Method for the production of gamma delta t cells for immunotherapy applications

Abstract

The present disclosure relates to a field of immunotherapy. Particularly, the present disclosure provides methods for production of gamma delta T (γδ T) cells and the γδ T cells produced therefrom.

Description

METHOD FOR THE PRODUCTION OF GAMMA DELTA T CELLS FOR IMMUNOTHERAPY APPLICATIONSField of the Invention

[0001] The present disclosure relates to the field of immunotherapy. In Particular, the present disclosure provides methods for producing gamma delta T (y5 T) cells and the y5 T cells produced therefrom.Background of the Invention

[0002] y5 T cells represent a distinct subset of T lymphocytes that present a promising avenue for T cell-based immunotherapy. Their capacity to produce significant amounts of cytokines with anti-tumor properties, along with characteristics of both innate and adaptive immune cells, makes them particularly appealing. Clinical trials utilizing a combination of in vivo and ex vivo stimulation of y5 T cells have often depended on agonists like aminobisphosphonates (n-BPs), which are associated with limited bioavailability and potential off-target toxicity (La-Beck NM, et al., Repurposing amino-bisphosphonates by liposome formulation for a new role in cancer treatment. Semin Cancer Biol 2021; 68: 175-185). Phosphoantigens (pAgs) can stimulate y5 T cell populations more effectively than conventional nitrogen-containing bisphosphonates (n-BPs) (Raverdeau M, Cunningham SP, Harmon C, Lynch L. yd T cells in cancer: a small population of lymphocytes with big implications. Clin Transl Immunology 2019; 8: e01080). Nonetheless, the clinical effectiveness of y5 T-cell immunotherapies is constrained by challenges related to the pharmacodynamic and pharmacokinetic profiles of frequently used agonists, the difficulty of infused T cells in penetrating solid tumors, and a limited understanding of y5 T-cell checkpoint inhibition.

[0003] Therefore, there is need to develop new therapeutics for improving y5 T-cell immunotherapies.Summary of the Invention

[0004] The present disclosure relates to a novel method for the production of V52+y5 T cells for therapeutic applications. The method includes isolation, expansion, and activation of V52+y5 T cells from peripheral blood mononuclear cells under optimized conditions.

[0005] In one aspect, the present disclosure provides a method for producing a cell population comprising enriched V52+y5 T cells, comprising:(a) providing peripheral blood mononuclear cells (PBMCs); optionally the V52+y5 T cells contained in the PBMCs are separated by immunomagnetic separation to obtain a V52+y5 T cell subpopulation;(b) culturing the PBMCs or V52+y5 T cell subpopulation in a medium containing zoledronic acid and one or more growth factors having interleukin-like activity to specifically enrich V52+y5 T cells to obtain an enriched V52+y5 T cell population; and(c) harvesting the expanded V52+y5 T cell population.

[0006] In a particular embodiment, the present disclosure provides a method for producing a cell population comprising enriched V52+y5 T cells, comprising:(al) providing peripheral blood mononuclear cells (PBMCs);(bl) culturing the PBMCs in a medium containing zoledronic acid and one or more growth factors having interleukin-like activity to specifically enrich V52+y5 T cells to obtain an enriched V52+y5 T cell population; and(cl) harvesting the expanded V52+y5 T cell population; or(a2) providing peripheral blood mononuclear cells (PBMCs);(i) separating V52+y5 T cells by immunomagnetic separation to obtain a V52+y5 T cell subpopulation;(b2) culturing the PBMCs in a medium containing zoledronic acid and one or more growth factors having interleukin-like activity to specifically enrich V52+y5 T cells to obtain an enriched V52+y5 T cell population; and(c2) harvesting the expanded V52+y5 T cell population; or

[0007] In one embodiment, after step (b), (bl) or (b2), the method further comprises a step (e) of separating the enriched V52+y5 T cell population by immunomagnetic separation to remove non-V52+y5 T cells.

[0008] In one embodiment, after step (b), (bl), (b2) or (e), the method further comprises a step (f) of expanding the enriched V52+y5 T cell population by culturing it for 7-28 days to obtain the expanded V52+y5 T cell population.

[0009] In one embodiment, the V52+y5 cells in the V52+y5 T cell population are unmodified.

[0010] In some embodiments, the PBMCs are obtained from a peripheral blood, umbilical cord blood, or fractions thereof.

[0011] In one embodiment, the zoledronic acid is at a concentration of about 1 to about 30 pM, about 1 to about 28 pM, about 1 to about 25 pM, about 1 to about 23 pM, about 1 toabout 20 gM, about 1 to about 18 gM, about 1 to about 15 gM, about 1 to about 13 gM, about 1 to about 10 gM, about 1 to about 8 gM, about 1 to about 5 gM, about 3 to about 30 gM, about 3 to about 28 gM, about 3 to about 25 gM, about 3 to about 23 gM, about 3 to about 20 gM, about 3 to about 18 gM, about 3 to about 15 gM, about 3 to about 13 gM, about 3 to about 10 gM, about 3 to about 8 gM, about 3 to about 5 gM, about 5 to about 30 gM, about 5 to about 28 gM, about 5 to about 25 gM, about 5 to about 23 gM, about 5 to about 20 gM, about 5 to about 18 gM, about 5 to about 15 gM, about 5 to about 13 gM, about 5 to about 10 gM, about 8 to about 30 gM, about 8 to about 28 gM, about 8 to about 25 gM, about 8 to about 23 gM, about 8 to about 20 gM, about 8 to about 15 gM, about 8 to about 12 gM, about 8 to about 10 gM, about 10 to about 30 gM, about 10 to about 28 gM, about 10 to about 25 gM, about 10 to about 23 gM, about 10 to about 20 gM, about 10 to about 18 gM, or about 10 to about 15 [iM.

[0012] In one embodiment, the medium is free of serum.

[0013] In one embodiment, the growth factor having interleukin-like activity is an interleukin.

[0014] In one embodiment, the concentration of the growth factor having interleukin-like activity ranges from about 5 ng / mL to about 200 ng / mL or about 50 HJ / mL to about 1,500 lU / mL.

[0015] In one embodiment, the concentration of the growth factor having interleukin-like activity ranges from about 5 ng / mL to about 200 ng / mL, about 5 ng / mL to about 180 ng / mL, about 5 ng / mL to about 150 ng / mL, about 5 ng / mL to about 100 ng / mL, about 5 ng / mL to about 80 ng / mL, about 5 ng / mL to about 70 ng / mL, about 5 ng / mL to about 60 ng / mL, about 5 ng / mL to about 50 ng / mL, about 5 ng / mL to about 40 ng / mL, about 5 ng / mL to about 30 ng / mL, about 5 ng / mL to about 20 ng / mL, about 10 ng / mL to about 200 ng / mL, about 10 ng / mL to about 180 ng / mL, about 10 ng / mL to about 150 ng / mL, about 10 ng / mL to about 100 ng / mL, about 10 ng / mL to about 80 ng / mL, about 10 ng / mL to about 70 ng / mL, about 10 ng / mL to about 60 ng / mL, about 10 ng / mL to about 50 ng / mL, about 10 ng / mL to about 40 ng / mL, about 10 ng / mL to about 30 ng / mL, about 20 ng / mL to about 200 ng / mL, about 20 ng / mL to about 180 ng / mL, about 20 ng / mL to about 150 ng / mL, about 20 ng / mL to about 100 ng / mL, about 20 ng / mL to about 80 ng / mL, about 20 ng / mL to about 70 ng / mL, about 20 ng / mL to about 60 ng / mL, about 20 ng / mL to about 50 ng / mL, about 20 ng / mL to about 40 ng / mL, about 20 ng / mL to about 30 ng / mL, about 30 ng / mL to about 200 ng / mL, about 30 ng / mL to about 180 ng / mL, about 30 ng / mL to about 150 ng / mL, about 30 ng / mL to about 100 ng / mL, about 30 ng / mL to about 80 ng / mL, about 30 ng / mL to about 70 ng / mL, about 30ng / mL to about 60 ng / mL, about 30 ng / mL to about 50 ng / mL or about 30 ng / mL to about 40 ng / mL.

[0016] In one embodiment, the concentration of the growth factor having interleukin-like activity ranges from about 50 lU / mL to about 1,500 ZU / mL, about 50 ZU / mL to about 1,400 ZU / mL, about 50 ZU / mL to about 1,300 ZU / mL, about 50 lU / mL to about 1,200 lU / mL, about 50 ZU / mL to about 1,100 lU / mL, about 50 ZU / mL to about 1,000 ZU / mL, about 70 lU / mL to about 1,500 lU / mL, about 70 ZU / mL to about 1,400 ZU / mL, about 70 ZU / mL to about 1,300 ZU / mL, about 70 ZU / mL to about 1,200 ZU / mL, about 70 lU / mL to about 1,100 lU / mL, about 70 ZU / mL to about 1,000 lU / mL, 80 ZU / mL to about 1,500 lU / mL, about 80 lU / mL to about 1,400 lU / mL, about 80 ZU / mL to about 1,300 lU / mL, about 80 ZU / mL to about 1,200 ZU / mL, about 80 ZU / mL to about 1,100 ZU / mL, about 80 lU / mL to about 1,000 ZU / mL, about 90 ZU / mL to about 1,500 ZU / mL, about 90 ZU / mL to about 1,400 lU / mL, about 90 ZU / mL to about 1,300 lU / mL, about 90 ZU / mL to about 1,200 lU / mL, about 90 ZU / mL to about 1,100 ZU / mL, about 90 ZU / mL to about 1,000 ZU / mL, about 100 lU / mL to about 1,500 ZU / mL, about 100 ZU / mL to about 1,400 ZU / mL, about 100 ZU / mL to about 1,300 ZU / mL, about 100 lU / mL to about 1,200 ZU / mL, about 100 ZU / mL to about 1,100 lU / mL or about 100 ZU / mL to about 1,000 lU / mL.

[0017] In some embodiments, the interleukin is IL-2, IL-4, IL-7, IL-15, IL-21, interleukin-ip or any combination thereof. In one further embodiment, the interleukin is IL-2, IL-15, or a combination of IL-2 and IL-5. In one further embodiment, the interleukin is IL-2 in a concentration of about 100 lU / mL to about 1,000 lU / mL. In one further embodiment, the interleukin is IL- 15 in a concentration of about 10 ng / mL to about 100 ng / mL. In one further embodiment, the interleukin is about 100 lU / mL to about 1,000 lU / mL of IL-2 in combination with about 10 ng / mL to about 100 ng / mL of IL-15.

[0018] In one embodiment, the medium containing IL-15 maintains a constant expansion of V52+y5 T-cells at a high level over time.

[0019] In one embodiment, the zoledronic acid mediated V52+y5 T-cell expansion can be increased by IL-15.

[0020] In one embodiment, the medium further comprises synthetic growth factors and amino acids tailored for T cell expansion. Examples of growth factor include, but are not limited to, neurotrophic factor (BDNF), epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF-1), erythropoietin (EPO), vascular growth factor (VEGF), transforming growth factor beta (TGF-P), nerve growth factor (NGF) and platelet derived growth factors (PDGF).

[0021] Examples of amino acid include, but are not limited to, glutamine (Gin), alanine (Ala), serine (Ser), leucine (Leu), methionine (Met), arginine (Arg), cysteine (Cys) and cystine (Cys-Cys).

[0022] In one embodiment, the step of expanding the V52+y5 T cells is conducted at 37°C and 5% CO2.

[0023] In one embodiment, the harvested V52+y5 T cells are cryopreserved in a solution containing DMSO and a protective cryoprotectant.

[0024] In one aspect, the present disclosure provides a cell population produced from the method described herein.

[0025] In one embodiment, the present disclosure provides a V52+y5 T cell population, comprising enriched V52+y5 T cells.

[0026] In some embodiments, the V52+y5 T cell population described herein comprises at least about 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% V52+y5 T cells with over about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% post-expansion viability.

[0027] In one embodiment, the V52+y5 T cell population described herein is of therapeutic grade.

[0028] In one embodiment, the V52+y5 T cell population described herein has low residual aP T cells. In some embodiments, the enriched y5 T cell population has residual aP T cell less than about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%.

[0029] In one embodiment, the V52+y5 T cell population described herein comprises at least about 95%, 96%, 97%, 98% or 99% V52+y5 T cells with over about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% post-expansion viability.

