about 10 pM, from about 3 pM to about 8 pM, or from about 4 pM to about 6 pM.

[0024] In some embodiments, the zoledronate concentration during activation is between about 1 pM to about 50 pM, such as about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 pM.

[0025] In some embodiments, the seeding density of the enriched y5 T cells during activation is from about 0.01 x 106cells / cm2to about 1 x 107cells / cm2, from about 0.1 x 106cells / cm2to about 5 x 106cells / cm2, from about 0.25 x 106cells / cm2to about 5 x 106cells / cm2, from about 0.5 x 106cells / cm2to about 5 x 106cells / cm2, from about 0.5 x 106cells / cm2to about 4 x 106cells / cm2, from about 0.5 x 106cells / cm2to about 3 x 106cells / cm2, from about 0.5 x 106cells / cm2to about 2.5 x 106cells / cm2, from about 0.5 x 106cells / cm2to about 2 x 106cells / cm2, from about 0.6 x 106cells / cm2to about 2 x 106cells / cm2, from about 0.7 x 106cells / cm2to about 2 x 106cells / cm2, from about 0.8 x 106cells / cm2to about 2 x 106cells / cm2, from about 0.9 x 106cells / cm2to about 2 x 106cells / cm2, from about 1 x 106cells / cm2to about 2 x 106cells / cm2, or from about 1.5 x 106cells / cm2to about 2 x 106cells / cm2.

[0026] In some embodiments, the seeding density of the enriched y5 T cells during activation is from about 0.5 x 106cells / cm2to about 3 x 106cells / cm2. In some embodiments, the seeding density of the enriched y5 T cells during activation is 1 x 106cells / cm2.

[0027] In some embodiments, the concentration of IL-2 during activation and / or expansion is from about 5 lU / ml to about 1000 lU / ml, from about 5 lU / ml to about 500 lU / ml, from about 5 lU / ml to about 400 lU / ml, from about 5 lU / ml to about 300 lU / ml, from about 5 lU / ml to about 200 lU / ml, from about 5 lU / ml to about 150 lU / ml, from about 5 lU / ml to about 100 lU / ml, from about 10 lU / ml to about 100 lU / ml, from about 20 lU / ml to about 100 lU / ml, from about 30 lU / ml to about 100 lU / ml, from about 40 lU / ml to about 100 lU / ml, from about 50 lU / ml to about 125 lU / ml, from about 20 lU / ml to about 80 lU / ml, from about 25 lU / ml to about 60 lU / ml. In some embodiments, the IL-2 concentration during activation and / or expansion is between about 5 lU / ml and about 200 lU / ml, such as about 5, 10, 25, 50, 75, 100, 125, 150, 175 or 200 lU / ml.

[0028] In some embodiments, the concentration of IL-15 during activation and / or expansion is from about 5 ng / ml to about 200 ng / ml. some embodiments, the concentration of IL-15 during activation and / or expansion is from about 5 ng / ml to about 1 pg / ml, from about 5 ng / ml to about 500 ng / ml, from about 5 ng / ml to about 400 ng / ml, from about 5 ng / ml to about 300 ng / ml, from about 5 ng / ml to about 200 ng / ml, from about 5 ng / ml to about 150 ng / ml, from about 10 ng / ml to about 100 ng / ml, from about 20 ng / ml to about 100 ng / ml, from about 30 ng / ml to about 100 ng / ml, from about 40 ng / ml to about 100 ng / ml, from about 20 ng / ml to about 80 ng / ml, from about 30 ng / ml to about 60 ng / ml, or from about 50 ng / ml to about 120 ng / ml.

[0029] In some embodiments, the concentration of IL-15 during activation and / or expansion is from about 50 ng / ml to about 100 ng / ml.

[0030] In some embodiments, the IL-15 concentration during activation and / or expansion is between about 10 ng / ml and about 200 ng / ml, such as about 10, 25, 50, 75, 100, 125, 150, 175 or 200 ng / ml.

[0031] In some embodiments, activation is in the presence of zoledronic acid at a concentration of about 1 pM to about 100 pM, IL-2 at a concentration from about 10 lU / ml to about200 I U / ml, and IL-15 at a concentration of about 10-500 ng / ml. In some embodiments, expansion is in the presence of IL-2 at a concentration from about 10 lU / ml to about 100 lU / ml and / or IL-15 at a concentration of about 10 - 200 ng / ml.

[0032] In some embodiments, cytokines used in activation and / or expansion may further include IL-18, IL-21, and IL-7. Preferably however, methods described herein exclude one or more of IL-12, IL-7. IL-21, IL-18, IL-19, IL-33, IL-4, IL-9, and / or IL-23 during expansion, activation, or both expansion and activation.

[0033] In some embodiments, activation takes no more than about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, 7 days, about 10 days, about 12 days, about 14 days, about 16 days, or about 20 days.

[0034] In some embodiments, activation takes from about 1 day to about 10 days, from about 1 day to about 9 days, from about 1 day to about 8 days, from about 1 day to about 7 days, from about 1 day to about 6 days, from about 1 day to about 5 days, from about 1 day to about 4 days, from 1 day to about 3 days, from 1 day to about 2 days from about 2 days to about 10 days, from about 2 days to about 8 days, from about 2 days to about 7 days, about 3 days to about 7 days, or about 4 to about 6 days.

[0035] Preferably, activation takes from about 1 day to about 2, 3, 4, 5, 6 or 7 days.

[0036] The method further includes expandingyST cells in the absence of an aminobisphosphonate, in particular zoledronate, and in the presence of a cytokine composition comprising or consisting of IL-2 and IL-15. Preferably, the cytokine composition is present in the cell culture medium. Preferably, the cytokine composition consists of IL-2 and IL-15, meaning that no other interleukins are added to the cell culture medium during expansion.

[0037] In some embodiments, expansion takes more than about 7 days, about 10 days, about 12 days, about 14 days, about 16 days, about 20 days, about 22 days, about 24 days, about 26 days, about 28 days, or about 30 days. In some embodiments, a duration of expansion is from about 7 days to about 30 days, from about 10 days to about 25 days, from about 12 days to about 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 days.

[0038] In some embodiments, the cell density of the y5 T cells during expansion is from about 0.1 x 106cells / cm2to about 5 x 106cells / cm2, from about 0.25 x 106cells / cm2to about 5 x 106cells / cm2, from about 0.5 x 106cells / cm2to about 5 x 106cells / cm2, from about 0.5 x 106cells / cm2to about 4 x 106cells / cm2, from about 0.5 x 106cells / cm2to about 3 x 106cells / cm2, from about 0.5x 106cells / cm2to about 2.5 x 106cells / cm2, from about 0.5 x 106cells / cm2to about 2 x 106cells / cm2, from about 0.6 x 106cells / cm2to about 2 x 106cells / cm2, from about 0.7 x 106cells / cm2to about 2 x 106cells / cm2, from about 0.8 x 106cells / cm2to about 2 x 106cells / cm2, from about 0.9 x 106cells / cm2to about 2 x 106cells / cm2, from about 1 x 106cells / cm2to about 2 x 106cells / cm2, or from about 1.5 x 106cells / cm2to about 2 x 106cells / cm2. In some embodiments, the cell density of the y5T cells during expansion is about 1 x 106cells / cm2.

[0039] In some embodiments, restimulation includes reseedin the cells during expansion into a culture vessel coated with anti-CD3 and anti-CD28 antibodies. In some embodiments, restimulation takes about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, 7 days, about 10 days, about 12 days, about 14 days, about 16 days, about 18 days or about 20 days.

[0040] In some embodiments, restimulation takes from about 2 days to about 12 days, from about 2 days to about 11 days, from about 2 days to about 10 days, from about 2 days to about 9 days, from about 2 days to about 8 days, from about 2 day to about 7 days, from about 2 days to about 6 days, from about 2 days to about 5 days, from about 2 days to about 4 days, or from about 2 days to about 3 days.

[0041] Preferably, restimulation takes from about 2 days to about 18 days.

[0042] The present disclosure further provides a population of expanded and activated y5 T cells prepared by the method of any described herein.

[0043] In some embodiments, the method of the invention includes transducing the y5 T cells with a recombinant viral vector. In some embodiments, the y5T cells are transduced after activation and are subsequently expanded and restimulated.

[0044] In some embodiments, the viral vector expresses an ap-TCR and optionally CD8.

[0045] In some aspects, the present disclosure provides transduced (i.e., engineered) y5 T cells prepared by the method of the present invention.

[0046] In some aspects, the present disclosure relates to a population of y5 T cells prepared by the method of the present disclosure, in which the concentration of the expanded y5 T cells is at least about 1 x 105total cells / ml, at least about 1 x 106total cells / ml, at least about 1 x 107total cells / ml, at least about 1 x 108total cells / ml, or at least about 1 x 109total cells / ml.

[0047] In some aspects, the present disclosure provides a method of treating cancer, comprising administering to a patient in need thereof an effective amount of the engineered y6 T cell prepared by the method of the present disclosure.

