GAMMA DELTA T CELLS AND USES THEREOF

Abstract

A method of preparing and using gamma delta T cells in the allogeneic or autologous treatment of subjects suffering from virus infection, fungal infection, protozoal infection and cancer.

Claims

1. A process to provide gamma delta T cells from a first subject to a second allogeneic subject, wherein the process comprises: culturing ex vivo the gamma delta T cells obtained from the first subject for a time sufficient to have the gamma delta T cells have a surface profile determined to have a reduced set of markers selected from the group consisting of: B7-H1/PD-L1, B7-DC/PD-L2, PD-1 and CTLA 4 prior to administering the cultured gamma delta T cells to the second allogeneic subject wherein the gamma delta T cell has a reset gamma T cell surface receptor profile associated with nave or partially nave gamma delta T cells when administered to the second allogeneic subject.

2. The process of claim 1, wherein the step of culturing the gamma delta T cell increases the number of gamma delta T cells in the sample relative to the other cell types in the sample.

3. The process of claim 1, wherein the culturing step comprises a further step of purifying gamma delta T cells in the sample from the other cell types in the sample.

4. The process of claim 3, wherein the purifying step uses an anti-gamma delta T cell antibody to purify and isolate the gamma delta T cells from the other cell types in the sample.

5. The process of claim 1, wherein the gamma delta T cells are cultured or purified to comprise 10% or more of the total number of cells present in the sample.

6. The process of claim 1, wherein the culturing step comprises one or more sub-steps to reduce or eliminate one or more gamma delta T cell surface receptor types present on the gamma delta T cells in the sample from the first subject.

7. The process of claim 1, wherein the culturing step includes a step that induces the expression, in the gamma delta T cells, of a surface receptor type(s) that was not present on the surface of the uncultured gamma delta T cells from the first subject or that induce an increase in the amount of expression of a cell surface receptor type(s) that was present on the surface of the gamma delta T cells from the first subject.

8. The process of claim 1 further comprising a step of monitoring the cell surface receptor profile of the gamma delta T cells in the sample.

9. A method of treating an infection or cancer in an individual comprising the step of providing the individual with gamma delta T cells obtained from a different allogeneic individual.

10. The method of claim 9, wherein the gamma delta T cells are provided by the process of claim 1.

11. (canceled)

12. The method of claim 9, wherein the individual is a first subject and the gamma delta T cells are obtained from a second subject that is a different allogeneic individual to the first subject, the method comprising administering gamma delta T cells from the second subject to the first subject, simultaneously, separately or sequentially with an immunosuppressive drug.

13. The method of claim 9, wherein the infection is at least one of a viral, a fungal or a protozoan infection.

14. (canceled)

15. (canceled)

16. The method of treating cancer or an infection as claimed in claim 9, wherein the infection is selected from the group consisting of: a virus, a fungi and a protozoa infection comprising administering to an individual in need of such treatment gamma delta T cells and an antibody immunotherapy.

17. A pharmaceutical composition comprising gamma delta T cells and an immunosuppressive drug.

18. The process of claim 1, wherein the cell surface receptors comprising at least one of B7-H1/PD-L1, B7-DC/PD-L2, PD-1 and CTLA-4 are absent or reduced on the cultured gamma delta T cells.

19. The process of claim 1, wherein the culturing step comprises culturing the gamma delta T cells from the first subject in the presence of one or more of the following: (i) a cytokine, IL-15 or IL-18; and (ii) an antibody targeting specific immune check-point inhibitor receptors or ligands of immune check-point inhibitors selected from the group of consisting of: CTLA-4, PD-1, PD-2, LAG3, CD80, CD86, B7-H3, B7-H4, HVEM, BTLA, KIR, TIM3, and AZaR.

20. The process of claim 1, wherein the process comprises challenging the gamma delta T cells with an antigen derived from a cancer, a bacterium, a fungi, a protozoa or a virus.

21. The process of claim 1, wherein the process comprises challenging the gamma delta T cells with an antigen derived from a virus, wherein the antigen is selected from an active or inactivated viral fragment, a peptide, a protein, an antigenic segment or the like from the virus.