[0030] In some embodiments, the V52+y5 T cells described herein express one or more CD3, CD45 and NKG2D in an intensity higher than about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99% or 99.5%.

[0031] In some embodiments, the V52+y5 T cells described herein express NKG2D in an intensity higher than about 95%, 96%, 97%, 98%, 98.5%, 99% or 99.5%. In a further embodiment, the V52+y5 T cells described herein express NKG2D in an intensity higher than about 98%.

[0032] In some embodiments, the V52+y5 T cells described herein express CD3 in an intensity higher than about 95%, 96%, 97%, 98%, 98.5%, 99% or 99.5%. In a further embodiment, the V52+y5 T cells described herein express CD3 in an intensity higher than about 99%.

[0033] In some embodiments, the V52+y5 T cells described herein express CD45 in an intensity higher than about 95%, 96%, 97%, 98%, 98.5%, 99% or 99.5%. In a further embodiment, the V52+y5 T cells described herein express CD45 in an intensity higher than about 98%.

[0034] In some embodiments, the V52+y5 T cells described herein express CD3, CD45 and NKG2D in an intensity higher than about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99% or 99.5%. In a further embodiment, the V52+y5 T cells described herein express CD3, CD45 and NKG2D in an intensity higher than about 95%, 96%, 97%, 98%, 98.5%, 99% or 99.5%.

[0035] In another aspect, the present disclosure provides a method of modulating an immune response in a subject, comprising administering the enriched V52+y5 T cell population described herein to the subject.

[0036] In another aspect, the present disclosure provides a method for use in immunotherapy in a subject, comprising administering the enriched V52+y5 T cell population described herein to the subject.

[0037] In some embodiments, the immunotherapy provides multifunctional immune modulation.

[0038] In some embodiments, the immunotherapy provides potent effector functionality and cytokine-producing capability of the expanded V52+y5 T cells upon tumor cell recognition.

[0039] In some embodiments, the immunotherapy is chimeric antigen receptor (CAR) T cell therapy.

[0040] In another aspect, the present disclosure provides a method for treating a cancer in a subject, comprising administering the enriched V52+y5 T cell population described herein to the subject.

[0041] In some embodiments, the cancer is sarcoma, basal cell skin cancer, lymphoma, leukemia, lymphoproliferative disorder, plasmacytoma, histiocytoma, adenoma, carcinomas of solid tissues, hypoxic tumor, genitourinary cancer, hematopoietic cancer, nervous system cancer, bile duct cancer, cervical cancer, squamous cell cancer, endometrial cancer, esophageal cancer, head and neck cancer, glioblastoma (GBM) (such as glioblastoma multiforme), kidney cancer, hepatocellular carcinoma, liver cancer, lung cancer, pancreatic cancer, melanoma, Merkel cell cancer, mesothelioma, stomach cancer, breast cancer or triple-negative breast cancer. In further embodiments, the cancer is breast cancer, lung cancer, hepatocellular carcinoma, liver cancer, pancreatic cancer or GBM.

[0042] In some further embodiments, the leukemia is chronic lymphocytic leukemia, chronic myelogenous leukemia, acute myelogenous leukemia, acute lymphoblastic leukemia, or T cell and B cell leukemias; the lymphoma is Hodgkin's lymphorma or non-Hodgkin lymphoma; and the genitourinary cancer is cervical cancer or bladder cancer.

[0043] In a further aspect, the present disclosure provides a method for treating an infectious disease in a subject, comprising administering the enriched V52+y5 T cell population described herein to the subject. In some embodiment, the infectious disease is bacterial infections such as those caused by mycobacteria (e.g. tuberculosis), viral infections such as those caused by herpes simplex viruses (HSV), human immunodeficiency viruses (HIV), the hepatitis viruses, SARS-CoV-2 viruses, papillomaviruses (HPV), Epstein-Barr viruses (EBV), measles viruses, cytomegaloviruses (CMV), hepatitis C viruses (HCV), or influenza viruses, and parasitic infections such as those caused by plasmodium (e.g. malaria).

[0044] In one embodiment, the infectious disease is a viral infection. In some embodiments, the viral infection is caused by human immunodeficiency virus (HIV), SARS-CoV-2, papillomavirus (HPV), herpes simplex virus (HSV), Epstein-Barr virus (EBV), measles virus, cytomegalovirus (CMV), hepatitis C virus (HCV), or influenza virus.

[0045] In a further aspect, the present disclosure provides a method for vaccinating an animal comprising administering the enriched V52+y5 T cell population described herein to the animal.Brief Description Of The Drawings

[0046] Figure 1 shows that zoledronic acid mediates V52+y5 T-cell expansion at different IL-2 concentrations depending on the concentrations of zoledronic acid.

[0047] Figure 2 shows the effect of different cytokine combinations on zoledronic acid mediated y5 T-cell expansion.

[0048] Figure 3 shows that zoledronic acid mediates V52+y5 T-cell expansion at different concentrations of IL-15.

[0049] Figure 4 shows constant long-term V52+y5 T-cell expansion by adding IL-15.

[0050] Figures 5 (A) to (E) show flow cytometric characterization of expanded V52+ y5 T cells. (A) Live-cell gating of cultured PBMC-derived cells. Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors and subjected to V52+y5 T-cell expansion. Zombie dye staining identified 95.65% of cells as viable, confirming high survival during the 14-day culture period. (B) Identification of CD45+CD3+T-cell gate. Analysis of live cells showed that 98.59% of the population consisted of CD45+CD3+lymphocytes, demonstratingsuccessful enrichment of T-lineage cells following culture. (C) Quantification of residual aP T cells. Within the CD3+fraction, TCRaP-cells accounted for 99.90%, with only 0.09% of the population expressing TCRaP+, confirming minimal residual aP T-cell contamination after the expansion process. (D) Enrichment of V52+y5 T cells. Among TCRaP-cells, V52+y5 T cells constituted 96.11% of the population, whereas V51+cells were undetectable (0.00%), indicating highly selective expansion of the V52subset. (E) Surface expression of NKG2D in expanded V52+y5 T cells. Expanded V52+y5 T cells exhibited high NKG2D expression (98.30%), indicating acquisition of an activated cytotoxic phenotype suitable for anti-tumor applications.

[0051] Figures 6 (A) to (C) show cytotoxic activity of expanded V52+y5 T cells against multiple human cancer cell lines.. (A) Cytotoxicity against MDA-MB231 human breast cancer cells. Expanded V52+y5 T cells were co-cultured with MDA-MB231 cells at effector-to-target (E / T) ratios of 0:1, 1:1, 2:1, 5:1, and 10:1 for 24 hours. Annexin V / PI staining demonstrated a dose-dependent increase in tumor cell apoptosis, with >80% apoptosis observed at an E / T ratio of 5:1 and sustained at 10:1. (B) Cytotoxicity against Huh-7 human liver cancer cells. Expanded V52+y5 T cells induced apoptosis in Huh-7 cells in an E / T ratio-dependent manner. At 5:1 and 10:1 ratios, >80% of tumor cells were apoptotic. Limited cell recovery at higher ratios was noted due to extensive cell death and fragmentation during co-culture. (C) Cytotoxicity against A549 human lung cancer cells. A549 tumor cells exhibited a higher resistance to V52+y5 T-cell-mediated cytotoxicity. Dose-dependent apoptosis was observed, with >80% apoptosis achieved at the 10:1 E / T ratio, indicating cell line-specific differences in susceptibility to V52+y5 T-cell killing.

[0052] Figures 7 (A) to (D) show the cytokine secretion profile of unmodified V52+y5 T cells following co-culture with tumor target cell. (A) IFN-y secretion at increasing effector-to-target ratios. Unmodified V52+y5 T cells were co-cultured with tumor cells at E:T ratios of 0:1, 0.5:1, 1:1, and 2:1 for 24 hours. ELISA quantification demonstrated a dose-dependent increase in IFN-y secretion, reaching levels approaching 300 pg / mL at the 2:1 ratio, indicating strong Thl-type activation. (B) IL-4 secretion at increasing effector-to-target ratios. IL-4 secretion progressively increased with higher E:T ratios, showing that V52+y5 T cells retain the capacity to produce Th2-associated cytokines following tumor cell stimulation. (C) IL-9 secretion at increasing effector-to-target ratios. IL-9 production exhibited a dose-dependent increase, with concentrations approaching 100 pg / mL at the 2:1 E:T ratio, indicating activation of additional effector cytokine pathways. (D) TNF-a secretion at increasing effector-to-targetratios. TNF-a levels increased in a ratio-dependent manner, almost reaching 40 pg / mL at the 2:1 ratio, consistent with a strong pro-inflammatory and cytotoxic activation profile.

[0053] Figures 8 (A) and (B) show in vivo anti-tumor efficacy of unmodified y5 T cells in a Huh7 hepatocellular carcinoma xenograft model. (A) Dose-dependent tumor growth inhibition following co-inoculation of V52+y5 T cells with Huh7 cells. (B) Therapeutic efficacy of systemically administered y5 T cells in established tumors.

[0054] Figure 9 shows in vivo anti-tumor efficacy of unmodified y5 T cells in an A549 lung cancer xenograft model. Unmodified V52+y5 T cells markedly inhibited tumor growth in the co-inj ection model.

[0055] Figures 10 (A) and (B) show cytotoxic activity of unmodified V52+y5 T cells against BxPC-3 pancreatic cancer cells measured by real-time cell analysis. (A) Real-time cell index profiles of BxPC-3 cells cultured alone or co-cultured with unmodified V52+y5 T cells at BxPC-3 :T ratios of 1:2, 1:3, and 1:4. (B) Quantitative comparison of cell indices at 24 and 48 hours after y5 T cell addition confirmed significant growth inhibition in all treatment groups, with near-complete suppression of BxPC-3 cell proliferation at ratios of 1:4.

[0056] Figures 11(A) to (D) show that y5 T cells exhibit superior cytotoxic effects against GBM cells (U87MG). (A) Representative flow cytometry plots of CD3+ / CD45+T cells showing the proportion of aP T cells (TCRaPQ and y5 T cells (TCRy5+). (B) y5 T cells exhibit a distinct TCRy5+population, whereas aP T cells predominantly express TCRaP+. (C) Quantification of viable GBM cells by luciferase assay after co-culture with aP or y5 T cells at 1:1 and 1:0.5 GBM:T cell ratios. (D) Cytotoxicity of y5 T cells and aP T cells against GBM cells. Statistical significance: *p < 0.001, p < 0.05, n.s = not significant.

[0057] Figures 12 (A) to (D) show in vivo anti-tumor efficacy of unmodified y5 T cells in an orthotopic glioblastoma (U87MG) mouse model. (A, B) Baseline tumor bioluminescence before V52+y5 T-cell administration (Day 0). An orthotopic glioblastoma model was generated by stereotactic implantation of U87MG-luc cells into the right striatum of immunodeficient mice. Bioluminescence imaging (BLI) was performed on Day 0 to confirm tumor engraftment and establish baseline tumor burden. (C, D) Tumor bioluminescence following intracranial V52+y5 T-cell administration (Day 2). Unmodified V52+y5 T cells were injected intracranially at the tumor site on Day 0, as indicated by the injection marker. BLI analysis on Day 2 demonstrated a substantial reduction in luminescence intensity relative to baseline, indicating early in vivo anti -tumor activity of the administered V52+y5 T cells.

[0058] Figures 13 (A) to (C) show inhibition of glioblastoma growth by co-injection of unmodified V52+y5 T cells in an orthotopic U87MG mouse model. (A) Representativebioluminescence imaging (BLI) of mice implanted orthotopically with U87MG-luc cells alone (U87L) or co-injected with unmodified V52+y5 T cells at U87:T ratios of 1:2, 1:4, and 1:8. (B) Quantitative BLI analysis showed significant reduction in photon flux in the GDT-treated groups compared with the control group (U87L). (C) Kaplan-Meier survival analysis demonstrated prolonged survival in mice receiving V52+y5 T cells, with complete tumor growth suppression in the 1:4 and 1:8 groups.Detailed Description of the Invention

[0059] When items are connected by the conjunction "and," it should not be interpreted as necessitating the presence of each individual item in the group; instead, it should be understood as "and / or," unless explicitly stated otherwise. Likewise, when items are linked by "or," it should not be construed as requiring that the items be mutually exclusive; rather, it should also be interpreted as "and / or," unless explicitly stated otherwise. Additionally, while items, elements, or components of the invention may be referred to in the singular form, the plural form is considered to be included within the scope unless there is a clear indication limiting it to the singular.