[0048] In some embodiments, the cancer is selected from the group consisting of acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancers, AIDS-related lymphoma, anal cancer, appendix cancer, astrocytomas, neuroblastoma, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancers, brain tumors, such as cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumors, visual pathway and hypothalamic glioma, breast cancer, bronchial adenomas, Burkitt lymphoma, carcinoma of unknown primary origin, central nervous system lymphoma, cerebellar astrocytoma, cervical cancer, childhood cancers, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorders, colon cancer, cutaneous T-cell lymphoma, desmoplastic small round cell tumor, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma, germ cell tumors, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gliomas, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular (liver) cancer, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell carcinoma, Kaposi sarcoma, kidney cancer, laryngeal cancer, lip and oral cavity cancer, liposarcoma, liver cancer, lung cancers, such as non-small cell and small cell lung cancer, lymphomas, leukemias, macroglobulinemia, malignant fibrous histiocytoma of bone / osteosarcoma, medulloblastoma, melanomas, mesothelioma, metastatic squamous neck cancer with occult primary, mouth cancer, multiple endocrine neoplasia syndrome, myelodysplastic syndromes, myeloid leukemia, nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, oral cancer, oropharyngeal cancer, osteosarcoma / malignant fibrous histiocytoma of bone, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, pancreatic cancer, pancreatic cancer islet cell, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal germinoma, pituitary adenoma, pleuropulmonary blastoma, plasma cell neoplasia, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma, renal pelvis and ureter transitional cell cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcomas, skin cancers, Merkel cell skin carcinoma, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, stomach cancer, T-cell lymphoma, throat cancer, thymoma, thymic carcinoma, thyroid cancer, trophoblastic tumor (gestational), cancers of unknown primarysite, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom’s macroglobulinemia, and Wilms tumor.

[0049] In some embodiments, the cancer is melanoma.BRIEF DESCRIPTION OF THE DRAWINGS

[0050] FIG.1A shows depletion of a|3 T cells from PBMC. PBMCs were incubated with biotin-conjugated ap TOR antibodies, followed by streptavidin-microbeads per manufacturer protocol. Samples were then passed through LS column to enrich for a|3 TCR-expressing cells. The column flow-through represents the a|3 TOR depleted fractions. After overnight culture, the a|3 TCR-enriched fraction and the a|3 TCR-depleted fraction were stained with fluorochrome-conjugated a|3 TCR antibody versus Vy9 antibody, followed by flow cytometry analysis.

[0051] FIGS.1B and 1C show minimal residual a|3T cells in Vy962T cell product. a|3 T cells were depleted from PBMC of normal donors (Donor A, Donor B, and Donor C) (FIG. 1 B) and (Donor 12 and Donor 13) (FIG. 1 C) using commercially available biotinylated anti a|3TCR antibody / streptavidin microbeads. a|3T cell-depleted PBMC were cultured with zoledronate / IL-2 / IL-15 for 14 days, followed by cell surface staining with respective fluorochrome conjugated antibodies, e.g., anti a|3 TCR antibodies and anti-yS TCR antibodies to assess for residual a|3 T cells and enriched y6 T cells by sub-gating on CD3.

[0052] FIG.1D shows cytokine profiling of Vy962 T cells. Vy962 cells were treated with Golgi Stop / Plug (i.e., protein transport inhibitors) for 6 hours prior to cell harvest. Cells were stained for surface V62 followed by fixation and permeabilization. Staining intracellular TNF-a, IL-17a, and IFN-ywere performed using fluorochrome-conjugated antibodies against TNF-a, IL-17a, and IFN-y.

[0053] FIG.1E shows depletion of a|3 T cells from a leukapheresis product, e.g., LeukoPak®, according to another embodiment of the disclosure. White blood cells including dendritic and progenitor cells from leukapheresis product may be incubated with biotin-conjugated a|3 TCR antibodies, followed by streptavidin-microbeads. Samples were then passed through LS column to enrich for a|3 TCR-expressing cells. The column flow-through represents the a|3 TCR depleted fractions. After overnight culture, the a|3 TCR-depleted fractions were stained with fluorochrome-conjugated apTCR antibody versus y6 TCR antibody, followed by flow cytometry analysis

[0054] FIG.2 shows effects of molecules on activation of Vy962 T cells. During the activation step, aminobisphosphonate, e.g., zoledronate (ZA), and cytokines, e.g., IL-2 and / or IL-15, may be present.

[0055] FIG.3 shows the effects of molecules on expansion of Vy952 T cells. During the expansion step, cytokines may continue to be present without aminobisphosphonate.

[0056] FIG.4A shows effects of cell density on T cell markers during expansion without restimulation. After activation in the presence of zoledronate, IL-2, and IL-15 for 14 days, Vy952 T cells were expanded by homeostatic cytokines, e.g., IL-2 and IL-15, in the absence of zoledronate at high density (e.g., 2 x 106total cells / ml) and at low density (e.g., 0.5 x 106total cells / ml). T cell markers, e.g., CD122, CD80, CD83, CD86, CD95, and CD95L, were analyzed.

[0057] FIG.4B shows the effects of cell density on cell death during expansion without restimulation. After activation in the presence of zoledronate, IL-2, and IL-15 for 14 days, T cells were expanded by homeostatic cytokines, e.g., IL-2 and IL-15, in the absence of zoledronate at high density (e.g., 2 x 106total cells / ml) and at low density (e.g., 0.5 x 106total cells / ml). Cell death was measured.

[0058] FIG.5 shows the effects of Amphotericin B on V52 T cells expressing IL-2Ra according to an embodiment of the disclosure. ap-TCR depleted PBMCs were cultured in Activation Medium supplemented with Zoledronate, IL-2, and IL-15 on Day 0. After 48 hours, Amphotericin B was added and 48 hours later, cells were harvested for flow cytometry based analysis of CD25 (or IL-2Ra) surface expression on CD3γVδ2 T cells. The use of Amphotericin B may not be required in the inventive method.

[0059] FIG.6 shows effects of Zoledronate (Zometa) on cell expansion in a comparative method. y5 T cell were expanded using Zoledronate (Zometa) in defined medium containing IL-2, IL-15, and Amphotericin B. The methods of the present disclosure may not require the use of Amphotericin B.

[0060] FIG.7 shows an exemplary time table for viral transduction into Vy952T cells. On Day 1, fresh PBMC were depleted of a|3 T cells and activated with Zoledronate in the presence of IL-2 and IL-15; 24 hours later (on Day 2), cells were transduced using respective viral supernatant at MOI of 1.5; GFP transgene expression was assessed by flow cytometry on Day 7 (i. e., day 5 posttransduction) for transduction efficiency; and on Day 12 (i.e., day 10 post-transduction) for persistent transgene expression.

[0061] FIG.8A shows viral transgene expression in Vy952 T cells using a y-retroviral vector. Different envelop protein-expressing viruses, e.g., green fluorescent protein (GFP)-expressing y-retrovirus (e.g., Gibbon Ape Leukemia Virus (GALV) pseudotype (for example, SEQ ID NO: 4), RD114TR pseudotype (SEQ ID NO: 1), and GFP-expressing lentivirus (e.g., VSV-G pseudotype (forexample, SEQ ID NO: 3)) were tested for their transduction efficiency into Vy962T cells at Day 5 and Day 10 post-transduction.

[0062] FIG.8B shows viral transgene, e.g., CD8a, expression in Vy962 T cells transduced by an RD114TR or a VSV-G pseudotyped lentiviral vector.

[0063] FIG.9A shows transduction ofyST cells with CD8a|3 using different transduction enhancers (RetroNectin® vs. Vectofusin-1®) during the transduction process. y6T cells obtained from 3 donors (Donor 1, Donor 2, and Donor 3) were transduced with a retrovirus encoding CD8a|3 in the presence of RetroNectin® (a fibronectin fragment coated onto plates) or VectoFusin-1® (a soluble cationic peptide), followed by flow cytometry to determine the % of CD8a|3+ cells.

[0064] FIG.9B shows transduction efficiency of Vy962 T cells with engineered viruses. Vy962 T cells were transduced without virus (Mock) or transduced with a|3TCR virus alone, with CD8 virus alone, or with a|3TCR virus + CD8 virus. Transduced cells were incubated with TAA / MHC-PE dextramer, anti-CD8 antibody, or NYESO-PE dextramer (negative control), followed by flow cytometry analysis.

[0065] FIG.10 shows fold-expansion of Vy962 T cells transduced with engineered viruses. Vy962 T cells (GD) or a|3T cells (AB) were transduced without virus (Mock), with a|3 TCR virus (TCR), with CD8 virus (CD8), or with CD8 + TCR, followed by measurement of fold expansion from day 7 to day 21 post-transduction.

[0066] FIG.11 A shows engineered Vy962 T cells according to an embodiment of the disclosure. Vy962 T cells transduced without virus (Mock) or with a|3 TCR retrovirus and CD8a|3 retrovirus (a|3TCR + CD8) were incubated with TAA / MHC complex, followed by flow cytometry analysis to detect Vy962 T cells that bind to TAA / MHC complex.

[0067] FIG.11 B shows effector functions of engineered Vy962 T cells according to an embodiment of the disclosure. Vy962 T cells transduced without virus (Mock) or with ap-TCR retrovirus and CD8a|3 retrovirus (a|3TCR + CD8) were incubated with target cells, followed by flow cytometry analysis to detect CD107a, an apoptosis marker.