Description

[0089] Embodiments of the invention will now be described by way of example only with reference to the accompanying figures in which

[0090] FIG. 1 (Panels A-E) illustrate immunophenotyping of starter culture PBMCs and following 14 days of expansion in culture to selectively activate and proliferate the T cell population (Vgamma9 Vdelta2) wherein flow cytometry immunophenotyping of cell populations is used at the start of the culturing process (day 0), using PBMCs isolated from human blood as the starting material and at the end of the selective expansion process (day 14). FIG. 1A is a histogram of isolated PBMCs on day 0 stained with anti-Vgamma9-FITC antibody to detect the percentage of T cells in starting population of PBMCs (1.3% of PBMCs are T cells). FIG. 1B is a dot plot analysis of the cell population after 14 days of selective culturing stained with anti-CD3 (T cells) and anti-Vgamma9 ( T cells (77.5% of T cells are T cells). FIG. 1C is a bright field image of isolated PBMCs on day 0. FIG. 1D is a bright field image of cell population after 14 days of expansion in culture. FIG. 1E is a Table indicating the percentages of T cells present within each cell culture population;

[0091] FIG. 2 (Panels A-D) illustrate the exponential growth of cells selectively expanded in culture to activate and proliferate the T cell population (Vgamma9 Vdelta2) wherein significant numbers of high purity T cells are generated by day 12 which are demonstrated to be potent effectors of cancer cell cytolysis using a panel of EBV-positive lymphoma cell lines in vitroFlow cytometry immunophenotyping of cell populations is used at the start of the culturing process (day 0), using PBMCs isolated from human blood as the starting material and later in the selective expansion process (day 12). FIG. 2A is a growth chart indicating the total number of viable cells in culture throughout the first 12 days of expansion with a total of 410.sup.9 cells achieved by day 12. FIG. 2B is the Flow cytometry analysis of starting PBMCs. FIG. 2C is the cell population following 12 days of selective expansion in culture demonstrating 3.1% (day 0) and 87.1% (day 12) T cells (anti-Vgamma9) respectively. FIG. 2D describes that T cells were incubated with five EBV positive target cells lines (BL2 B95-8, BL30 B95-8, BL74 B95-8, Raji and 1B4) at an effector: target cell ratio of 5:1 for 16 hours T cell elicited cytolysis was measured using the non-radioactive Cytotox96 assay and is expressed as a percentage of maximum target cell lysis; and

[0092] FIG. 3 (Panels A-B) illustrate an antibody-mediated purification method employed to isolate discrete cellular phenotypes from a heterogeneous cell population wherein in this example, cells have been selected with a pan-anti- T cell receptor antibody to obtain a T cell population in extremely high purity. FIG. 3A is a Flow cytometry immunophenotyping analysis of the cell population prior to purification. FIG. 3B is a flow cytometry immunophenotyping analysis of the cell population following purification using an anti- T cell receptor-FITC conjugated antibody demonstrates that T cells are obtained at 99.7% purity from a 45% T cell starting material.

[0093] Gamma Delta T cells may be culture expanded using the technique outlined by Nicol A. J. et. al., 2011 Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation using Ficoll-Paque (GE Healthcare, Buckinghamshire, UK) and V9V2 T cells selectively proliferated by culture of PBMCs in RPMI 1640 media (Lonza, Walkersville, Md., USA) supplemented with 10% human AB plasma (Lonza), L-glutamine (2 mM; Lonza) and gentamycin (40 g; Pfizer, Bentley, WA, Australia). Recombinant human IL-2 (700 IU ml-1; Novartis, Basel, Switzerland) and zoledronate (1 M; Novartis) were added on day 0 and additional IL-2 (350IU m1-1) was added every 2-3 days during the culture period. After 7-14 days culture, purified effector cell populations containing 70-95% V9V2 cells were obtained for in vitro functional assessment by depletion of CD4+, CD8+ and CD56+ cells using miniMACS (Miltenyi Biotec, Bergisch Gladbach, Germany).

[0094] The autologous treatment of patients with solid tumours with ex vivo expanded V9V2 cells has been demonstrated to provide clinical benefit (Noguchi et al., 2011). Additionally, allogeneic treatment with HLA-matched, ex vivo expanded TCR-positive cytotoxic T lymphocytes (CTLs) has proven to be efficacious in the treatment of EBV-PTLD (Hague T et al., 2007). The present inventors consider therefore that the treatment of cancer and viral infections with allogeneic gamma delta T cells is both feasible and likely to provide demonstrable therapeutic benefit to the patient.

[0095] Although the invention has been particularly shown and described with reference to particular examples, it will be understood by those skilled in the art that various changes in the form and details may be made therein without departing from the scope of the present invention.

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