[0060] As used herein, the terms "a," "an," and "the" should be interpreted to encompass both singular and plural forms, unless specified otherwise. Therefore, "a," "an," and "the" (along with their grammatical variations, as applicable) refer to one or more entities.

[0061] The term "pharmaceutically acceptable" as used in this context refers to compounds, materials, compositions, and / or dosage forms that, according to sound medical judgment, are appropriate for contact with the tissues of a subject (whether human or nonhuman animal) without causing excessive toxicity, irritation, allergic reactions, or other complications, while maintaining a reasonable benefit / risk ratio. Additionally, each carrier, excipient, and similar component must be deemed "acceptable" in terms of compatibility with the other ingredients in the formulation. Suitable carriers and excipients can be identified in standard pharmaceutical references.

[0062] The term "treatment" is understood as meaning to lessen or decrease at least one sign, symptom, indication, or effect of a specific disease or condition. As used herein, "prevention" is understood to mean limiting, reducing the rate or degree of onset, or inhibiting the development of at least one sign or symptom of a disease or condition.

[0063] As used herein, the term "subject" is any animal that can benefit from the administration of a compound or composition as disclosed herein. In some embodiments, the subject is a mammal, for example, a human, a primate, a dog, a cat, a horse, a cow, a pig, a rodent, such as a rat or mouse. Typically, the mammal is a human.

[0064] The term "effective amount" as referenced here refers to the quantity of the cells that is sufficient to positively impact the condition being treated, while remaining low enough to minimize the risk of serious side effects, all within the bounds of prudent medical judgment.

[0065] The present disclosure relates to novel methods for the isolation and the selective in vitro! ex vivo expansion and differentiation of y5 T cells (such as V52+y5 T cells), and their clinical application. Accordingly, the present disclosure provides a scalable and reproducible method for producing therapeutic-grade V52+y5 T cells described herein suitable for clinical immunotherapy applications. The method begins with isolating PBMCs using Ficoll-Paque density gradient centrifugation. Optionally, the isolated PBMCs are enriched for V52+y5 T cells using immunomagnetic beads. The selected cells are then cultured in a serum-free medium supplemented with zoledronic acid and cytokines (such as IL-2, IL-4, IL-7, IL-15, IL-21, interleukin- ip or any combination thereof) to promote cell growth and functional activation. Optionally, the enriched V52+y5 T cell population can be treated with immunomagnetic beads to remove the non- V52+y5 T cells to further enrich the V52+y5 T cells. The enriched V52+y5 T cell populations described herein cells are further expanded, harvested, washed, and cryopreserved under Good Manufacturing Practices GMP) conditions.

[0066] The PBMCs may be isolated from a sample of blood using techniques known in the art such as density gradient centrifugation. Optionally, the V52+y5 T cell subpopulation is selected from the isolated PBMCs using immunomagnetic separation to obtain V52+y5 T cell population having the selected V52+y5 T cell subpopulation. Immunomagnetic separation (IMS) is a tool that can efficiently isolate cells from bodily fluid or cultured cells. A mixture of cell population will be placed in a magnetic field where the cells then attach to super paramagnetic beads. Antibodies coating paramagnetic beads will bind to antigens present on the surface of cells thus capturing the cells and facilitating the concentration of these bead-attached cells.

[0067] Gamma delta (y5) T cells are considered the archetype of unconventional T cells and constitute a relatively small fraction of T cells found in peripheral blood. They are characterized by the presence of heterodimeric T-cell receptors (TCRs) made up of y and 5 chains, distinguishing them from the more familiar CD4+ helper T cells and CD8+ cytotoxic T cells, which express aP TCRs. The process of (thymic) selection for y5 T cells remains largely unclear. In general, y5 T cells are enriched in epithelial and mucosal tissues where they are thought to serve as the first line of defense against pathogenic challenge. The majority of y5 T cells are activated in an MHC-independent manner, in striking contrast to MHC -restricted aP T cells. The antigens recognized by most y5 T cells are still unknown.

[0068] y5 T cells exhibit significant functional versatility upon recognizing infected or transformed cells. They produce various cytokines (such as IFN-y, TNF-a, and IL-17) and chemokines (including RANTES, IP- 10, and lymphotactin), engage in the cytolysis of these target cells through mechanisms involving performing, granzymes, and TRAIL, and interact with a range of other cell types, including epithelial cells, monocytes, dendritic cells, neutrophils, and B cells. Moreover, y5 T cells can recognize and eliminate a wide range of cancers without the need for MHC restriction, underscoring their promise for universal immunotherapy. This stands in contrast to aP T-cell mediated immunotherapy, which is restricted by MHC.

[0069] The method of the present disclosure and the cell population produced therefrom provide an enriched V52+y5 T cell subpopulation. V52+y5 T cells form the predominant human y8 T-cell population in peripheral blood and mediate T-cell receptor (TCR)-dependent anti¬ microbial and anti-tumor immunity.

[0070] A medium containing zoledronic acid and one or more growth factors having interleukin-like activity is used to culture the y5 T cell population having selected V52+y5 T cell subpopulation to enrich the V52+y5 T cells. The concentration of zoledronic acid is, for example, about 1 to about 30 pM, about 1 to about 28 pM, about 1 to about 25 pM, about 1 to about 23 pM, about 1 to about 20 pM, about 1 to about 18 pM, about 1 to about 15 pM, about 1 to about 13 pM, about 1 to about 10 pM, about 1 to about 8 pM, about 1 to about 5 pM, about 3 to about 30 pM, about 3 to about 28 pM, about 3 to about 25 pM, about 3 to about 23 pM, about 3 to about 20 pM, about 3 to about 18 pM, about 3 to about 15 pM, about 3 to about 13 pM, about 3 to about 10 pM, about 3 to about 8 pM, about 3 to about 5 pM, about 5 to about 30 pM, about 5 to about 28 pM, about 5 to about 25 pM, about 5 to about 23 pM, about 5 to about 20 pM, about 5 to about 18 pM, about 5 to about 15 pM, about 5 to about 13 pM, about 5 to about 10 pM, about 8 to about 30 pM, about 8 to about 28 pM, about 8 to about 25 pM, about 8 to about 23 pM, about 8 to about 20 pM, about 8 to about 15 pM, about 8 to about 12 pM, about 8 to about 10 pM, about 10 to about 30 pM, about 10 to about 28 pM, about 10 to about 25 pM, about 10 to about 23 pM, about 10 to about 20 pM, about 10 to about 18 pM, or about 10 to about 15 pM.

[0071] The concentration of the growth factor having interleukin-like activity ranges from about 5 ng / mL to about 200 ng / mL or about 50 lU / mL to about 1,500 lU / mL. In one embodiment, the concentration of the growth factor having interleukin-like activity ranges from about 5 ng / mL to about 200 ng / mL, about 5 ng / mL to about 180 ng / mL, about 5 ng / mL to about 150 ng / mL, about 5 ng / mL to about 100 ng / mL, about 5 ng / mL to about 80 ng / mL,about 5 ng / mL to about 70 ng / mL, about 5 ng / mL to about 60 ng / mL, about 5 ng / mL to about 50 ng / mL, about 5 ng / mL to about 40 ng / mL, about 5 ng / mL to about 30 ng / mL, about 5 ng / mL to about 20 ng / mL, about 10 ng / mL to about 200 ng / mL, about 10 ng / mL to about 180 ng / mL, about 10 ng / mL to about 150 ng / mL, about 10 ng / mL to about 100 ng / mL, about 10 ng / mL to about 80 ng / mL, about 10 ng / mL to about 70 ng / mL, about 10 ng / mL to about 60 ng / mL, about 10 ng / mL to about 50 ng / mL, about 10 ng / mL to about 40 ng / mL, about 10 ng / mL to about 30 ng / mL, about 20 ng / mL to about 200 ng / mL, about 20 ng / mL to about 180 ng / mL, about 20 ng / mL to about 150 ng / mL, about 20 ng / mL to about 100 ng / mL, about 20 ng / mL to about 80 ng / mL, about 20 ng / mL to about 70 ng / mL, about 20 ng / mL to about 60 ng / mL, about 20 ng / mL to about 50 ng / mL, about 20 ng / mL to about 40 ng / mL, about 20 ng / mL to about 30 ng / mL, about 30 ng / mL to about 200 ng / mL, about 30 ng / mL to about 180 ng / mL, about 30 ng / mL to about 150 ng / mL, about 30 ng / mL to about 100 ng / mL, about 30 ng / mL to about 80 ng / mL, about 30 ng / mL to about 70 ng / mL, about 30 ng / mL to about 60 ng / mL, about 30 ng / mL to about 50 ng / mL or about 30 ng / mL to about 40 ng / mL.

[0072] Alternatively, the concentration of the growth factor having interleukin-like activity ranges from about 50 lU / mL to about 1,500 lU / mL, about 50 lU / mL to about 1,400 lU / mL, about 50 lU / mL to about 1,300 lU / mL, about 50 lU / mL to about 1,200 lU / mL, about 50 lU / mL to about 1,100 lU / mL, about 50 lU / mL to about 1,000 lU / mL, about 70 lU / mL to about 1,500 lU / mL, about 70 lU / mL to about 1,400 lU / mL, about 70 lU / mL to about 1,300 lU / mL, about 70 lU / mL to about 1,200 lU / mL, about 70 lU / mL to about 1,100 lU / mL, about 70 lU / mL to about 1,000 lU / mL, 80 lU / mL to about 1,500 lU / mL, about 80 lU / mL to about 1,400 lU / mL, about 80 lU / mL to about 1,300 lU / mL, about 80 lU / mL to about 1,200 lU / mL, about 80 lU / mL to about 1,100 lU / mL, about 80 lU / mL to about 1,000 lU / mL, about 90 lU / mL to about 1,500 lU / mL, about 90 lU / mL to about 1,400 lU / mL, about 90 lU / mL to about 1,300 lU / mL, about 90 lU / mL to about 1,200 lU / mL, about 90 lU / mL to about 1,100 lU / mL, about 90 lU / mL to about 1,000 lU / mL, about 100 lU / mL to about 1,500 lU / mL, about 100 lU / mL to about 1,400 lU / mL, about 100 lU / mL to about 1,300 lU / mL, about 100 lU / mL to about 1,200 lU / mL, about 100 lU / mL to about 1,100 lU / mL or about 100 lU / mL to about 1,000 lU / mL.

[0073] Preferably, the medium is free of serum. Examples of the medium include, but are not limited to, RPMI medium, TexMACS medium, IMDM medium, DMEM medium, CTS OpTMizer medium, KBM581 medium, AIM-V medium or X-VIVO medium, which can be used for culture by adding zoledronic acid and one or more growth factors having interleukinlike activity.

[0074] Optionally, V52+y5 T cells in the y5 T cell population having enriched V52+y5 T cell subpopulation can be further enriched by using immunomagnetic separation to remove the non-V52+y5 T cells in the population or subpopulation.

[0075] The y5 T cell population having an enriched V52+y5 T cell subpopulation is further expanded for 7-28 days and then the resulting cell population is harvested.

[0076] The cell population produced from the method of the present disclosure comprises an enriched V52+y5 T cell subpopulation. For example, the enriched y5 T cell population comprises at least about 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% V52+T cells with over about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% post-expansion viability.

[0077] The V52+y5 T cell population of the present disclosure may provide a clinical grade cell line bank capable of proliferation and differentiation for use in a large number of patients. In certain embodiments, the V52+y5 T cells that have been collected and processed can be stored in a cell bank for future applications. These cells may be preserved using cryopreservation agents like DMSO or CryoStor™ and maintained at controlled freezing rates in liquid nitrogen. The y5 T cells can be organized into unitized storage, with specific units or dosages prepared for single or multiple treatment sessions.

[0078] The present disclosure also encompass V52+y5 T cells derived from the methods described herein for the purpose of modulating an immune response, treating infections, or addressing cancer as previously detailed.