[0068] FIG.11C shows effector functions of engineered Vy962 T cells. Vy962 T cells transduced without virus (Mock) orwith a|3TCR retrovirus and CD8a|3 retrovirus (a|3TCR + CD8) were incubated with target cells, followed by flow cytometry analysis to detect IFN-y release.

[0069] FIG.11 D shows cytolytic activity of engineered Vy962 T cells. Vy962 T cells transduced without virus (Mock) or with ap TCR retrovirus and CD8ap retrovirus (apTCR + CD8) were incubated with target cells, followed by flow cytometry analysis to detect apoptotic cells.

[0070] FIG.11 E shows prolonged cytolytic activity of engineered y6 T cells. Cytolytic activity was evaluated in real-time during an 84-hour co-culture assay. Target positive A375-RFP tumor cells were incubated without T cells (tumor cells) or with non-transduced cells (apT cells and y6T cells), with apT cells transduced with ap-TCR virus and CD8ap virus (apTCR + apT cells), or with y6 T cells transduced with ap-TCR virus and CD8ap virus (apTCR + y6 T cells), followed by IncuCyte® live cell analysis to measure target cell growth.

[0071] FIG.12A shows a schematic of an engineered virus. CD8 / CD4 chimeric receptor-T2A-truncated CSF1 R contains CD8a extracellular domain linked to CD4 transmembrane and intracellular domain.

[0072] FIG.12B shows a schematic of an engineered virus. CD8 / CD4 chimeric receptor-T2A-CSF1 R / 41 BB chimeric receptor contains CSF1 R extracellular domain linked downstream from chimeric CD8 / CD4 protein.

[0073] FIG.13 shows allogenic T cell therapy. Allogenic T cell therapy may include collecting y6 T cells from healthy donors, engineeringyST cells by viral transduction with exogenous genes of interest, such as exogenous TCRs, followed by cell expansion, harvesting the expanded engineered y6 T cells, which may be cryopreserved as “off-the-shelf” T-cell products, before infusing into patients.

[0074] FIG.14 shows an exemplary y6 T cell manufacturing process. y6T cell manufacturing may include collecting or obtaining white blood cells, e.g. from a buffy coat, PBMC, ora leukapheresis product (e.g. comprising white blood cells, plasma, and platelets), depleting apT cells from PBMC or leukapheresis product, followed by activation, transduction, and expansion of y6T cells.

[0075] FIG. 15 shows the effects of phospho-Vitamin C on y5T cells. y5T cells were manufactured from healthy donor starting material using different media in the presence or absence of phospho Vitamin C (pVC). Manufacturing metrics including A) harvest total cell viability (FIG. 15A), B) cumulative y5T cells fold expansion (from day 0 to harvest) (FIG. 15B), C) purity of y5 T cells, and V51 and V52 subsets (FIG. 15C) and D) frequency of NK cells as measured by flow cytometry (FIG. 15D) are shown. Pooled data from n = 9 healthy donors (5 donors repeated 2-5x). E) y5T cells were co-cultured with T98g-RFP+ tumor cell line at an E: T ratio of 8:1 for 14 days, every 3-5 days T cells were rechallenged with fresh tumor cells and tumor fold growth (normalized to day 0) was analyzed using IncuCyte® live-cell analysis system (FIG. 15 E). n = 3 healthy donors. Data shown as mean ± SEM. ns-not significant, **p<0.01 by one-way ANOVA (A, B, D) or two-way ANOVA (C, E) with Tukey’s test for multiple comparisons.

[0076] FIG.16 shows the effects of restimulation with anti-CD3 and anti-CD28 antibodies on y6 T cells. y6 T cells were manufactured from healthy donor starting material using media + pVC with standard zoledronate and cytokine concentrations. Cells were harvested either at the end of standard manufacturing (standard harvest) or a later time point with or without restimulating using aCD3 / CD28 monoclonal antibodies. A) Schematic representation of study design. Manufacturing metrics including B) harvest total cell viability, C) cumulative y6T cells fold expansion (from day 0 to harvest) and D) purity of y6 T cells, and V61 and V62 subsets as measured by flow cytometry are shown. Pooled data from n = 6 healthy donors (1 repeated 2x). Data shown as mean ± SEM. ns-not significant, *p<0.05, **p<0.01 by one-way ANOVA (B, C) or two-way ANOVA (D) with Tukey’s test for multiple comparisons.DETAILED DESCRIPTION

[0077] The present disclosure provides methods that are suitable for large-scale manufacturing of y6T cells. The y6T cells are useful in allogeneic T cell therapy but are difficult to obtain in sufficient quantities. The restimulation step of the inventive method helps overcoming these problems and can produce large-scale, optionallyGMP-grade, engineered Vy962 T cells for research or therapy.

[0078] Embodiments of the present disclosure may include methods that can maximize the yield of y6T cells while minimizing the presence of residual a|3T cells in the final allogeneic products.

[0079] An exemplaryapproach for adoptive allogeneic T cell therapy with “off-the-shelf” y6T cells is shown in FIG. 13. This approach may include collecting y6 T cells from healthy donors, activating and engineering y6 T cells, followed by cell expansion, harvesting the expanded engineered y6 T cells, which may be cryopreserved as “off-the-shelf” T-cell products, and infusing the y6 T cells into patients. This approach may eliminate the need for personalized T cell manufacturing.

[0080] y6 T cells may be enriched from a subject or from a sample of a subject. In some embodiments, a sample may be a peripheral blood sample, a cord blood sample, a tumor, a stem cell precursor, a tumor biopsy, a tissue, a lymph, or from epithelial sites of a subject directlycontactingthe external milieu or derived from stem precursor cells. Preferably, the sample is a leukapheresis product. Preferably, y6 T cells may be directly enriched in the sample, for example, by depleting (i.e., removing) a0TCR+ cells from the sample. The a0 TCR+ cells or ap TCR+ fraction are also referred to herein as a0T cells (although they might also comprise other, smaller fractions of cell types, such as NKT cells). The term “enriching” as used herein refers to increasing the relative numbers of y6 T cells in the sample, preferably by removing other, unwanted cell types, typically a0 TCR+ cells, via positive and / or negative selection of (e,g,, a0 T cell) cell surface markers. Alternatively, y6 T cells may be enriched from a sample by positive selection for y6 T cell surface markers. The enriched y6 T cells (or: enriched y6 T cell fraction) may still contain other cell types, that are either irrelevant or even helpful during y6 T cell manufacturing. For instance, other cell types in the enriched y6 T cell fraction may include monocytes.

[0081] Alternatively, y6 T cells s may be isolated (i.e., purified) from the sample, e.g. via positive and / or negative selection of cell surface markers. For instance, y6 T cells can be isolated from a sample based on positive or negative expression of CD2, CD3, CD4, CD8, CD24, CD25, CD44, Kit, TCR a, TCR 0, TCR a, TCR 6, NKG2D, CD70, CD27, CD30, CD16, CD337 (NKp30), CD336 (NKp46), 0X40, CD46, CCR7, and other suitable cell surface markers.

[0082] Peripheral blood mononuclear cells can be collected from a subject, for example, with an apheresis machine, includingthe Ficoll-Paque™ PLUS (GE Healthcare) system, oranother suitable device / system. y6T-cell(s), ora desired subpopulation of y6 T-cell(s), can be enriched from the collected sample with, for example, with flow cytometry techniques.

[0083] In some embodiments, y6 T cells may be enriched or isolated from a sample that is cultured in vitro. In some embodiments, whole PBMC populations, without prior depletion of specific cell populations, such as monocytes, a0 T-cells, B-cells, and NK cells, can be activated and expanded. In some embodiments, enriched y6 T cell populations can be generated priorto their specific activation and expansion. In some embodiments, activation, expansion and restimulation of y6T cells may be performed without the presence of native or engineered antigen presenting cells (APCs). In some embodiments, the isolation and expansion of y6T cells can be performed in the absence of y6T cell mitogens, including antibodies specific to y6 TCR, and other y6 TCR activating agents, including lectins or phosphoantigens.

[0084] In some embodiments, y6 T cells are enriched from a leukapheresis sample of a subject, for example, a human subject. In some embodiments, y6 T cells are not isolated from peripheral blood mononuclear cells (PBMC).

[0085] To enrich y6 T cells by depleting a|3 T cells from PBMC or a leukapheresis product, a|3 TCR-expressing cells may be separated from the PBMC or the leukapharesis product by magnetic separation, e.g., using CliniMACS® magnetic beads coated with anti-ap TCR antibodies, followed by cryopreserving a|3 TCR-T cells depleted fraction. To manufacture “off-the-shelf” T-cell products, cryopreserved a|3 TCR-T cells depleted fraction may be thawed and activated in small / mid-scale, e.g., 24 to 4-6 well plates orT75 / T175 flasks, or in large scale, e.g., 50 ml-100 liter bags, in the presence of aminobisphosphonate, interleukin 2 (IL-2) and interleukin 15 (IL-15), for 1 - 10 days, e.g., 2 -6 days.