[0079] For example, a method for treating a subject's infection or cancer is described, which involves administering the V52+y5 T cell population of the present disclosure. In this approach, the V52+y5 T cell population of the present disclosure is utilized to address various conditions, including viral, bacterial, fungal, or protozoan infections, as well as cancers in a subject.

[0080] In embodiments, the virus can be hepatitis B, hepatitis C, influenza (for example, a pandemic influenza virus from birds or pigs, such as H5N1, H7N3, H7N7, H7N9 and H9N2 (bird subtype) or H1N1, H1N2, H2N1, H3N1, H3N2H2N3 (pig subtype)), herpes variant, cytomegalovirus (CMV), Epsteiner-Bar virus, varicella, papillomavirus, Ebola, varicellazoster virus, or natural pox.

[0081] In embodiments, the cancer can be sarcoma, basal cell skin cancer, lymphoma, leukemia, lymphoproliferative disorder, plasmacytoma, histiocytoma, adenoma, carcinomas of solid tissues, hypoxic tumor, genitourinary cancer, hematopoietic cancer, nervous system cancer, bile duct cancer, cervical cancer, squamous cell cancer, endometrial cancer, esophagealcancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, melanoma, Merkel cell cancer, mesothelioma, stomach cancer, breast cancer or triple-negative breast cancer.

[0082] In certain embodiments, subjects receiving the V52+y5 T cell population of the present disclosure may also be given immunosuppressive agents either simultaneously, continuously, or at separate times. The use of immunosuppressive agents may assist in mitigating any negative systemic reactions to the gamma delta T cells.

[0083] The present disclosure presents a method for vaccinating an animal by administering an effective dose of the V52+y5 T cell population of the present disclosure. These cells can be obtained through the method described in the present disclosure. This vaccine is suitable for use in immunocompromised patients or subjects at an increased risk of developing infectious diseases or cancer.

[0084] Embodiments

[0085] Embodiment 1: A method for producing a cell population comprising enriched V52+y5 T cells, comprising:providing peripheral blood mononuclear cells (PBMCs); optionally the V52+y5 T cells contained in the PBMCs are separated by immunomagnetic separation to obtain a V52+y5 T cell subpopulation;culturing the PBMCs or V52+y5 T cell subpopulation in a medium containing zoledronic acid and one or more growth factors having interleukin-like activity to specifically enrich V52+y5 T cells to obtain an enriched V52+y5 T cell population; andharvesting the expanded V52+y5 T cell population.

[0086] Embodiment 2: A method for producing a cell population comprising enriched V52+y5 T cells, comprising:(al) providing peripheral blood mononuclear cells (PBMCs);(bl) culturing the PBMCs in a medium containing zoledronic acid and one or more growth factors having interleukin-like activity to specifically enrich V52+y5 T cells to obtain an enriched V52+y5 T cell population; and(cl) harvesting the expanded V52+y5 T cell population; or(a2) providing peripheral blood mononuclear cells (PBMCs);(ii) separating V52+y5 T cells by immunomagnetic separation to obtain a V52+y5 T cell subpopulation;(b2) culturing the PBMCs in a medium containing zoledronic acid and one or more growth factors having interleukin-like activity to specifically enrich V52+y5 T cells to obtain an enriched V52+y5 T cell population; and(c2)harvesting the expanded V52+y5 T cell population

[0087] Embodiment 3: The method of any one of preceding embodiments, wherein after step (b), (bl) or (b2), the method further comprises a step (e) of separating the enriched V52+y5 T cell population by immunomagnetic separation to remove non-V52+y5 T cells.

[0088] Embodiment 4: The method of any one of preceding embodiments, wherein after step (b) , (bl), (b2) or (e), the method further comprises a step (f) of expanding the enriched V52+y5 T cell population by culturing it for 7-28 days to obtain the expanded V52+y5 T cell population.

[0089] Embodiment 5: The embodiments of any one of preceding embodiments, wherein the V52+y5 cells in the V52+y5 T cell population are unmodified.

[0090] Embodiment 6: The method of any one of preceding embodiments, wherein the PBMCs are obtained from a peripheral blood or umbilical cord blood or fractions thereof.

[0091] Embodiment 7: The method of any one of preceding embodiments, wherein the zoledronic acid is at a concentration of about 1 to about 30 pM, about 1 to about 28 pM, about 1 to about 25 pM, about 1 to about 23 pM, about 1 to about 20 pM, about 1 to about 18 pM, about 1 to about 15 pM, about 1 to about 13 pM, about 1 to about 10 pM, about 1 to about 8 pM, about 1 to about 5 pM, about 3 to about 30 pM, about 3 to about 28 pM, about 3 to about 25 pM, about 3 to about 23 pM, about 3 to about 20 pM, about 3 to about 18 pM, about 3 to about 15 pM, about 3 to about 13 pM, about 3 to about 10 pM, about 3 to about 8 pM, about 3 to about 5 pM, about 5 to about 30 pM, about 5 to about 28 pM, about 5 to about 25 pM, about 5 to about 23 pM, about 5 to about 20 pM, about 5 to about 18 pM, about 5 to about 15 pM, about 5 to about 13 pM, about 5 to about 10 pM, about 8 to about 30 pM, about 8 to about 28 pM, about 8 to about 25 pM, about 8 to about 23 pM, about 8 to about 20 pM, about 8 to about 15 pM, about 8 to about 12 pM, about 8 to about 10 pM, about 10 to about 30 pM, about 10 to about 28 pM, about 10 to about 25 pM, about 10 to about 23 pM, about 10 to about 20 pM, about 10 to about 18 pM, or about 10 to about 15 pM.

[0092] Embodiment 7: The method of any one of preceding embodiments, wherein the medium is free of serum.

[0093] Embodiment 8: The method of any one of preceding embodiments, wherein the growth factor having interleukin-like activity is interleukin.

[0094] Embodiment 9: The method of any one of preceding embodiments, wherein the concentration of the growth factor having interleukin-like activity ranges from about 5 ng / mL to about 200 ng / mL or about 50 lU / mL to about 1,500 lU / mL.

[0095] Embodiment 10: The method of any one of preceding embodiments, wherein the concentration of the growth factor having interleukin-like activity ranges from about 5 ng / mL to about 200 ng / mL, about 5 ng / mL to about 180 ng / mL, about 5 ng / mL to about 150 ng / mL, about 5 ng / mL to about 100 ng / mL, about 5 ng / mL to about 80 ng / mL, about 5 ng / mL to about 70 ng / mL, about 5 ng / mL to about 60 ng / mL, about 5 ng / mL to about 50 ng / mL, about 5 ng / mL to about 40 ng / mL, about 5 ng / mL to about 30 ng / mL, about 5 ng / mL to about 20 ng / mL, about 10 ng / mL to about 200 ng / mL, about 10 ng / mL to about 180 ng / mL, about 10 ng / mL to about 150 ng / mL, about 10 ng / mL to about 100 ng / mL, about 10 ng / mL to about 80 ng / mL, about 10 ng / mL to about 70 ng / mL, about 10 ng / mL to about 60 ng / mL, about 10 ng / mL to about 50 ng / mL, about 10 ng / mL to about 40 ng / mL, about 10 ng / mL to about 30 ng / mL, about 20 ng / mL to about 200 ng / mL, about 20 ng / mL to about 180 ng / mL, about 20 ng / mL to about 150 ng / mL, about 20 ng / mL to about 100 ng / mL, about 20 ng / mL to about 80 ng / mL, about 20 ng / mL to about 70 ng / mL, about 20 ng / mL to about 60 ng / mL, about 20 ng / mL to about 50 ng / mL, about 20 ng / mL to about 40 ng / mL, about 20 ng / mL to about 30 ng / mL, about 30 ng / mL to about 200 ng / mL, about 30 ng / mL to about 180 ng / mL, about 30 ng / mL to about 150 ng / mL, about 30 ng / mL to about 100 ng / mL, about 30 ng / mL to about 80 ng / mL, about 30 ng / mL to about 70 ng / mL, about 30 ng / mL to about 60 ng / mL, about 30 ng / mL to about 50 ng / mL or about 30 ng / mL to about 40 ng / mL.

[0096] Embodiment 11: The method of any one of preceding embodiments, wherein the concentration of the growth factor having interleukin-like activity ranges from about 50 lU / mL to about 1,500 lU / mL, about 50 lU / mL to about 1,400 lU / mL, about 50 lU / mL to about 1,300 lU / mL, about 50 lU / mL to about 1,200 lU / mL, about 50 lU / mL to about 1,100 lU / mL, about 50 lU / mL to about 1,000 lU / mL, about 70 lU / mL to about 1,500 lU / mL, about 70 lU / mL to about 1,400 lU / mL, about 70 lU / mL to about 1,300 lU / mL, about 70 lU / mL to about 1,200 lU / mL, about 70 lU / mL to about 1,100 lU / mL, about 70 lU / mL to about 1,000 lU / mL, 80 lU / mL to about 1,500 lU / mL, about 80 lU / mL to about 1,400 lU / mL, about 80 lU / mL to about 1,300 lU / mL, about 80 lU / mL to about 1,200 lU / mL, about 80 lU / mL to about 1,100 lU / mL, about 80 lU / mL to about 1,000 lU / mL, about 90 lU / mL to about 1,500 lU / mL, about 90 lU / mL to about 1,400 lU / mL, about 90 lU / mL to about 1,300 lU / mL, about 90 lU / mL to about 1,200 lU / mL, about 90 lU / mL to about 1,100 lU / mL, about 90 lU / mL to about 1,000 lU / mL, about 100 lU / mL to about 1,500 lU / mL, about 100 lU / mL to about 1,400 lU / mL, about 100 lU / mL to about 1,300 lU / mL, about 100 lU / mL to about 1,200 lU / mL, about 100 lU / mL to about 1,100 lU / mL or about 100 lU / mL to about 1,000 lU / mL.

[0097] Embodiment 12: The method of any one of preceding embodiments, wherein the interleukin is IL-2, IL-4, IL-7, IL-15, IL-21, interleukin- ip or any combination thereof.

[0098] Embodiment 13: The method of any one of preceding embodiments, wherein the interleukin is IL-2, IL- 15, or a combination of IL-2 and IL-5.

[0099] Embodiment 14: The method of any one of preceding embodiments, wherein the interleukin is IL-2 in a concentration of about 100 HJ / mL to about 1,000 lU / mL.

[0100] Embodiment 15: The method of any one of preceding embodiments, wherein the interleukin is IL- 15 in a concentration of about 10 ng / mL to about 100 ng / mL.

[0101] Embodiment 16: The method of any one of preceding embodiments, wherein the interleukin is about 100 HJ / mL to about 1,000 lU / mL of IL-2 in combination with about 10 ng / mL to about 100 ng / mL of IL-15.

[0102] Embodiment 17: The method of any one of preceding embodiments, wherein the medium containing IL-15 maintains a constant expansion of V52+y5 T-cells at a high level over time.

[0103] Embodiment 18: The method of any one of preceding embodiments, wherein the zoledronic acid mediated V52+y5 T-cell expansion can be increased by IL-15.

[0104] Embodiment 19: The method of any one of preceding embodiments, which comprises the steps of:providing peripheral blood mononuclear cells (PBMCs); optionally the V52+y5 T cells contained in the PBMCs are separated by immunomagnetic separation to obtain a V52+y5 T cell subpopulation;culturing the PBMCs or V52+y5 T cell subpopulation in a medium containing zoledronic acid at a concentration of about 5 pM to about 10 pM, about 5 pM or about 10 pM, and IL- 12 at a concentration of about 100 lU / mL to about 1000 lU / mL, about 100 lU / mL or about 1000 lU / mL to specifically enrich V52+y5 T cells to obtain an enriched V52+y5 T cell population; andharvesting the expanded V52+y5 T cell population.

[0105] Embodiment 20: The method of any one of preceding embodiments, which comprises the steps of:providing peripheral blood mononuclear cells (PBMCs); optionally the V52+y5 T cells contained in the PBMCs are separated by immunomagnetic separation to obtain a V52+y5 T cell subpopulation;culturing the PBMCs or V52+y5 T cell subpopulation in a medium containing zoledronic acid at a concentration of about 5 pM to about 10 pM, about 5 pM or about 10 pM, and IL- 12 at aconcentration of about 100 lU / mL to about 1000 lU / mL, about 100 lU / mL or about 1000 lU / mL to specifically enrich V52+y5 T cells to obtain an enriched V52+y5 T cell population; optionally separating the enriched V52+y5 T cell population by immunomagnetic separation to remove non-V52+y5 T cells;expanding the enriched V52+y5 T cell population by culturing it for 7-28 days to obtain the expanded V52+y5 T cell population; andharvesting the expanded V52+y5 T cell population.