[0086] In some embodiments, the method of the invention does not include the use of y5 T cell antigens during activation, expansion and / or restimulation, including prenyl-pyrophosphates, such as isopentenyl pyrophosphate (IPP), alkyl-amines, metabolites of human microbial pathogens, metabolites of commensal bacteria, methyl-3-butenyl-1 -pyrophosphate (2M3B1 PP), (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), ethyl pyrophosphate (EPP), farnesyl pyrophosphate (FPP), dimethylallyl phosphate (DMAP), dimethylallyl pyrophosphate (DMAPP), ethyl-adenosine triphosphate (EPPPA), geranyl pyrophosphate (GPP), geranylgeranyl pyrophosphate (GGPP), isopentenyl-adenosine triphosphate (IPPPA), monoethyl phosphate (MEP), monoethyl pyrophosphate (MEPP), 3-formyl-1-butyl-pyrophosphate (TUBAg 1), X-pyrophosphate (TUBAg 2), 3-formyl-1-butyl-uridine triphosphate (TUBAg 3), 3-formyl-1-butyl-deoxythymidine triphosphate (TUBAg 4), monoethyl alkylamines, allyl pyrophosphate, crotoyl pyrophosphate, dimethylallyl-y-uridine triphosphate, crotoyl-y-uridine triphosphate, allyl-y-uridi ne triphosphate, ethylamine, isobutylamine, sec-butylamine, iso-amylamine and nitrogen containing bisphosphonates.

[0087] In some embodiments, the method includes an additional step of transducing the y5T-cells with a vector to generate engineered y5 T-cells. For instance, y6T cell can be engineered to provide a universal allogeneic therapy that recognizes an antigen of choice in vivo. To this end, y6 T cells may be transduced with a suitable vector, e.g. a viral vector. Genetic engineering of the y5 T-cells may include stably integrating a construct expressing a tumor recognition moiety, such as a|3 TCR, y5 TCR, chimeric antigen receptor (CAR), or an antigen binding fragment thereof

[0088] In some embodiments, the viral vector is selected from parvoviruses, such as adeno-associated viruses (AAVs) and retroviruses, such as lentivi ruses.

[0089] Preferably, engineered (transduced) y5 T cells are expanded and restimulated ex vivo without stimulation by an antigen presenting cell or aminobisphosphonate. In some embodiments, a population of engineered y6 T cells may be expanded and restimulated ex vivo without antigen stimulation by an antigen presenting cell, an antigenic peptide, a non-peptide molecule, ora smallmolecule compound, such as an aminobisphosphonate. In some embodiments, a population of engineered y6 T cells can be expanded in less than 60 days, less than 48 days, less than 36 days, less than 24 days, less than 12 days, less than 10 days, less than 8 days, less than 7 days, less than 6 days or less than 5 days.

[0090] In some embodiments, the present disclosure provides methods for the ex vivo manufacturing of a population of engineered y6 T-cells for adoptive transfer therapy without coculture with antigen-presenting cells (APCs), y6 T cell antigens, and aminophosphates during activation, expansion and / or restimulation.

[0091] In some embodiments, a y6 T-cell population can be expanded in vitro in fewer than 36 days, fewer than 35 days, fewer than 34 days, fewer than 33 days, fewer than 32 days, fewer than 31 days, fewer than 30 days, fewer than 29 days, fewer than 28 days, fewer than 27 days, fewer than 26 days, fewer than 25 days, fewer than 24 days, fewer than 23 days, fewer than 22 days, fewer than 21 days, fewer than 20 days, fewer than 19 days, fewer than 18 days, fewer than 17 days, fewer than 16 days, fewer than 15 days, fewer than 14 days, fewer than 13 days, fewer than 12 days, fewer than 11 days, fewer than 10 days, fewer than 9 days, fewer than 8 days, fewer than 7 days, fewer than 6 days, fewer than 5 days, fewer than 4 days, or fewer than 3 days.

[0092] The expansion of the engineered y6 T cells may be carried out in small / mid-scale, e.g., flasks / G-Rex, or in large scale, e.g., 50 ml-100-liter bags, for 7-35 days, e.g., 14-28 days. The expanded engineered y6 T cells may then be cryopreserved as “off-the-shelf” T-cell products for infusion into patients.

[0093] In a preferred embodiment, the method includes providing a blood sample from a human subject, such as a leukapheresis sample, and depleting a|3 TCR+ cells from the sample to obtain a y5 T cell enriched fraction, activate the y6 T cells for 1 -2 days with

[0094] 5, 10, 15, 20, 25, 50, or 75 lU / ml IL-2,

[0095] 5, 10, 15, 20, 25, 50, or75 ng / ml IL-15 and

[0096] 1, 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 pM zoledronate,

[0097] optionally in the presence of 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or 500 pg / ml L-ascorbic acid 2-phosphate;

[0098] transduce the activated y6 T cells with a suitable viral vector;

[0099] expand the transduced y6 T cells for 12 to 28 days in the presence of

[0100] 5, 10, 15, 20, 25, 50, or75 lU / ml IL-2,

[0101] 5, 10, 15, 20, 25, 50, or75 ng / ml IL-15 and optionally

[0102] 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or 500 pg / ml L-ascorbic acid 2-phosphate;

[0103] wherein expanding comprises restimulating the y5 T cells with plate-bound anti-CD3 and anti-CD28 antibodies on day 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20or21 after activation.

[0104] In such embodiments, activated and transduced y5 T cells can for instance be expanded in the presence of IL-2 and IL-15 and optionally L-ascorbic acid 2-phosphate (but not zoledronate) for about 8 days, reseeded onto anti-CD3 / anti-CD28 coated culture vessels (i.e., restimulated), and expanded foran additional period of time, such as about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 days to achieve a total expansion of from about 12 days to about 28 days.

[0105] In another preferred embodiment, the method includes providing a blood sample from a human subject, such as a leukapheresis sample, and depleting a|3 TCR+ cells from the sample to obtain a y5 T cell enriched fraction, activate the y5 T cells for 5-7 days with

[0106] 5, 10, 15, 20, 25, 50, or75 lU / ml IL-2,

[0107] 5, 10, 15, 20, 25, 50, or75 ng / ml IL-15 and

[0108] 1, 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 pM zoledronate,

[0109] optionally in the presence of 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or 500 pg / ml L-ascorbic acid 2-phosphate;

[0110] transduce the activated y5 T cells with a suitable viral vector;

[0111] expand the transduced y5 T cells for about 3-5 days in the presence of

[0112] 5, 10, 15, 20, 25, 50, or75 lU / ml IL-2,

[0113] 5, 10, 15, 20, 25, 50, or75 ng / ml IL-15 and optionally

[0114] 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or 500 pg / ml L-ascorbic acid 2-phosphate;

[0115] wherein expanding comprises restimulating the y5 T cells with plate-bound anti-CD3 and anti-CD28 antibodies on day 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20or21 after activation.

[0116] In such embodiments, activated and transduced y6 T cells can for instance be expanded in the presence of IL-2 and IL-15 and optionally L-ascorbic acid 2-phosphate (but not zoledronate) for about 3-4 days, reseeded onto anti-CD3 / anti-CD28 coated culture vessels (i.e., restimulated), and expanded foran additional period of time, such as about 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 days to achieve a total expansion of from about 12 days to about 28 days.Methods of Treatment

[0117] The engineered y6 T cells can be provided in the form of a pharmaceutical composition, optionally further comprising at least one pharmaceutically acceptable excipient, such as carriers and buffers. Such pharmaceutical compositions may be administered for prophylactic and / or therapeutic treatments. In therapeutic applications, pharmaceutical compositions can be administered to a subject already suffering from a disease or condition in an amount sufficient to cure or at least partially arrest the symptoms of the disease or condition. Pharmaceutical compositions can also be administered to lessen a likelihood of developing, contracting, or worsening a condition. Effective amounts of a population of engineered y5 T-cells for therapeutic use can vary based on the severity and course of the disease or condition, previous therapy, the subject's health status, weight, and / or response to the drugs, and / or the judgment of the treating physician.

[0118] In a further aspect, the disclosure provides the engineered y6 T cells for use in a method of treating a subject in need of treatment for a condition, for example, a cancer described herein.

[0119] In a further aspect, the disclosure provides a method of treating a subject in need of treatment for a condition, for example, a cancer described herein, comprising administering the engineered y6 T cells.

[0120] A method of treating a condition in a subject with y6 T cells may include administering to the subject a therapeutically-effective amount of engineered y6 T cells. y6 T cells may be administered at various regimens (e.g., timing, concentration, dosage, spacing between treatment, and / or formulation). A subject can also be preconditioned with, for example, chemotherapy, radiation, or a combination of both, prior to receiving engineered y6 T cells of the present disclosure. A population of engineered y6 T cells may also be frozen or cryopreserved prior to being administered to a subject. A population of engineered y6 T cells can include two or more cells that express identical, different, or a combination of identical and different tumor recognition moieties.For instance, a population of engineered T-cells can include several distinct engineered Y<5 T cells that are designed to recognize different antigens, or different epitopes of the same antigen.