[0106] Embodiment 21: The method of any one of preceding embodiments, which comprises the steps of:providing peripheral blood mononuclear cells (PBMCs); optionally the V52+y5 T cells contained in the PBMCs are separated by immunomagnetic separation to obtain a V52+y5 T cell subpopulation;culturing the PBMCs or V52+y5 T cell subpopulation in a medium containing zoledronic acid at a concentration of about 5 pM to about 10 pM, about 5 pM or about 10 pM, and about 100 lU / mL to about 1000 lU / mL, about 100 lU / mL or about 1000 lU / mL of IL-12 in combination with about 100 lU / mL to about 1000 lU / mL, about 100 lU / mL or about 1000 lU / mL of IL-15 to specifically enrich V52+y5 T cells to obtain an enriched V52+y5 T cell population; and harvesting the expanded V52+y5 T cell population.

[0107] Embodiment 22: The method of any one of preceding embodiments, which comprises the steps of:providing peripheral blood mononuclear cells (PBMCs); optionally the V52+y5 T cells contained in the PBMCs are separated by immunomagnetic separation to obtain a V52+y5 T cell subpopulation;culturing the PBMCs or V52+y5 T cell subpopulation in a medium containing zoledronic acid at a concentration of about 5 pM to about 10 pM, about 5 pM or about 10 pM, and about 100 lU / mL to about 1000 lU / mL, about 100 lU / mL or about 1000 lU / mL of IL-12 in combination with about 100 lU / mL to about 1000 lU / mL, about 100 lU / mL or about 1000 lU / mL of IL-15 to specifically enrich V52+y5 T cells to obtain an enriched V52+y5 T cell population; optionally separating the enriched V52+y5 T cell population by immunomagnetic separation to remove non-V52+y5 T cells;expanding the enriched V52+y5 T cell population by culturing it for 7-28 days to obtain the expanded V52+y5 T cell population; andharvesting the expanded V52+y5 T cell population.

[0108] Embodiment 23: The method of any one of preceding embodiments, wherein the medium further comprises synthetic growth factors and amino acids tailored for T cell expansion; preferably the growth factor is neurotrophic factor (BDNF), epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF-1), erythropoietin (EPO), vascular growth factor (VEGF), transforming growth factor beta (TGF-P), nerve growth factor (NGF) or platelet derived growth factors (PDGF); preferably the amino acid is glutamine (Gin), alanine (Ala), serine (Ser), leucine (Leu), methionine (Met), arginine (Arg), cysteine (Cys) or cystine (Cys-Cys).

[0109] Embodiment 24: The method of any one of preceding embodiments, wherein the step of expanding the V52+y5 T cells is conducted under 37°C and 5% CO2.

[0110] Embodiment 25: The method of any one of preceding embodiments, wherein the harvested V52+y5 T cells are cryopreserved in a solution containing DMSO and a protective cryoprotectant.

[0111] Embodiment 26: A cell population produced from a method of any one of preceding embodiments.

[0112] Embodiment 27: A V52+y5 T cell population, comprising enriched V52+y5 T cells.

[0113] Embodiment 28: The V52+y5 T cell population of Embodiment 27, wherein the V52+y5 T cell population comprises at least about 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% V52+y5 T cells with over about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% post-expansion viability.

[0114] Embodiment 29: The V52+y5 T cell population of Embodiment 26, 27 or 28, the V52+y5 T cell population described herein is in therapeutic-grade.

[0115] Embodiment 30: The V52+y5 T cell population of any one of Embodiments 26 to 29, wherein the V52+y5 T cell population has low residual aP T cells.

[0116] Embodiment 31: The V52+y5 T cell population of any one of Embodiments 26 to 30, wherein the enriched y5 T cell population has residual aP T cell less than about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%.

[0117] Embodiment 32: The V52+y5 T cell population of any one of Embodiments 26 to 31, wherein the V52+y5 T cell population comprises at least about 95%, 96%, 97%, 98% or 99% V52+y5 T cells with over about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% post-expansion viability.

[0118] Embodiment 33: The V52+y5 T cell population of any one of Embodiments 26 to 32, wherein the V52+y5 T cells express one or more CD3, CD45 and NKG2D in an intensity higher than about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99% or 99.5%.

[0119] Embodiment 34: The V52+y5 T cell population of any one of Embodiments 26 to 33, wherein the V52+y5 T cells express NKG2D in an intensity higher than about 95%, 96%, 97%, 98%, 98.5%, 99% or 99.5%. In a further embodiment, the V52+y5 T cells described herein express NKG2D in an intensity higher than about 98%.

[0120] Embodiment 35: The V52+y5 T cell population of any one of Embodiments 26 to 34, wherein the V52+y5 T cells express CD3 in an intensity higher than about 95%, 96%, 97%, 98%, 98.5%, 99% or 99.5%. In a further embodiment, the V52+y5 T cells described herein express CD3 in an intensity higher than about 99%.

[0121] Embodiment 36: The V52+y5 T cell population of any one of Embodiments 26 to 35, wherein the V52+y5 T cells express CD45 in an intensity higher than about 95%, 96%, 97%, 98%, 98.5%, 99% or 99.5%. In a further embodiment, the V52+y5 T cells described herein express CD45 in an intensity higher than about 98%.

[0122] Embodiment 37: The V52+y5 T cell population of any one of Embodiments 26 to 36, wherein the V52+y5 T cells express CD3, CD45 and NKG2D in an intensity higher than about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99% or 99.5%. In a further embodiment, the V52+y5 T cells described herein express CD3, CD45 and NKG2D in an intensity higher than about 95%, 96%, 97%, 98%, 98.5%, 99% or 99.5%.

[0123] Embodiment 38: A method of modulating an immune response in a subject, comprising administering the enriched V52+y5 T cell population of any one of Embodiments 26 to 37 to the subject.

[0124] Embodiment 39: A method for use in immunotherapy in a subject, comprising administering the enriched V52+y5 T cell population of any one of Embodiments 26 to 37 to the subject.

[0125] Embodiment 40: The method of Embodiment 39, wherein the immunotherapy provides multifunctional immune modulation.

[0126] Embodiment 41: The method of Embodiment 39 or 40, wherein the immunotherapy provides potent effector functionality and cytokine-producing capability of the expanded V52+y5 T cells upon tumor cell recognition.

[0127] Embodiment 42: The method of Embodiment 39, 40 or 41, wherein the immunotherapy is chimeric antigen receptor (CAR) T cell therapy.

[0128] Embodiment 43: A method for treating a cancer in a subject, comprising administering the enriched V52+y5 T cell population described herein to the subject.

[0129] Embodiment 44: The method of Embodiment 43, wherein the cancer is sarcoma, basal cell skin cancer, lymphoma, leukemia, lymphoproliferative disorder, plasmacytoma,histiocytoma, adenoma, carcinomas of solid tissues, hypoxic tumor, genitourinary cancer, hematopoietic cancer, nervous system cancer, bile duct cancer, cervical cancer, squamous cell cancer, endometrial cancer, esophageal cancer, head and neck cancer, glioblastoma (GBM) (such as glioblastoma multiforme), kidney cancer, hepatocellular carcinoma, liver cancer, lung cancer, pancreatic cancer, melanoma, Merkel cell cancer, mesothelioma, stomach cancer, breast cancer, triple-negative breast cancer.

[0130] Embodiment 45: The method of Embodiment 43 or 44, wherein the cancer is breast cancer, lung cancer, hepatocellular carcinoma, liver cancer, pancreatic cancer or GBM.

[0131] Embodiment 46: The method of Embodiment 43 or 44, wherein the leukemia is chronic lymphocytic leukemia, chronic myelogenous leukemia, acute myelogenous leukemia, acute lymphoblastic leukemia, or T cell and B cell leukemias; the lymphoma is Hodgkin's lymphoma or non-Hodgkin lymphoma; and the genitourinary cancer is cervical cancer or bladder cancer.

[0132] Embodiment 47: A method for treating an infectious disease in a subject, comprising administering the enriched V52+y5 T cell population described herein to the subject. In some embodiment, the infectious disease is bacterial infections such as those caused by mycobacteria (e.g. tuberculosis), viral infections such as those caused by herpes simplex viruses (HSV), human immunodeficiency viruses (HIV), the hepatitis viruses, SARS-CoV-2 viruses, papillomaviruses (HPV), Epstein-Barr viruses (EBV), measles viruses, cytomegaloviruses (CMV), hepatitis C viruses (HCV), or influenza viruses, and parasitic infections such as those caused by plasmodium (e.g. malaria).

[0133] Embodiment 48: The method of Embodiment 47, wherein the infectious disease is a viral infection.

[0134] Embodiment 49: The method of Embodiment 48, wherein the viral infection is caused by human immunodeficiency virus (HIV), SARS-CoV-2, papillomavirus (HPV), herpes simplex virus (HSV), Epstein-Barr virus (EBV), measles virus, cytomegalovirus (CMV), hepatitis C virus (HCV), or influenza virus.

[0135] Embodiment 50: A method for vaccinating an animal comprising administering the enriched V52+y5 T cell population described herein to the animal.

[0136] The present invention is described in greater detail in the examples presented below, which are preceded by a brief description of the figures. It goes without saying however, that these examples are given by way of illustration of the subject of the invention and do not constitute in any manner a limitation thereto.EXAMPLES

[0137] Example 1 Zoledronic acid mediates y6 T-cell expansion at different IL-2 concentrations depending on the concentrations of zoledronic acid

[0138] Materials and Methods

[0139] PBMC isolation: Peripheral blood mononuclear cells (PBMCs) were isolated from fresh human peripheral blood or leukopak collected from healthy donors using Ficoll-Paque (Cytiva) density-gradient centrifugation. After separation, PBMCs were washed twice with PBS and resuspended in KBM581 (Kohjin Bio) medium containing 10 % human platelet lysate (HPL; EliteGro).

[0140] Initial cell seeding: PBMCs were seeded at 1 x 106cells / mL in culture flasks.

[0141] Zoledronic acid stimulation: Zoledronic acid (ZOL; Novartis) stock solution was diluted in the culture medium to final concentrations of 5 pM and 10 pM.

[0142] IL-2 supplementation: Recombinant human IL-2 (Akron Bio) was added to the culture at the following concentrations: 100 lU / mL, 1000 lU / mL.

[0143] Culture conditions: Cells were incubated at 37°C, 5% CO2 for 14 days. Cell density was maintained between 0.5-2 * 106cells / mL by adding fresh medium every 2-3 days. ZOL and IL-2 were added at Day 0. Fresh IL-2 was replenished every 2-3 days during culture.

[0144] Flow cytometric analysis: On Day 14, cells were harvested and stained with: anti-CD3 (Sony), anti-V52 (Sony), Zombie B550 viability dye (Sony). Cells were analyzed using a Sony S A3800 Spectral Analyzer. V52+y5 T-cell expansion was quantified as: Fold expansi on= Total y5 T cells on Day 14 / Total y5 T cells on Day 0.

[0145] It is shown that V52+y5 T-cell expansion in the presence of zoledronic acid was achieved at different IL-2 concentrations. Furthermore, lower concentration of zoledronic acid to V52+y5 T-cells resulted in greater V52+y5 T-cell expansion, indicating that the induction of V52+y5 T-cell expansion by zoledronic acid is dose dependent (Figure 1).

[0146] Example 2 Effect of different cytokine combinations on zoledronic acid mediated y6 T-cell expansion

[0147] Materials and Methods

[0148] PBMC isolation: PBMCs were isolated from fresh human peripheral blood or leukopak collected from healthy donors using Ficoll-Paque density-gradient centrifugation. After separation, PBMCs were washed twice with PBS and resuspended in KBM581 medium containing 10 % HPL.

[0149] Cell seeding: PBMCs were seeded at 1 x 106cells / mL in culture flasks.

[0150] ZOL stimulation: ZOL stock solution was diluted in culture medium to reach a final concentration of 5 pM, added on Day 0.

[0151] Cytokine supplementation conditions: Three cytokine conditions were tested: (1) IL-2 alone: IL-2 (1000 lU / mL), (2) IL-15 (Akron Bio) alone: IL-15 (10 ng / mL), (3) IL-2 + IL-15 combination: IL-2 (1000 HJ / mL) + IL-15 (10 ng / mL).

[0152] Culture conditions: Cells were incubated at 37°C, 5% CO2 for 14 days. Cell density was maintained between 0.5-2 * 106cells / mL by adding fresh medium every 2-3 days. ZOL and cytokines were added at Day 0. Fresh cytokines were replenished every 2-3 days during culture.

[0153] Flow cytometric analysis: On Day 14, cells were harvested and stained with: anti-CD3, anti-V52, Zombie B550 viability dye. Cells were analyzed using a Sony SA3800 Spectral Analyzer. V52+y5 T-cell expansion was quantified as: Fold expansion= Total y5 T cells on Day 14 / Total y5 T cells on Day 0.

[0154] It is shown that combining IL-2 with IL-15 can significantly enhance the zoledronic acid mediated V52+y5 T-cell expansion (Figure 2).

[0155] Example 3: Zoledronic acid mediated y6 T-cell expansion at different concentrations of IL-15

[0156] Materials and Methods

[0157] PBMC isolation: PBMCs were isolated from fresh human peripheral blood or leukopak collected from healthy donors using Ficoll-Paque density-gradient centrifugation. After separation, PBMCs were washed twice with PBS and resuspended in KBM581 medium containing 10 % HPL.

[0158] Cell seeding: PBMCs were seeded at 1 x 106cells / mL in culture flasks.

[0159] ZOL stimulation: ZOL stock solution was diluted in culture medium to reach a final concentration of 5 pM, added on Day 0.

[0160] IL-15 supplementation: Recombinant human IL-15 was added at three different concentrations: 10 ng / mL, 30 ng / mL, 100 ng / mL.

[0161] Culture conditions: Cells were maintained at 37°C, 5% CO2. Total culture duration was 14 days. ZOL and IL-15 were added at Day 0. Fresh IL-15 was replenished every 2-3 days. Fresh KBM581 medium was added every 2-3 days to maintain a density of 0.5-2 x 106cells / mL.

[0162] Flow cytometric analysis: On Day 14, cells were harvested and stained with: anti-CD3, anti-V52, Zombie B550 viability dye. Cells were analyzed using a Sony SA3800 Spectral Analyzer. V52+y5 T-cell expansion was quantified as: Fold expansion= Total V52+y5 T cells on Day 14 / Total V52+y5 T cells on Day 0.

[0163] Zoledronic acid mediated V52+y5 T-cell expansion can be increased by IL-15 in a dose dependent manner (Figure 3).

[0164] Example 4 Constant long-term y6 T-cell expansion by adding IL-15

[0165] Materials and Methods

[0166] PBMC isolation: PBMCs were isolated from fresh human peripheral blood or leukopak collected from healthy donors using Ficoll-Paque density-gradient centrifugation. After separation, PBMCs were washed twice with PBS and resuspended in KBM581 medium containing 10 % HPL.

[0167] Cell seeding: PBMCs were seeded at 1 x 106cells / mL in culture flasks.

[0168] ZOL stimulation: ZOL stock solution was diluted into culture medium to reach a final concentration of 5 pM, added on Day 0.

[0169] Cytokine supplementation for long-term expansion: IL-2 (1000 lU / mL), IL-15 (10 ng / mL).

[0170] Culture conditions: Cells were incubated at 37°C, 5% CO2 for 28 days. Cell density was maintained between 0.5-2 * 106cells / mL by adding fresh medium every 2-3 days. ZOL and cytokines were added at Day 0. Fresh cytokines were replenished every 2-3 days during culture.

[0171] Cell counting and calculation of V52+y5 T cell numbers: Total cell numbers were quantified on Days 0, 4, 7, 10, 14, 17, 21, 25, and 28 using NucleoCounter NC-200 automated cell counter. In parallel, an aliquot of cells from each timepoint was analyzed by flow cytometry to determine the proportion of V52+y5 T cells within the culture. Cells were stained with anti-CD3, anti-V52, Zombie B550 viability dye, and the percentage of viable V52+y5 T cells was recorded. Cells were analyzed using a Sony S A3800 Spectral Analyzer. The absolute number of V52+y5 T cells at each timepoint was calculated according to the following formula: Absolute V52+y5 T cell count = Total viable cells x Percentage of V52+y5 T cells.

[0172] It was possible to maintain a constant expansion of V52+y5 T-cells at a high level over long period of time by adding IL- 15 during the cultivation (Figure 4).

[0173] Example 5: Isolation and Expansion of GDT Cells

[0174] Materials and Methods

[0175] PBMC isolation: PBMCs were isolated from fresh human peripheral blood or leukopak collected from healthy donors using Ficoll-Paque density-gradient centrifugation. After separation, PBMCs were washed twice with PBS and resuspended in KBM581 medium containing 10 % HPL.

[0176] Cell seeding: PBMCs were seeded at 1 x 106cells / mL in culture flasks.

[0177] ZOL stimulation: ZOL stock solution was diluted in culture medium to reach a final concentration of 5 pM, added on Day 0.

[0178] IL-2 supplementation: Recombinant human IL-2 was added at 1000 lU / mL.

[0179] Culture conditions: Cells were incubated at 37°C, 5% CO2 for 14 days. Cell density was maintained between 0.5-2 * 106cells / mL by adding fresh medium every 2-3 days. ZOL and IL-2 were added at Day 0. Fresh IL-2 was replenished every 2-3 days during culture.

[0180] aP T cell Depletion: After 14 days of expansion, cells were harvested and subjected to TCRaP depletion using the aP T-cell Depletion Kit (Miltenyi Biotec) following the manufacturer’s protocol.

[0181] Flow Cytometry Analysis: Cells were stained with Zombie B550 viability dye (Sony), anti-CD3 (Sony), anti-CD45 (Sony), anti-TCRaP (Sony), anti-V52 (Sony), anti-V51 (Miltenyi Biotec), anti-NKG2D (Sony). Cells were analyzed using a Sony S A3800 Spectral Analyzer. Gating strategy matched Figure 5, including: Live cells (Zombie"), CD3+CD45+T cells, TCRaP" population, V51"V52+subsets, andNKG2D expression within V52+cells.

[0182] Peripheral blood mononuclear cells PBMCs) were isolated from healthy donors and enriched for V52+ T cells using immunomagnetic separation. The cells were cultured in KBM581 supplemented with IL-2 1000 lU / mL) and zoledronic acid 5 pM). After 14 days of expansion, flow cytometry analysis revealed a 10,000-fold increase in cell numbers with a viability of >90%. The expanded cells exhibited high surface expression of NKG2D, indicating a potent cytotoxic phenotype. Additionally, the expanded cell population demonstrated low residual aP T cell contamination, ensuring enhanced safety for allogeneic applications. These cells exhibited robust anti-tumor activity against various cancer cell lines, confirming their immunotherapeutic potential for clinical use (Figure 5).

[0183] Example 6 Functional Analysis of Expanded GDT Cells

[0184] Materials and Methods

[0185] Tumor Cell Preparation: MDA-MB-231 (ATCC) breast cancer cells, HuH-7 (JCRB) liver cancer cells, and A549 (ATCC) lung cancer cells were expanded according to each cell line’s recommended conditions. MDA-MB-231 cells were cultured in Leibovitz’s L-15 medium supplemented with 10% fetal bovine serum (FBS) and maintained under CCL-free incubation, whereas HuH-7 cells were maintained in low-glucose DMEM with 10% FBS and incubated at 37°C with 5% CO2. A549 cells were cultured in F-12K medium supplemented with 10% FBS under the same CO2 conditions. All cell lines were confirmed to be free of mycoplasma contamination before use. For cytotoxicity assays, each tumor cell line wasseeded at a density of 3 x io5cells per well in 6-well plates and allowed to attach for approximately 16 hours before V52+y5 T cell addition.

[0186] Co-Culture Setup with V52+y5 T Cells: Expanded V52+y5 T cells were washed and resuspended in complete medium prior to being added to the tumor cells. Co-cultures were prepared at effector-to-target ratios of 0:1, 1:1, 2:1, 5:1, and 10:1, corresponding to the addition of 0, 3 x 105, 6 x io5, 1.5 x 106, and 3 x 106V52+y5 T cells per well, respectively. Following V52+y5 T-cell addition, the plates were incubated for 24 hours at 37°C under the appropriate incubation conditions for each tumor type.

[0187] Cell Harvesting and Staining Procedure: After the 24-hour co-culture period, both suspended and adherent cells were collected for apoptosis assessment. Suspended cells were harvested first, and adherent tumor cells were washed twice with PBS and dissociated using 0.25% trypsin at 37°C for approximately three minutes. The detached cells were washed twice with PBS and combined with the suspended fraction. To distinguish tumor cells from V52+y5 T cells, the mixed cell suspension was incubated with an APC / Cy7-conjugated anti-CD45 antibody for 20 minutes on ice in the dark, followed by washing and resuspension in Annexin V binding buffer. Annexin V-FITC and propidium iodide were then added, and the cells were incubated for 15 minutes at room temperature before flow cytometry analysis. Samples were analyzed using a CytoFLEX S flow cytometer, and tumor cells were identified as CD45-negative events. A minimum of 10,000 tumor cell events were acquired for each condition.

[0188] Flow Cytometry Analysis of Apoptosis: Apoptotic states were defined based on Annexin V and propidium iodide staining patterns, with viable cells identified as Annexin V / PI", early apoptotic cells as Annexin V+ / PI , late apoptotic cells as Annexin V+ / PI+, and dead cells as Annexin V / PI+. The proportion of cells in each apoptosis category was quantified within the CD45" tumor cell population. These analyses enabled direct comparison of V52+y5 T cell-mediated cytotoxicity across different E:T ratios and tumor cell types.

[0189] Expanded GDT cells were co-cultured with MDA-MB231 cells human breast cancer cells), Huh-7 cells human liver cancer cells), and A549 cells human lung cancer cells) at effector-to-target E / T) ratios of 0:1, 1:1, 2:1, 5:1, and 10:1. After 24 hours of co-culture, Annexin V / PI staining was performed to evaluate tumor cell apoptosis. The analysis demonstrated a dose-dependent cytotoxic effect across all three cancer cell lines. For MDA-MB231 and Huh-7 cells, significant tumor cell death was observed at an E / T ratio of 5:1, where over 80% of the cancer cells underwent apoptosis. This effect was further confirmed at a 10:1 ratio, showing consistent and robust cytotoxic activity. In contrast, A549 cells required a higher E / T ratio, with over 80% of the cells undergoing apoptosis only at a 10:1 ratio. Theseresults highlight the potent and selective anti-tumor capabilities of the expanded GDT cells against multiple cancer types, supporting their potential in cancer immunotherapy applications (Figure 6).

[0190] Example 7 Cytokine secretion profile of unmodified y6 T cells following co-culture with tumor target cells

[0191] Materials and Methods

[0192] Co-Culture of V52+y5 T Cells with Tumor Target Cells: Unmodified V52+y5 T cells were co-cultured with tumor target cells to evaluate cytokine secretion following tumor recognition. Tumor cells were seeded one day prior to effector addition to ensure stable adherence and were cultured under standard conditions until use. Expanded V52+y5 T cells were washed and resuspended in complete medium before being added to the tumor cells at effector-to-target (E:T) ratios of 0:1, 0.5:1, 1:1, and 2:1. The co-cultures were maintained for 24 hours at 37°C and 5% CO2 to allow interaction between effector and target cells and subsequent cytokine release into the culture supernatant.

[0193] Collection of Supernatants for Cytokine Quantification: Following the 24-hour coculture period, cell culture supernatants were collected carefully to avoid disturbing the cell layer. The supernatants were transferred into sterile tubes and centrifuged briefly to remove cell debris. Clarified supernatants were then stored at -80°C until cytokine analysis. This ensured preservation of cytokine integrity and prevented degradation prior to ELISA measurement.