[0121] y6T cells may be used to treat various conditions. In some embodiments, engineered Y<5 T cells of the present disclosure may be used to treat a cancer, including solid tumors and hematologic malignancies. Non-limiting examples of cancers include: acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancers, AIDS-related lymphoma, anal cancer, appendix cancer, astrocytomas, neuroblastoma, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancers, brain tumors, such as cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumors, visual pathway and hypothalamic glioma, breast cancer, bronchial adenomas, Burkitt lymphoma, carcinoma of unknown primary origin, central nervous system lymphoma, cerebellar astrocytoma, cervical cancer, childhood cancers, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorders, colon cancer, cutaneous T-cell lymphoma, desmoplastic small round cell tumor, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma, germ cell tumors, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gliomas, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular (liver) cancer, Hodgkin lymphoma, Hypopharyngeal cancer, intraocular melanoma, islet cell carcinoma, Kaposi sarcoma, kidney cancer, laryngeal cancer, lip and oral cavity cancer, liposarcoma, liver cancer, lung cancers, such as non-small cell and small cell lung cancer, lymphomas, leukemias, macroglobulinemia, malignant fibrous histiocytoma of bone / osteosarcoma, medulloblastoma, melanomas, mesothelioma, metastatic squamous neck cancer with occult primary, mouth cancer, multiple endocrine neoplasia syndrome, myelodysplastic syndromes, myeloid leukemia, nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, oral cancer, oropharyngeal cancer, osteosarcoma / malignant fibrous histiocytoma of bone, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, pancreatic cancer, pancreatic cancer islet cell, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal germinoma, pituitary adenoma, pleuropulmonary blastoma, plasma cell neoplasia, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma, renal pelvis and ureter transitional cell cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcomas, skin cancers, Merkel cell skin carcinoma, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, stomach cancer, T-cell lymphoma, throat cancer, thymoma, thymic carcinoma, thyroid cancer, trophoblastic tumor (gestational), cancers of unknown primarysite, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom’s macroglobulinemia, and Wilms tumor.

[0122] In some embodiments, engineered y6 T cells of the present disclosure may be used to treat an infectious disease. In some embodiments, engineered y6 T cells of the present disclosure may be used to treat an infectious disease, an infectious disease may be caused a virus. In some embodiments, engineered y6 T cells of the present disclosure may be used to treat an immune disease, such as an autoimmune disease.

[0123] Treatment with y6 T cells of the present disclosure may be provided to the subject before, during, and after the clinical onset of the condition. Treatment may be provided to the subject after 1 day, 1 week, 6 months, 12 months, or 2 years after clinical onset of the disease. Treatment may be provided to the subject for more than 1 day, 1 week, 1 month, 6 months, 12 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years or more after clinical onset of disease. Treatment may be provided to the subject for less than 1 day, 1 week, 1 month, 6 months, 12 months, or 2 years after clinical onset of the disease. Treatment may also include treating a human in a clinical trial. A treatment can include administering to a subject a pharmaceutical composition comprising engineered y6 T cells of the present disclosure.

[0124] During most bone marrow transplants, a combination of cyclophosphamide with total body irradiation may be conventionally employed to prevent rejection of the hematopoietic stem cells (HSC) in the transplant by the subject's immune system. In some embodiments, incubation of donor bone marrow with interleukin-2 (IL-2) ex vivo may be performed to enhance the generation of killer lymphocytes in the donor marrow. Interleukin-2 (IL-2) is a cytokine that may be necessary for the growth, proliferation, and differentiation of wild-type lymphocytes. Current studies of the adoptive transfer of y6 T-cells into humans may require the co-administration of y6 T-cells and interleukin-2. However, both low- and high-dosages of IL-2 can have highly toxic side effects. IL-2 toxicity can manifest in multiple organs / systems, most significantly the heart, lungs, kidneys, and central nervous system. In some embodiments, the disclosure provides a method for administrating engineered y6T cells to a subject without the co-administration of a native cytokine or modified versions thereof, such as IL-2, IL-15, IL-12, IL-21. In some embodiments, engineered y6 T cells can be administered to a subject without co-administration with IL-2. In some embodiments, engineered y6 T cells may be administered to a subject during a procedure, such as a bone marrow transplant without the co-administration of IL-2.

[0125] Methods of Administration

[0126] One or multiple engineered y6 T cell populations may be administered to a subject in any order or simultaneously. If simultaneously, the multiple engineered y6 T cell can be provided in a single, unified form, such as an intravenous injection, or in multiple forms, for example, as multiple intravenous infusions. Engineered y6 T-cells can be packed together or separately, in a single package or in a plurality of packages. One or all of the engineered y6 T cells can be given in multiple doses. If not simultaneous, the timing between the multiple doses may vary to as much as about a week, a month, two months, three months, four months, five months, six months, or about a year. In some embodiments, engineered y6 T cells can expand within a subject's body, in vivo, after administration to a subject. Engineered y6 T cells can be frozen to provide cells for multiple treatments with the same cell preparation. Engineered y6 T cells, 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 engineered y6 T cells and compositions comprising the same.

[0127] In some embodiments, a method of treating a cancer comprises administering to a subject a therapeutically-effective amount of engineered y6 T cells, in which the administration treats the cancer. In some embodiments, the therapeutically-effective amount of engineered y6 T cells may be 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 engineered y6 T cells may be administered for at least one week. In some embodiments, the therapeutically-effective amount of engineered y6 T cells may be administered for at least two weeks.

[0128] Engineered y6 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 an engineered y6 T-cell can vary. For example, engineered y6 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. Engineered y6 T-cells can be administered to a subject during or as soon as possible after the onset of the symptoms. The administration of engineered y6T 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, such as by any route described herein using any formulation described herein. In some embodiments, the administration of engineered y6 Tcells of the present disclosure may be an intravenous administration. One or multiple dosages of engineered y5 T cells can be administered as soon as is practicable after the onset of a cancer, an infectious disease, an immune disease, sepsis, orwith a bone marrow transplant, and fora length of time necessary for the treatment of the immune 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 engineered y6 T cells can be administered years after onset of the cancer and before or after other treatments. In some embodiments, engineered y6 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.

[0129] Preservation

[0130] In some embodiments, y6 T cells may be formulated in freezing media and placed in cryogenic storage units such as liquid nitrogen freezers (-196°C) or ultra-low temperature freezers (-65°C, -80°C, -120°C, or-150°C) for long-term storage of at least about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 3 years, or at least 5 years. The freeze media can contain dimethyl sulfoxide (DMSO), and / or sodium chloride (NaCl), and / or dextrose, and / or dextran sulfate and / or hydroxyethyl starch (HES) with physiological pH buffering agents to maintain pH between about 6.0 to about 6.5, about 6.5 to about 7.0, about 7.0 to about 7.5, about 7.5 to about 8.0 or about 6.5 to about 7.5. The cryopreserved y5 T cells can be thawed and further processed by stimulation with antibodies, proteins, peptides, and / or cytokines as described herein. The cryopreserved y5 T-cells can be thawed and genetically transduced with viral vectors (including retroviral, adeno-associated virus (AAV), and lentiviral vectors) or non-viral means (including RNA, DNA, e.g., transposons, and proteins) as described herein. The transduced y5 T cells can be further cryopreserved to generate cell banks in quantities of at least about 1, 5, 10, 100, 150, 200, 500 vials at about at least 101, 102, 103, 104, 105, 106, 107, 108, 109, or at least about 1010 cells per mL in freeze media. The cryopreserved cells may retain their functionality and can be thawed and further stimulated and expanded. In some embodiments, thawed cells can be stimulated and expanded in suitable closed vessels, such as cell culture bags and / or bioreactors,to generate quantities of cells as allogeneic cell product. Cryopreserved y5 T cells can maintain their biological functions for at least about 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 15 months, 18 months, 20 months, 24 months, 30 months, 36 months, 40 months, 50 months, or at least about 60 months under cryogenic storage condition. In some embodiments, no preservatives may be used in the formulation. Cryopreserved y5 T-cells can be thawed and infused into multiple patients as allogeneic off-the-shelf cell product.

[0131] In some embodiments, engineered y5 T-cell described herein may be present in a composition in an amount of at least 1 *103total cells / ml, at least 2* 103total cells / ml, at least 3×103total cells / ml, at least 4*103total cells / ml, at least 5*103total cells / ml, at least 6* 103total cells / ml, at least 7*103total cells / ml, at least 8*103total cells / ml, at least 9*103total cells / ml, at least 1×104total cells / ml, at least 2x104total cells / ml, at least 3x104total cells / ml, at least 4x104total cells / ml, at least 5x104total cells / ml, at least 6x 104total cells / ml, at least 7x104total cells / ml, at least 8x104total cells / ml, at least 9x104total cells / ml, at least 1×105total cells / ml, at least 2x105total cells / ml, at least 3x105total cells / ml, at least 4x105total cells / ml, at least 5x105total cells / ml, at least 6x 105total cells / ml, at least 7x 105total cells / ml, at least 8x105total cells / ml, at least 9x105total cells / ml, at least 1×106total cells / ml, at least 2x106total cells / ml, at least 3x106total cells / ml, at least 4x106total cells / ml, at least 5x106total cells / ml, at least 6x106total cells / ml, at least 7x 106total cells / ml, at least 8x106total cells / ml, at least 9x106total cells / ml, at least 1×107total cells / ml, at least 2x107total cells / ml, at least 3x107total cells / ml, at least 4x107total cells / ml, at least 5x107total cells / ml, at least 6x107total cells / ml, at least 7x107total cells / ml, at least 8X107total cells / ml, at least 9X107total cells / ml, at least 1×108total cells / ml, at least 2x108total cells / ml, at least 3x108total cells / ml, at least 4x108total cells / ml, at least 5x108total cells / ml, at least 6x108total cells / ml, at least 7x108total cells / ml, at least 8x108total cells / ml, at least 9×108total cells / ml, at least 1×109total cells / ml, or more, from about 1×103total cells / ml to about at least 1×108total cells / ml, from about 1 xio5total cells / ml to about at least 1×108total cells / ml, or from about 1 xio6total cells / ml to about at least 1×108total cells / ml.