[0194] Cytokine Detection by ELISA: The concentrations of IFN-y, IL-4, IL-9, and TNF-a in the collected supernatants were quantified using commercially available human ELISA kits according to the manufacturer’s instructions. Standard curves were generated for each cytokine, and sample concentrations were calculated based on optical density measurements obtained using a microplate reader. Each sample was measured in technical replicates to ensure data accuracy and reproducibility.

[0195] Unmodified y5 T cells were co-cultured with tumor cells at various effector-to-target (E:T) ratios (0:1, 0.5:1, 1:1, and 2:1) for 24 hours. The concentrations of IFN-y, IL-4, IL-9, and TNF-a in the supernatants were quantified by ELISA. A dose-dependent increase in cytokine secretion was observed with increasing E:T ratios. Specifically, y5 T cells produced high levels of IFN-y (>300 pg / mL) and TNF-a (>40 pg / mL), indicating robust Thl-type activation, as well as detectable IL-4 and IL-9 secretion, suggesting multifunctional immune modulation. These data confirm the potent effector functionality and cytokine-producing capability of the expanded y5 T cells upon tumor cell recognition (Figure 7).

[0196] Example s In vivo anti-tumor efficacy of unmodified y6 T cells in a Huh7 hepatocellular carcinoma xenograft model.

[0197] Tumor Cell Preparation and Xenograft Establishment: Human hepatocellular carcinoma Huh-7 cells were expanded under standard culture conditions and harvested during logarithmic growth. Cells were washed with phosphate-buffered saline and resuspended in a 1:1 mixture of serum-free medium and Matrigel. Each mouse received a subcutaneous inoculation of 2 x 106Huh-7 cells in a 200-pL injection volume. Following implantation, tumor growth was monitored by caliper measurements, and tumor volume was calculated using the formula 0.5 x length x width2. Animals were observed daily to ensure stable tumor engraftment prior to treatment initiation.

[0198] Co-Inoculation Study for Dose-Dependent Tumor Inhibition: To evaluate the prophylactic anti-tumor effects of V52+y5 T cells, freshly expanded V52+y5 T cells were mixed directly with Huh-7 cells at the time of implantation. The mixtures were prepared at effector-to-target ratios of 0:1, 2:1, and 4:1, maintaining a constant tumor cell number of 2 x 106cells per injection. Tumor growth was measured throughout the study period. Mice receiving Huh-7 cells alone exhibited continuous and rapid tumor progression, whereas coadministration of V52+y5 T cells resulted in significant and ratio-dependent suppression of tumor formation. Complete inhibition of detectable tumor growth was observed in mice receiving V52+y5 T cells at the 4: 1 ratio, demonstrating potent anti-tumor activity at the site of implantation.

[0199] Systemic V52+y5 T-Cell Administration in Established Tumors: A separate cohort was used to evaluate the therapeutic efficacy of V52+y5 T cells against established tumors. Treatment began when tumor sizes reached approximately 100 mm3, typically on Day 21 after implantation. Mice received intravenous injections of V52+y5 T cells via the tail vein at doses of either 5 x 106or 2 x 107cells per administration. Injections were performed once every three days in a recurring schedule. Tumor volumes were recorded throughout the treatment period. Animals treated systemically with V52+y5 T cells showed clear inhibition of tumor growth compared to PBS-treated controls, with the higher V52+y5 T-cell dose producing the most pronounced suppression. Representative excised tumors confirmed the reductions in tumor size observed in vivo.

[0200] Each mouse received 2x]06Huh7 cells subcutaneously, either alone (E:T = 0:1) or co-injected with y5 T cells at E:T ratios of 2:1 and 4:1. Tumor volume was monitored over time. Mice receiving y5 T cells showed marked suppression of tumor growth in a dosedependent manner, with complete inhibition observed at E:T = 4:1. Treatment was initiatedwhen tumors reached approximately 100 mm3(Day 21). Mice were injected with 5*106or 2*107y5 T cells intravenously once every three days. Representative tumor images (top) and tumor growth curves (bottom) show significant tumor regression in the y5 T cell-treated groups compared to PBS controls. These data demonstrate that unmodified y5 T cells exert potent anti-tumor activity against hepatocellular carcinoma both in prophylactic and therapeutic settings (Figure 8).

[0201] Example 9 In vivo anti-tumor efficacy of unmodified y6 T cells in an A549 lung cancer xenograft model.

[0202] Establishment of the A549 Xenograft Model: Human A549 lung adenocarcinoma cells were expanded under standard culture conditions and harvested during logarithmic growth. The cells were washed with phosphate-buffered saline, resuspended in serum-free medium, and mixed with an equal volume of Matrigel to support tumor establishment. Each immunodeficient mouse received a subcutaneous injection of 2 x 106A549 cells in a total volume of 200 pL. Tumor implantation sites were monitored three times weekly, and tumor size was measured using calipers. Tumor volume was calculated using the standard formula 0.5 x length x width2.

[0203] Co-Injection Study for Evaluation of Local V52+y5 T-Cell Activity: To evaluate the direct anti-tumor activity of unmodified V52+y5 T cells at the site of tumor initiation, V52+y5 T cells were freshly expanded and resuspended in complete medium prior to injection. A549 tumor cells were combined with V52+y5 T cells at an A549:T-cell ratio of 1:2, maintaining the tumor cell inoculum at 2 x 106cells per mouse. The mixtures were injected subcutaneously into the flank region, and tumor progression was monitored at regular intervals. Mice receiving A549 cells alone exhibited progressive tumor growth, whereas those co-injected with V52+y5 T cells demonstrated rapid and sustained inhibition of tumor formation. Tumor regression was observed as early as Day 17, and near-complete disappearance of measurable tumor masses occurred by Day 24. The difference in tumor volume between treated and untreated groups was statistically significant (***p < 0.001), demonstrating potent V52+y5 T-cell-mediated suppression of A549 tumor growth.

[0204] A549 cells (2x 106) were subcutaneously implanted in immunodeficient mice, either alone or co-injected with V52+y5 T cells at an A549:T ratio of 1:2. Tumor volume was measured every three to four days. Mice receiving y5 T cells showed a rapid and sustained suppression of tumor growth, with near-complete tumor regression by Day 24 compared with the A549-alone group (***p < 0.001). These findings demonstrate that unmodified V52+y5 T cells exhibit potent in vivo cytotoxic activity against human lung cancer through local deliveryroutes. The unmodified V52+y5 T cells markedly inhibited tumor growth in the co-inj ection model (Figure 9).

[0205] Example 10: Cytotoxic activity of unmodified y6 T cells against BxPC-3 pancreatic cancer cells measured by real-time cell analysis.

[0206] Preparation of BxPC-3 Pancreatic Cancer Cells: BxPC-3 human pancreatic cancer cells were cultured under standard conditions in complete growth medium until they reached logarithmic phase. Cells were harvested, washed with phosphate-buffered saline, and resuspended in fresh culture medium prior to seeding. For real-time cytotoxicity monitoring, cells were plated into E-Plate 16 plates compatible with the xCELLigence Real-Time Cell Analysis system. A total of 1 x 104BxPC-3 cells were added per well, and the plates were inserted into the xCELLigence station to allow continuous impedance-based monitoring. Cells were permitted to attach and proliferate until a stable baseline cell index was achieved.

[0207] Co-Culture of BxPC-3 Cells with V52+y5 T Cells: Unmodified V52+y5 T cells were expanded ex vivo and prepared for co-culture by washing and resuspending them in complete medium. Once BxPC-3 cells reached stable adhesion, V52+y5 T cells were added directly into the wells at tumor-to-effector ratios of 1:2, 1:3, and 1:4. Co-cultures were maintained at 37°C with continuous impedance recording for up to 80 hours. Control wells containing only BxPC-3 cells were monitored in parallel to provide baseline proliferation profiles. The xCELLigence system recorded real-time changes in cell index, which reflect alterations in tumor cell adhesion, proliferation, and viability in response to V52+y5 T-cell-mediated cytotoxicity.

[0208] Real-Time Monitoring and Data Acquisition: Impedance values were collected automatically at predefined intervals throughout the 80-hour monitoring period. The cell index signal increased steadily in control wells containing only BxPC-3 cells, reflecting normal tumor cell proliferation. In contrast, wells containing V52+y5 T cells displayed a rapid decline in cell index shortly after effector addition, indicating disruption of tumor cell adherence and viability. The magnitude and rate of decrease varied in accordance with the effector-to-target ratio, with higher V52+y5 T cell numbers producing more pronounced cytotoxic effects.

[0209] The impedance-based xCELLigence system continuously monitored cell growth over 80 hours. BxPC-3 cells cultured alone showed progressive proliferation, whereas coculture with y5 T cells resulted in a rapid and sustained decrease in cell index, indicating potent cytotoxicity. Quantitative comparison of cell indices at 24 and 48 hours after y5 T cell addition confirmed significant growth inhibition in all treatment groups, with near-complete suppression of BxPC-3 cell proliferation at ratios of 1:4. These data demonstrate thatunmodified y5 T cells exert strong, dose-dependent cytotoxic effects against pancreatic cancer cells in vitro (Figure 10).

[0210] Example 11 y6 T cells exhibit superior cytotoxic effects against GBM cells (U87MG).

[0211] Preparation and Culture of GBM Cells: U87MG-luc human glioblastoma cells were obtained from ATCC and maintained in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum and 100 pg / mL penicillin / streptomycin. Cells were cultured under standard incubation conditions until they reached logarithmic growth. Prior to co-culture experiments, U87MG-luc cells were seeded into 96-well plates at densities optimized for luciferase-based viability assays and were allowed to adhere overnight.

[0212] Preparation and Expansion of y5 T Cells and aP T Cells: Peripheral blood mononuclear cells (PBMCs) were isolated from fresh human peripheral blood or leukopak collections obtained from healthy donors using Ficoll-Paque density-gradient centrifugation. After separation, PBMCs were washed twice with phosphate-buffered saline and resuspended in KBM581 medium supplemented with ten percent human platelet lysate. The cells were seeded at an initial density of 1 x 106cells per milliliter in tissue culture flasks. Zoledronic acid was prepared from a concentrated stock solution and added to the culture medium on Day 0 at a final concentration of 5 pM. Recombinant human IL-2 was added at 1000 lU / mL at the start of culture and replenished every two to three days thereafter. Cultures were maintained at 37°C in a humidified incubator with 5% CO2 for a total of 14 days, during which the cell density was kept between 0.5 x 106and 2 x 106cells per milliliter by periodic addition of fresh medium. At the end of the 14-day expansion period, the mixed T-cell population was subjected to TCRaP depletion using a commercially available aP T-cell depletion kit following the manufacturer’s instructions. During magnetic separation, cells that bound to the aP-specific microbeads remained attached within the column matrix and were subsequently collected as the aP T-cell fraction. In contrast, cells that did not express TCRaP passed freely through the column and were recovered as the y5 T-cell-enriched flow-through fraction. These y5 T cells were used for all downstream cytotoxicity and functional assays, while the retained aP T cells served as the comparator population in the experiments.

[0213] Flow Cytometry Phenotyping of aP and y5 T-Cell Populations: Flow cytometry was performed to distinguish aP and y5 T-cell subsets within the CD3+ / CD45+T-cell population. Staining included TCRaP antibodies, and representative plots demonstrated that aP T cells contained predominantly TCRaP+cells, whereas y5 T cells formed a distinctTCRaP" population. This confirmation ensured that downstream cytotoxicity comparisons reflected true biological differences between the two T-cell types.

[0214] Co-Culture of T Cells with U87MG-luc GBM Cells: To assess cytotoxic activity, U87MG-luc cells were co-cultured with either y5 T cells or aP T cells at tumor-to-effector ratios of 1:1 and 1:0.5. T cells were added directly to the wells containing U87MG-luc cells, and co-cultures were incubated for 24 hours under standard conditions. Control wells containing U87MG-luc cells alone were included to establish baseline viability and caspase activation levels.

[0215] Luciferase-Based Viability Measurement: At the end of the 24-hour co-culture period, U87MG-luc cell viability was assessed using luminescence-based quantification on the GloMax® Discover Multimode Reader, as described in the experimental documentation. Luminescence measurements were normalized to control wells to determine relative survival of GBM cells following exposure to y5 or aP T cells. This assay enabled sensitive detection of GBM cell viability based on luciferase signal intensity reduction, which directly correlates with cytotoxicity.