[0132] In some embodiments, methods described herein may be used to produce autologous or allogenic products.

[0133] The present invention may be better understood by reference to the following examples, which are not intended to limit the scope of the claims.EXAMPLES

[0134] Example 1

[0135] Processing a leukapheresis product

[0136] A leukapheresis product, e.g., LeukoPak®, may be processed as follows: one end of a LeukoPak® bag may be swabbed with alcohol swab and cut with razor blade to drain into a flask. The volume may be diluted to between approximately 500 ml with Hank's solution and then aliquoted into 16-29 tubes with 50 ml capacity, 30 ml per tube. The tubes may be spun at 400 g for 30 minutes with no brake and no acceleration. White liquid may be aspirated, and new 50 ml tubes may be filled up halfway and topped off with 25 ml PBS. This procedure may be repeated 2 additional times for a total of 3 washes. Cells may be counted before the last wash using a hemocytometer. The yield may be between 30-60 tubes of 1 *108cells / tube.

[0137] Example 2

[0138] Depleting cells

[0139] FIG. 1 A shows depletion of a|3 T cells from PBMC. Post-ficolled PBMCs were incubated with biotin-conjugated a|3 TCR antibodies, followed by streptavidin-microbeads per manufacturer protocol. Samples were then passed through a LS column to enrich for a|3 TCR-expressing cells. The column flow-through represents the a|3 TCR depleted fractions. After overnight culture, the a|3 TCR-enriched fractions and the a|3 TCR-depleted fractions were stained with fluorochrome-conjugated a|3TCR antibody versus Vy9 antibody, followed by flow cytometry analysis. The data shows, while the a|3 TCR-enriched fractions contains almost none (0%) Vy952 cells, almost all Vy952 cells are enriched (45%) in the a|3 TCR-depleted fractions.

[0140] Similarly, cells from leukapheresis product may be incubated with biotin-conjugated a|3 TCR antibodies, followed by streptavidin-microbeads per manufacturer protocol. Samples were then passed through LS column to enrich for a|3 TCR-expressing cells. The column flow-through represents the a|3 TCR depleted fractions. After overnight culture, the a|3 TCR-depleted fractions were stained with fluorochrome-conjugated a|3TCR antibody versus y5 TCR antibody, followed by flow cytometry analysis. FIG. 1 E shows, while the starting cells in leukapheresis product contain minimum (4.14%) Vy952 cells, almost all Vy952 cells are enriched (95.5%) in the a|3 TCR-depleted fractions. In some embodiments, using the afore-mentioned methods, Vy952 cells may be enriched more than about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0141] FIG. 1 B and 1C show minimal residual a|3T cells in Vy952T cell product a|3 T cells were depleted from PBMC of normal donors (Donor A, Donor B, Donor C, Donor 12, and Donor 13) usingbioti nylated anti a|3 TCR antibody / streptavidin microbeads. a|3 T cell-depleted PBMC were cultured with zoledronate / IL-2 / IL-15 for 14 days, followed by cell surface staining with respective fluorochrome conjugated antibodies, e.g., anti a|3 TCR antibodies and anti-yS TCR antibodies to assess for residual a|3 T cells and enriched y6 T cells by sub-gating on CD3. These results show a|3 T cell-depleted y6T cells were enriched, i.e., 90.1% (Donor A), 78.6% (Donor B), 28.9% (Donor C), 89% (Donor 12), and 74% (Donor 13). In addition, a|3T cell-depleted y6T cells contain minimal residual a|3T cells, i.e., 0.02% (Donor A), 0.05% (Donor B), 0.2% (Donor C), 0.4% (Donor 12), and 1% (Donor 13). Minimal residual a|3 T cells is important because, for example, in haploidentical hematopoietic stem cell transplantation (HSCT), residual a|3 T cells ranging from 0.2-0.6% did not result in chronic graft versus host disease (GVHD), thus, making these a|3 T cell-depleted y5 T cells safe allogeneic products.

[0142] FIG. 1 D shows cytokine profiling of Vy952 cells. a|3T cell-depleted PBMC cultured with zoledronate / IL-2 / IL-15 were treated with Golgi Stop / Plug (i.e., protein transport inhibitors) for 6 hours prior to cell harvest. Cells were stained for surface V52 followed by fixation and permeabilization. Staining intracellular TNF-a, IL-17a, and IFN-ywere performed using fluorochrome-conjugated antibodies against TNF-a, IL-17a, and IFN-y. These results show TNF-a, IL-17a, and IFN-ywere expressed in 12%, 0.2%, and 4% of Vy952 cells, respectively. IL-15-mediated inhibition of IL-17 commitment is shown by low amount of IL-17-producing Vy952 T cells, e.g., 0.2%.

[0143] Examples

[0144] Activation and Expansion of T Cell-Depleted PBMC

[0145] Maximal T cell activation, proliferation, and survival without commitment to anergy may require three signals: signal 1 elicited through TCR, signal 2 elicited through co-stimulatory molecules, and signal 3 elicited through growth factor signaling.

[0146] FIGS. 2 and 3 show, respectively, an activation step and expansion step. The activation step and expansion step may be two sequential steps. For example, during the activation step (FIG.2), aminobisphosphonate, e.g., zoledronate (ZA), and cytokines, e.g., IL-2 and / or IL-15, are present. Whereas, during the expansion step (FIG. 3), cytokines continue to be present without aminobisphosphonate. For instance, the activation step (FIG. 2) may occur during the first 14 days, when aminobisphosphonate, is added. The expansion step (FIG. 3) may be from Day 15 onward because, at the end of Day 14, activated cells may be collected by removing all medium, which contains aminobisphosphonate, and replace with medium with cytokines in the absence ofaminobisphosphonate or phosphoantigen, e.g., IPP. In contrast, the conventional protocol for Vy952 zoledronate-mediated production often refers Day 1-14 as activation / expansion because, under such conditions, cells could not be kept alive beyond day 14. In the present disclosure, Day 1-14 may be referred to as the “activation step because aminobisphosphonate, e.g., zoledronate is present, and Day 15-onward is referred to as expansion step because cells can be kept alive and expanded beyond the conventional 14-day process.

[0147] FIG. 2 shows signal 1 elicited through y6 TCR / IPP interaction induced by aminobisphosphonate, which may include pamidronic acid, alendronic acid, zoledronic acid, risedronic acid, ibandronic acid, incadronic acid, a salt thereof and / or a hydrate thereof; or phosphoantigens, e.g., (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), isoprenoid pyrophosphates (farnesyl pyrophosphate (FPP), geranylgeranyl pyrophosphate (GGPP), isopentenyl pyrophosphate (IPP), and dimethylallyl diphosphate (DMAPP)), to activate Vy962 T cells for proliferation. For example, Zoledronate (Zoledronic acid (ZA) or Zometa) inhibits the mevalonate pathway in monocytes (Mo), leading to accumulation of phosphoantigens, such as IPP, displayed on monocytes (ZA-Mo) to activate the y6 TCR / CD3 and induce proliferation via PKC signaling, thereby serving as signal 1. IPP perse can also act directly on y6 T cells by binding to the ySTCR, thereby obviating the need for monocytes.

[0148] As described herein, a|3 T cell depleted PBMCs or a|3 T cell depleted leukapheresis product may be cultured at high cell density during Zoledronate treatment to facilitate engagement of co-stimulatory molecules presenton myeloid cells as well as the expanding y6T cells, thereby enhancing activation, proliferation, and survival of Vy962 T cells. For example, exogenous IL-2 (10-1000 U / ml, preferably 25-500 U / ml) may be added to a|3 depleted PBMCs or a|3 T cell depleted leukapheresis product, in which CD4 helper T cells (to secrete IL-2) are among the a|3 T cells depleted cells, to sustain survival and proliferation during activation and expansion. Exogenous IL-15 (10-1000 ng / ml, preferably 20-500 ng / ml) may be used in high cell density culture to maximize Vy952 activation by inhibiting the development of IL-17-producing Vy952 T cells, as shown in FIG.1 D (middle panel), enhancing y5T cell proliferation, and promoting the differentiation of naive Vy952 T cells to effector cells. High density culture, thus, exploits the reciprocal costimulatory action between y5 T cells, which express CD28, CD86, CD83, CD80 on y5 T cells. For example, CD28 costimulatory molecule, which interacts with CD86 and / or CD80, can enhance y5 T cells survival and proliferation.

[0149] To determine the effects of cell density on T cell phenotypes and cell death, after activation in the presence of zoledronate, IL-2, and IL-15 for 14 days, Vy952T cells were expanded by homeostatic cytokines, e.g., IL-2 and IL-15, in the absence of zoledronate at high density (e.g., 2 x 106total cells / ml) and at low density (e.g., 0.5 x 106total cells / ml) followed byT cell marker analysis. FIG. 4A shows immunophenotyped markers, e.g., CD122, CD80, CD83, CD86, CD95, and CD95L, were not affected in Vy952 T cells by cell density as there is no significant difference between marker expressions in Vy952 T cells cultured at high density and at low density. However, FIG. 4B shows a significant reduction in cell death (20%) during expansion at high density as compared with that (51%) at low density. These results suggest that expanding Vy952 T cells at high density, e.g., at least 1 x 106total cells / ml, may promote cell survival by reducing cell death.