[0216] Caspase 3 / 7 Activity Assay for Cytotoxicity Assessment: To further characterize cell death mechanisms, supernatants were collected from co-cultures and mixed with reagents from the Gio-Caspase 3 / 7 Assay Kit based on the manufacturer’s instructions. After incubation, caspase activity was measured using the GloMax® Discover platform. Increased caspase 3 / 7 levels indicated apoptosis induced by T-cell cytotoxic effects. Comparison between y5 T cell-treated and aP T cell-treated groups provided additional mechanistic insight into differential killing efficiency (Figure 11).

[0217] Example 12 In vivo anti-tumor efficacy of unmodified y6 T cells in an orthotopic glioblastoma (U87MG) mouse model

[0218] Establishment of the Orthotopic Glioblastoma Model: An orthotopic glioblastoma model was generated using immunodeficient NOD.CB17-Prkdcscld / NCrCrl mice. U87MG-luc human glioblastoma cells were maintained in DMEM supplemented with 10% fetal bovine serum and 100 pg / mL penicillin / streptomycin prior to implantation. Tumor implantation was performed stereotactically under anesthesia, with I I04U87MG-luc cells delivered into the right striatum in a total injection volume of 5 pL. Following implantation, mice were monitored for recovery and tumor establishment.

[0219] Bioluminescence Imaging for Tumor Confirmation: To verify tumor engraftment, bioluminescence imaging was conducted on Day 0. Mice received an intraperitoneal injection of D-luciferin (15 mg / mL, 200 pL), followed by imaging on the IVIS Lumina III XRMSsystem (PerkinElmer) to quantify baseline tumor burden. Luminescent signal intensity was used as the reference point for subsequent response assessments.

[0220] Intracranial Administration of V52+y5 T Cells: Unmodified V52+y5 T cells were expanded ex vivo under serum-free conditions and prepared fresh on the day of administration. The V52+y5 T cells were delivered intracranially at the established tumor site immediately following baseline imaging on Day 0. The injection was performed using the same stereotactic coordinates employed for tumor implantation to ensure direct delivery into the tumor mass. The administered dose was selected based on prior optimization studies evaluating V52+y5 T-cell persistence and functional activity in the intracranial compartment.

[0221] Follow-Up Tumor Imaging and Assessment of Anti-Tumor Activity: To evaluate the early therapeutic effects of V52+y5 T-cell treatment, bioluminescence imaging was repeated on Day 2 using the same luciferin dosage and imaging parameters as baseline. A marked reduction in tumor-associated luminescence was observed relative to Day 0 measurements, indicating a substantial decrease in viable tumor cell burden within 48 hours of V52+y5 T-cell administration. The observed suppression of bioluminescent signal demonstrated rapid and robust anti-tumor activity mediated by intracranially delivered V52+y5 T cells.

[0222] An orthotopic glioblastoma model was established by stereotactic implantation of U87MG-luc cells into the right striatum of immunodeficient mice. On Day 0, baseline bioluminescence imaging (BLI) was performed to confirm successful tumor engraftment. Unmodified y5 T cells (GDT) were administered intracranially at the tumor site, as indicated by the injection marker. Follow-up BLI on Day 2 demonstrated a marked reduction in luminescence intensity compared to baseline, indicating early anti-tumor activity of the administered GDT cells (Figure 12).

[0223] Example 13 Inhibition of glioblastoma growth by co-injection of unmodified y6 T cells in an orthotopic U87MG mouse model.

[0224] Establishment of the Orthotopic Glioblastoma Co-Injection Model: Human U87MG-luc glioblastoma cells were maintained in DMEM supplemented with 10% fetal bovine serum until use. For orthotopic implantation, mice were anesthetized and positioned in a stereotactic frame. Tumor cells were prepared at a concentration of 1 x 104cells in a 5-pL suspension. To evaluate the prophylactic anti-tumor effects of V52+y5 T cells, freshly expanded V52+y5 T cells were mixed directly with U87MG-luc cells immediately prior to implantation. The mixtures were prepared at U87MG:T-cell ratios of 1:2, 1:4, and 1:8. A total volume of 5 pL was delivered stereotactically into the right striatum using the samecoordinates employed for U87MG-luc implantation alone. All mice were monitored during recovery and observed for signs of discomfort or neurological impairment.

[0225] Longitudinal Bioluminescence Imaging (BLI) for Tumor Monitoring: Tumor progression was evaluated non-invasively through serial bioluminescence imaging. Mice received intraperitoneal injections of D-luciferin (15 mg / mL, 200 pL) prior to imaging sessions. BLI was performed on Days 10, 16, 23, 30, and 38 post-implantation using the IVIS Lumina III XRMS system. Photon flux (radiance) measurements were captured under identical imaging conditions for all timepoints to ensure comparability. In the U87MG-luc-only cohort, robust and progressive increases in luminescence were observed, confirming rapid tumor expansion. In contrast, mice co-injected with V52+y5 T cells exhibited markedly reduced or completely absent luminescent signals, indicating inhibition of tumor formation.

[0226] Quantitative Analysis of Tumor Burden: Radiance values were quantified from acquired BLI images using Living Image analysis software. Mice in the U87MG-luc-only group demonstrated high photon flux values beginning at Day 10, with further escalation at later timepoints. A dose-dependent reduction in photon emission was observed in the V52+y5 T-cell co-injection groups, with the 1:4 and 1:8 groups showing no detectable signal throughout the entire study period. These findings confirmed that co-administration of V52+y5 T cells at the time of tumor inoculation effectively prevents tumor establishment in the orthotopic brain environment.

[0227] Survival Monitoring and Analysis: All mice were monitored daily for survival, weight loss, and neurological symptoms. Kaplan-Meier survival curves were generated to compare outcomes among treatment groups. Consistent with imaging results, mice in the U87MG-luc-only group exhibited progressive tumor-associated morbidity, resulting in significantly shorter survival. In contrast, survival was prolonged in all V52+y5 T-cell-treated groups, with the 1:4 and 1:8 groups exhibiting complete inhibition of tumor development and no tumor-related mortality during the study window. The survival benefit correlated directly with the degree of tumor growth suppression observed by BLI.

[0228] Images were acquired on Days 10, 16, 23, 30, and 38 after implantation. Robust tumor growth was observed in the U87L group, while co-injection with y5 T cells dose-dependently suppressed tumor formation. At E:T ratios of 1:4 and 1:8, no detectable bioluminescent signal was observed throughout the study period. These results indicate that co-administration of unmodified y5 T cells at the time of tumor cell inoculation effectively prevents glioblastoma establishment in vivo (Figure 13).

Claims

ClaimsWhat is claimed is:

1. A method for producing a y5 T cell population comprising enriched V52+y5 T cells, comprising:(a) providing peripheral blood mononuclear cells (PBMCs); optionally the V52+y5 T cells contained in the PBMCs are separated by immunomagnetic separation to obtain a V52+y5 T cell subpopulation;(b) culturing the PBMCs or V52+y5 T cell subpopulation in a medium containing zoledronic acid and one or more growth factors having interleukin-like activity to specifically enrich V52+y5 T cells to obtain an enriched V52+y5 T cell population; and(c) harvesting the expanded V52+y5 T cell population.

2. The method of Claim 1, wherein after step (b), the method further comprises a step (e) of separating the enriched V52+y5 T cell population by immunomagnetic separation to remove non-V52+y5 T cells.

3. The method according to any one of the proceeding claims, wherein after step (b), the method further comprises a step (f) of expanding the enriched V52+y5 T cell population by culturing it for 7-28 days to obtain the expanded V52+y5 T cell population.

4. The method according to any one of the proceeding claims, wherein the cells in the V52+y5 T cell population are unmodified.

5. The method according to any one of the proceeding claims, wherein the PBMCs are obtained from a peripheral blood, umbilical cord blood, or fractions thereof.

6. The method according to any one of the proceeding claims, wherein the zoledronic acid is at a concentration of about 1 to about 30 pM.

7. The method according to any one of the proceeding claims, wherein the medium is free of serum.

8. The method according to any one of the proceeding claims, wherein the concentration of the growth factor having interleukin-like activity ranges from about 5 ng / mL to about 200 ng / mL or about 50 lU / mL to about 1,500 lU / mL.

9. The method according to any one of the proceeding claims, wherein the growth factor having interleukin-like activity is an interleukin.

10. The method according to any one of the proceeding claims, wherein the interleukin is IL-2, IL-4, IL-7, IL- 15, IL-21 interleukin- ip or any combination thereof.

11. The method according to any one of the proceeding claims, wherein the interleukin is IL-2, IL- 15, or a combination of IL-2 and IL-5.

12. The method according to any one of the proceeding claims, wherein the medium containing IL-15 maintains a constant expansion of y5 T-cells at a high level over time.

13. The method according to any one of the proceeding claims, wherein the zoledronic acid mediated y5 T-cell expansion can be increased by IL-15.

14. The method according to any one of the proceeding claims, wherein further comprises synthetic growth factors and amino acids tailored for T cell expansion.

15. The method according to any one of the proceeding claims, wherein the step of expanding the y5 T cells is conducted under 37°C and 5% CO2.

16. A cell population produced from a method according to any one of the proceeding claims.

17. An enriched y5 T cell population, comprising an enriched V52+y5 T cell subpopulation, wherein the enriched y5 T cell population comprises at least about 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% V52+T cells with over about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% post-expansion viability.

18. The cell population of Claim 16 or 17, wherein the y5 T cells are of therapeutic grade.

19. The cell population according to any one of Claims 16 to 18, wherein the enriched y5 T cell population has low residual aP T cells.

20. The cell population of according to any one of Claims 16 to 19, wherein the V52+y5 T cells express one or more CD3, CD45 and NKG2D in an intensity higher than about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99% or 99.5%.

21. The cell population according to any one of Claims 16 to 20, wherein the V52+y5 T cells express NKG2D in an intensity higher than about 95%, 96%, 97%, 98%, 98.5%, 99% or 99.5%.

22. The cell population according to any one of Claims 16 to 21, wherein the V52+y5 T cells described herein express CD3 in an intensity higher than about 95%, 96%, 97%, 98%, 98.5%, 99% or 99.5%.

23. The cell population according to any one of Claims 16 to 22, wherein the V52+y5 T cells described herein express CD45 in an intensity higher than about 95%, 96%, 97%, 98%, 98.5%, 99% or 99.5%.

24. The cell population according to any one of Claims 16 to 23, wherein the V52+y5 T cells described herein express CD3, CD45 and NKG2D in an intensity higher than about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99% or 99.5%.

25. The cell population according to any one of Claims 16 to 24 for modulating an immune response in a subject.

26. The cell population according to any one of Claims 16 to 24 for use in immunotherapy in a subject.

27. The cell population according to any one of Claims 16 to 24 for treating a cancer in a subject.

28. The cell population according to Claim 27 for treating a cancer, wherein the cancer is sarcoma, basal cell skin cancer, lymphoma, leukemia, lymphoproliferative disorder, plasmacytoma, histiocytoma, adenoma, carcinomas of solid tissues, hypoxic tumor, genitourinary cancer, hematopoietic cancer, nervous system cancer, bile duct cancer, cervical cancer, squamous cell cancer, endometrial cancer, esophageal cancer, head and neck cancer, glioblastoma (GBM) (such as glioblastoma multiforme), kidney cancer, hepatocellular carcinoma, liver cancer, lung cancer, pancreatic cancer, melanoma, Merkel cell cancer, mesothelioma, stomach cancer, breast cancer, or triple-negative breast cancer.

29. The cell population according to any one of Claims 16 to 24 for treating an infectious disease in a subject.

30. The method of Claim 29, wherein the infectious disease is bacterial infections such as those caused by mycobacteria (e.g. tuberculosis), viral infections such as those caused by herpes simplex viruses (HSV), human immunodeficiency viruses (HIV), the hepatitis viruses, SARS-CoV-2 viruses, papillomaviruses (HPV), Epstein-Barr viruses (EBV), measles viruses, cytomegaloviruses (CMV), hepatitis C viruses (HCV), or influenza viruses, and parasitic infections such as those caused by plasmodium (e.g. malaria).

31. A method for vaccinating an animal comprising administering the enriched y5 T cell population of any one of Claims 16 to 24 to a subject.

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