[0150] FIG. 6 shows y5 T cell expansion using Zoledronate (Zometa) in defined medium, which contains IL-2, IL-15, and Amphotericin B. Fold increase in absolute number of y5T cells is 3,350-fold, 11,060-fold, and 31,666-fold for Donor 20 from Day 0 to Day 17, from Day 0 to Day 22, and from Day 0 to Day 29, respectively. Similarly, fold increase in absolute number of y5 T cells is 4,633-fold, 12,320-fold, and 32,833-fold for Donor 21 from Day 0 to Day 17, from Day 0 to Day 22, and from Day 0 to Day 29, respectively. In contrast, as noted above, classic Vy952 T cell expansion protocol, at best, could yield only a 100-fold increase in total Vy952 T cells within 14 days, thereafter, the expansion rate decreases, which may be caused by an increase of cell death. With the methods described herein, fold increase in absolute number of y5 T cells after expansion on Day 29 as compared with that of Day 0 may be from about 1000-fold to about 40,000-fold, from about 3000-fold to about 35,000-fold, from about 5000-fold to about 35,000-fold, from about 6000-fold to about 35,000-fold, from about 7000-fold to about 35,000-fold, from about 8000-fold to 30,000-fold, from about 10,000-fold to about 35,000-fold, from about 15,000-fold to about 35,000-fold, from about 20,000-fold to about 35,000-fold, from about 25,000-fold to about 35,000-fold, from about 30,000-fold to about 35,000-fold, more than about 10,000 fold, more than about 15,000 fold, more than about 20,000 fold, more than about 25,000 fold, more than about 30,000 fold, more than about 40,000 fold, or more than about 40,000 fold.

[0151] Example 4

[0152] TCR engineering of Vy952 T cells with retroviral vectors

[0153] FIG. 7 shows a time table for viral transduction into Vy952 T cells. Fresh leukocytes are depleted of a|3T cells and activated with Zoledronate in the presence of IL-2 and IL-15 for a minimum of 1 day or maximum of 7 days. y5 T cells can be transduced between 24 to 168 hours after activation using viral supernatant expressing an a|3TCR and / or CD8.

[0154] To determine whether Vy952T cells prepared bythe methods of the present disclosure are suitable for viral transfection with viruses expressing different envelop proteins, green fluorescent protein (GFP)-expressing y5 -retrovirus (e.g., Gibbon Ape Leukemia Virus (GALV) pseudotype (SEQ ID NO: 4) and RD114TR pseudotype (SEQ ID NO: 1)) and GFP-expressing lentivirus (e.g., VSV-G pseudotype (for example, SEQ ID NO: 3)) were tested for their transduction efficiency into these Vy962 T cells. In addition, CD8a-expressing lentivirus (LV) pseudotyped with VSV-G and RD114TRwere tested for their transduction efficiency into Vy962T cells.

[0155] FIG. 8A shows, day 5 post-transduction, GFP expression is 12% for GALV pseudotype, 34% for RD114TR pseudotype, and 46% for VSV-G pseudotype.

[0156] FIG. 8B shows, day 4 post-transduction, CD8a expression on Vy962T cells ranged from 63.2% (using 2.18 pl LV) up to 95.8% (using 35 pl LV) when Vy952 T cells were transduced with LV pseudotyped with RD114TR.

[0157] Examples

[0158] Engineering y5 T cells expressing a|3TCR and CD8

[0159] Engineered y5 T-cells of the disclosure may be used to treat a subject in need of treatment for a condition. To engineer y5 T cells that express a|3TCR specifically binding to a TAA / MHC complex, a|3TCR -expressingy-retrovirus (a|3TCR virus) was generated. Because y5T cells may not express CD8, y5 T cells may need CD8 in addition to a|3TCR to recognize TAA / MHC-I complexes on cell membrane of target cells, e.g., cancer cells. To that end, CD8-expressingy-retrovirus (CD8 virus) was generated.

[0160] To determine transduction efficiency of Vy952 T cells with engineered y- retroviruses, Zoledronate-activated Vy952 T cells were transduced with a|3TCR virus and / or CD8 virus at MOI of 3 in defined medium supplemented with IL-2 and IL-15. Transduction efficiency was measured at 96-hours post-transduction by staining with TAA / MHC-PE dextramer (or negative control NYESO-PE dextramer), followed by CD3, CD8a, and V52 staining. Acquisition on MacsQuant was followed by analysis gating on CD3 population.

[0161] Transduction may be performed in the presence of transduction enhancers to increase transduction efficiency by physically reducing electrostatic repulsion between the negatively charged cell and the virion and therefore increasing cell-virion interaction. To this end, two transduction enhancers were tested during y5T cell transduction with a retrovirus encoding CD8ap. RetroNectin® (a fibronectin fragment coated onto plates) and VectoFusin-1® (a soluble cationic peptide) were tested. FIG. 9A shows, while RetroNectin® resulted in higher transductionefficiencies (mean 49.7%), VectoFusin-1 ® was also able to transduce y6 T cells at mean transduction efficiency of 27.5%.

[0162] FIG. 9B shows 71% of Vy952T cells transduced with a|3TCR virus alone and 49% of Vy952 T cells transduced with both a|3TCR virus and CD8 virus identified byTAA / MHC-PE dextramer staining, as compared with negative control NYESO-PE dextramer staining, i.e., transduced with apTCR virus alone (1.6%) and both a|3TCR virus and CD8 virus (2%). These results indicate a|3TCR, which specifically binds a TAA / MHC complex, was readily presented on cell surface of the Vy952 T cells transduced with a|3TCR virus. In addition, 7.6% of Vy952 T cells transduced with CD8 virus alone and 6.8% of Vy952 T cells transduced with both a|3TCR virus and CD8 virus were identified by CD8a staining, as compared with mock (no virus) (4%) and transduced with a|3TCR virus alone (4.4%). These data show Vy952 T cells prepared by Zoledronate, IL-2, and IL-15-mediated activation and expansion can be used to express TAA-specificTCRs and CD8 by viral transduction.

[0163] To determine the fold-expansion of Vy952 T cells after viral transduction, Vy952 T cells (GD) or ap T cells (AB) were transduced with a|3TCR virus (TCR) and / or CD8 virus (CD8) followed by measurement of fold expansion from day 7 to day 21 post-transduction. FIG. 10 shows, without transduction, Vy952 T cells (GD Mock) (1,040-fold) generally have higher fold expansion than a|3T cells (AB Mock) (289-fold). After transduction with a|3TCR alone, Vy952 T cells (GD + TCR) have 517-fold, which is higher than that of a|3 T cells (AB + TCR) (211 -fold). After transduction with CD8 alone (GD + CD8) or CD8 + ap-TCR (GD + CD8 + TCR), Vy952 T cells have 620-fold and 540-fold expansion, respectively. These results indicate that Vy952 T cells possess better capacity for cell expansion than a|3T cells, in general, and for viral transduction.

[0164] ap-TCR-expressingVy952T cells, in which ap-TCR specifically binds to TAA / MHC complex, were generated by transducing Vy952 T cells with a|3TCR retrovirus and CD8a|3 retrovirus. FIG. 11Ashows, as compared with Vy952T cells without viral transduction (Mock), 34.9%ofVy952 T cells transducing with a|3TCR retrovirus and CD8a|3 retrovirus (a|3TCR + CD8) stained positive by TAA / MHC-dextramer (TAA / MHC-dex) and anti-CD8 antibody (CD8), indicating the generation of Vy952 T cells expressing both a|3TCR and CD8a|3 on cell surface (a|3TCR +CD8a|3 engineered Vg9d2 T cells).

[0165] To determine cytolytic activity of engineered Vy952 T cells, a|3TCR +CD8a|3 engineered Vy952 T cells were exposed to target cells, e.g., A375 cell line, which is a human malignant melanoma cell line having TAA / MHC complex presented on cell surface. Four functional assays: (1) CD107a degranulation, (2) IFN-y release, (3) apoptosis of A375 cells after 6 hours, and (4) cytotoxic effect of engineered y5 T cells on A375 after longterm co-culture.

[0166] The principle of CD107a degranulation assay is based on killing of target cells via a granule-dependent pathway that utilizes pre-formed lytic granules located within the cytoplasm of cytotoxic cells. The lipid bilayer surrounding these granules contains lysosomal associated membrane glycoproteins (LAMPs), including CD107a (LAMP-1 ). Rapidly upon recognition of target cells via the T cell receptor complex, apoptosis-inducing proteins like granzymes and perforin are released into the immunological synapse, a process referred to as degranulation. Thereby, the transmembrane protein CD107a is exposed to the cell surface and can be stained by specific monoclonal antibodies. FIG. 11 B shows, as compared with Vy962 T cells without viral transduction (Mock), 23.1% of Vy962 T cells transduced with a|3TCR retrovirus and CD8a|3 retrovirus (a|3TCR + CD8) incubated with target cells, e.g., A375 cells, stained positive by anti-CD107a antibody, indicating that a|3TCR +CD8a|3 engineered Vg9d2 T cells are cytolytic by carrying out degranulation, when exposed to A375 cells.

[0167] IFN-y release assays measure the cell mediated response to antigen-presenting cells, e.g., A375 cells, through the levels of IFN-y released, when TCR of T cells specifically binds to peptide / MHC complex of antigen-presenting cells on cell surface. FIG. 11C shows, as compared with Vy962T cells without viral transduction (Mock), 19.7% of Vy962T cells transduced with a|3TCR retrovirus and CD8a|3 retrovirus (a|3TCR + CD8) stained positive by anti-IFN-y antibody, indicating that apTCR +CD8a|3 engineered Vy962 T cells are cytolytic by releasing IFN-y, when exposed to A375 cells.

[0168] Cytolytic activity were evaluated at 24 hours post-exposure to A375 cells by gating on apoptosis of non-CD3T cells, i.e., A375 cells. Apoptosis was assessed by staining the harvested culture with live / dead dye. FIG. 11 D shows, as compared with Vy962T cells without viral transduction (Mock), a|3TCR +CD8a|3 engineered Vy962 T cells (a|3TCR + CD8) induced apoptosis in 70% of A375 cells, indicating that a|3TCR +CD8a|3 engineered Vy962 T cells are cytolytic by killing A375 cells.

[0169] Cytolytic activity was also evaluated in real-time during an 84-hour co-culture assay. Non-transduced and a|3TCR+CD8ap transduced y6 T cells were co-culture with target positive A375-RFP tumor cells at an effector to target ratio of 3:1. Lysis of target positive A375-RFP tumor cells was assessed in real time by IncuCyte® live cell analysis system (Essen BioScience). Tumor cells alone and non-transduced and a|3TCR transduced a|3 T cells were used as negative and positive controls, respectively. As shown in FIG. 11 E, while non-transduced y6 T cells showed cytotoxic potential due to intrinsic anti-tumor properties of y6T cells, a|3TCR+CD8ap transduced y6T cells showed similar cytotoxic potential as compared to a|3TCR transduced a|3 T cells, indicating that a|3TCR+CD8ap transduced y5 T cells can be engineered to target and kill tumor cells.

[0170] These data indicate engineered Vy952 T cells produced by the methods of the present disclosure are functional and can be used to kill target cells, e.g., cancer cells, in a TAA peptidespecific manner.

[0171] In an aspect, TAA peptides described herein that are capable of use with the methods and embodiments described herein include, for example, those TAA peptides described in U. S. Publication 20160187351, U. S. Publication 20170165335, U. S. Publication 20170035807, U. S. Publication 20160280759, U. S. Publication 20160287687, U. S. Publication 20160346371, U. S. Publication 20160368965, U. S. Publication 20170022251, U. S. Publication 20170002055, U. S. Publication 20170029486, U. S. Publication 20170037089, U. S. Publication 20170136108, U. S. Publication 20170101473, U. S. Publication 20170096461, U. S. Publication 20170165337, U. S. Publication 20170189505, U. S. Publication 20170173132, U. S. Publication 20170296640, U. S. Publication 20170253633, and U. S. Publication 20170260249, the contents of each of these publications and sequence listings described therein are herein incorporated by reference in their entireties.Example 7:

[0172] Adding phospho Vitamin C as a supplement

[0173] y5 T cells were isolated, activated and expanded as described in Example 3 in the presence or absence of phospho Vitamin C and harvested. As shown in FIG. 15, the addition of phospho Vitamin C to media enhances the viability, expansion, purity and tumor killing ability of y<5 T cells.Example 8:

[0174] Restimulation with anti-CD3 and anti-CD28 antibodies

[0175] y5T cells were isolated, activated and expanded as described in Example 3 in the presence or absence of phospho Vitamin C and harvested with or without restimulating with anti-CD3 and anti-CD28 antibodies. As shown in FIG. 16, restimulation further enhances y5T cell expansion.

[0176] Taken together, the examples demonstrate the feasibility of achieving up to ~5000xy6 fold expansion of y5T cells without relying on feeder cells. To accomplish this, various additiveprocess improvements were made including: Optimizing media by adding supplements such as phospho vitamin C and restimulating y6T cells with aCD3 / CD28 antibodies.

[0177] All references cited in this specification are herein incorporated by reference as though each reference was specifically and individually indicated to be incorporated by reference. The citation of any reference is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such reference by virtue of prior invention.

[0178] It will be understood that each of the elements described above, or two or more together may also find a useful application in other types of methods differing from the type described above. Without further analysis, the foregoing will so fully reveal the gist of the present disclosure that others can, by applying current knowledge, readily adapt it for various applications without omitting features that, from the standpoint of prior art, fairly constitute essential characteristics of the generic or specific aspects of this disclosure set forth in the appended claims. The foregoing embodiments are presented by way of example only; the scope of the present disclosure is to be limited only by the following claims.

Claims

CLAIMSWhat is claimed is:

1. An in vitro method of activating and expanding Y<5 T cells comprisingactivating y6 T cells in the presence of an aminobisphosphonate, human recombinant interleukin 2 (IL-2), and human recombinant interleukin 15 (IL-15), expanding the activated Y<5 T cells in the absence of an aminobisphosphonate and in the presence of interleukin 2 (IL-2) and interleukin 15 (IL-15),wherein expanding comprises restimulating the expanded Y<5 T cells with a suitable Y<5 T cell stimulation agent.

2. The method of claim 1, wherein the Y<5 T cell stimulation agent is selected from an anti-CD3 antibody and an anti-CD28 antibody, or a combination thereof.

3. The method of claim 1 or 2, wherein said restimulation takes about 2 to about 18 days.

4. The method of any one of the preceding claims, wherein said anti-CD3 antibody and / or said an anti-CD28 antibody are immobilized.

5. The method of any one of the preceding claims, wherein said anti-CD3 antibody concentration and / or said an anti-CD28 antibody concentration during restimulation is between about 0.1 pg / ml and about 2 pg / ml, such as about 0.25, O.5., 0.75, 1.0, 1.25, 1.5, 1.75 or 2 pg / ml.

6. The method of any one of the preceding claims, wherein activation and / or expansion is further in the presence of L-ascorbic acid or a derivative thereof.

7. The method of claim 6, wherein said derivative is L-ascorbic acid 2-phosphate (pVC).

8. The method of claim 6 or 7, wherein L-ascorbic acid 2-phosphate is used at a concentration of 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975 or 1000 pg / mL.

9. The method of any one of the preceding claims, wherein the aminobisphosphonate is selected from the group of pamidronic acid, alendronic acid, zoledronic acid, risedronic acid, ibandronic acid, incadronic acid, a salt thereof and / or a hydrate thereof.

10. The method of anyone of the preceding claims, wherein the aminobisphosphonate is zoledronic acid.

11. The method of claim 10, wherein the zoledronate concentration during activation is between about 1 pM to about 50 pM, such as about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 pM.

12. The method of anyone of the preceding claims, wherein activation is in the presence of zoledronic acid and a cytokine composition consisting of IL-2 and IL-15.

13. The method of anyone of the preceding claims, wherein the IL-2 concentration during activation and / or expansion is between about 5 lU / ml and about 500 lU / ml, such as about 5, 10, 15, 20, 25, 50, 75, 100, 125, 150, 175 or 200 lU / ml.

14. The method of anyone of the preceding claims, wherein the IL-15 concentration during activation and / or expansion is between about 5 ng / ml and about 200 ng / ml, such as about 5, 10, 15. 20, 25, 50, 75, 100, 125, 150, 175 or 200 ng / ml.

15. The method of any one of the preceding claims, wherein activation and / or expansion is in the presence of IL-2 at a concentration from about 10 lU / ml to about 50 I U / ml and / or IL-15 at a concentration of about 10-50 ng / ml.

16. The method of anyone of the preceding claims, wherein activation takes about 1 day to about 7 days.

17. The method of any one of the preceding claims, wherein expansion takes about 12 days to about 28 days.

18. The method of any one of the preceding claims, further comprising transducing the activated y5 T cells with a viral vector.

19. The method of claim 18, wherein the viral vector is a retroviral vector, a lentiviral vector, an adeno-associated virus (AAV), or a transposon.

20. The method of any one of claims 18 or 19, wherein the viral vector encodes CD8 and / or an a0-TCR.

21. The method of any one of claims 18 to 20, wherein the transducing is in the presence of a transduction enhancer.

22. The method of any one of claims 18to21, further comprising enriching the y5 T cells from a sample, wherein said sample is optionally a blood sample, PBMC or a leukapheresis sample.

23. An engineered y6T cell prepared by the method of any one of claims 18 to 22.

24. A population of expanded and activated y6T cells prepared by the method of any one of the preceding claims.

25. A population of expanded y5T cells prepared by the method of any one of the preceding claims, wherein the density of the expanded y6 T cells is at least about 1 x 105total cells / ml, at least about 1 x 106total cells / ml, at least about 1 x 107total cells / ml, at least about 1 x 108total cells / ml, or at least about 1 x 109total cells / ml.

26. A method of treating cancer, comprising administering to a patient in need thereof an effective amount of the engineered y6 T cell of claim 23.

Images