γδ T cell expansion procedure

Abstract

A method for expanding a population of γδ T-cells is provided in which isolated activated Peripheral Blood Mononuclear Cells (PBMCs) are cultured in a medium comprising transforming growth factor beta (TGF-β) under conditions in which the production of effector γδ T-cells having therapeutic activity against malignant disease is favored. The use of TGF-β in the production of effector cells in particular Vγ9Vδ2 T-cells is also described and claimed.

Claims

1. A method of treating cancer, the method comprising: (a) expanding a population of effector γδ T-cells comprising: culturing isolated Peripheral Blood Mononuclear Cells (PBMCs) in a medium comprising: (i) transforming growth factor beta (TGF-β), (ii) interleukin-2 (IL-2), and (iii) an activator for Vγ9Vδ2 T-cells, wherein the medium is serum-free; and (b) administering to a cancer patient effective amounts of the population of effector γδ T-cells and cytarabine.

2. The method of claim 1, wherein the population of effector γδ T-cells and cytarabine are administered sequentially.

3. The method of claim 2, wherein cytarabine is administered before the population of effector γδ T-cells.

4. The method of claim 1, wherein the population of effector γδ T-cells and cytarabine are administered concurrently.

5. The method of claim 1, further comprising administering to the cancer patient a cytokine.

6. The method of claim 5, wherein the cytokine is IL-2.

7. The method of claim 6, wherein the IL-2 is administered concurrently with the population of effector γδ T-cells.

8. The method of claim 1, wherein no additional cytokines are present in the medium.

9. The method of claim 1, wherein the activator is an aminobisphosphonate.

10. The method of claim 9, wherein the aminobisphosphonate is zoledronic acid, alendronic acid, pamidronic acid, ibandronic acid, or a salt thereof.

11. The method of claim 10, wherein the aminobisphosphonate is zoledronic acid or a salt thereof.

12. The method of claim 1, wherein the effector γδ T-cells are human Vγ9Vδ2 T-cells.

13. The method of claim 1, wherein the PBMCs are human PBMCs.

14. The method of claim 13, wherein the PBMCs are from a healthy human.

15. The method of claim 13, wherein the PBMCs are from a human patient.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a schematic view showing the mevalonate pathway. Phosphoantigens (PAg) recognized by Vγ9Vδ2 T-cells include DMAPP, IPP and Apppl. Points of inhibition of the pathway by aminobisphosphonates and statins are indicated by circles where IPP=Isopentenyl diphosphate and DMAPP=Dimethylallyl diphosphate.

(2) FIG. 2A shows the results of ex-vivo expansion of Vγ9Vδ2 T-cells using a comparative method (Method 1). After culture using conditions described above, the percentage of γδ T-cells per 20 ml blood sample was evaluated at initiation of the culture period and after 15 days. FIG. 2B shows the results of ex-vivo expansion of Vγ9Vδ2 T-cells using a comparative method (Method 1). After culture using conditions described above, the absolute number of γδ T-cells per 20 ml blood sample was evaluated at initiation of the culture period and after 15 days. FIG. 2C presents expression of the expected Vγ9 and Vδ2 T-cell receptor subunits as determined by flow cytometry. FIG. 2D shows pooled representative immunophenotypic data of γδ T-cells, expanded ex-vivo for 15 days from healthy donors and women with newly diagnosed EOC (donor number indicated in brackets). FIG. 2E shows representative immunophenotypic data of γδ T-cells, expanded ex-vivo for 15 days from healthy donors and women with newly diagnosed EOC (donor number indicated in brackets).

(3) FIG. 3A shows the total cell number of γδ T-cells after expansion of Vγ9Vδ2 T-cells in various media, with and without human AB serum using comparative method 1 after 14 days of culture. FIG. 3B shows the % γδ T-cells present after expansion of Vγ9Vδ2 T-cells in various media, with and without human AB serum using comparative method 1 after 14 days of culture. FIG. 3C shows the yield of γδ T-cells after expansion of Vγ9Vδ2 T-cells in various media, with and without human AB serum using comparative method 1 after 14 days of culture.

(4) FIGS. 4A-F show the results of cytotoxicity assays using cells expanded using the comparative method 1 in an assay against a range of ovarian cancer cell lines as follows: FIG. 4(A) IGROV-1; FIG. 4(B) KOC7C; FIG. 4(C) PEO1; FIG. 4(D) PEA; FIG. 4(E) SKOV-3; FIG. 4(F) TOV-21G.

(5) FIGS. 5A-B show the results obtained using a method to expand Vγ9Vδ2 T-cells ex-vivo in accordance with the invention. FIG. 5A shows enrichment of Vγ9Vδ2 T-cells (mean±SEM, n=13 independent replicates). FIG. 5B shows expansion of Vγ9Vδ2 T-cells (mean±SEM, n=13 independent replicates). Percentage γδ T-cells present at the beginning and end of manufacture are also shown (mean±SD, n=10). *p=0.03 by Mann Whitney test.

(6) FIGS. 6A-I show the comparative anti-tumor activity of method 1 and method 2-expanded γδ T-cells. After expansion of γδ T-cells for weeks using either method 1 or 2, cytotoxicity assays were established in triplicate at a 5:1 effector:target ratio in 96 well plates. Tumor cells were cultured with the indicated aminobisphosphonates for 24 hours prior to undertaking the cytotoxicity assay. After overnight co-culture with Vγ9Vδ2 T-cells, residual tumor cell viability was measured by MTT or luciferase assay. Data show mean±SEM tumor cell killing from 2-5 independent replicate experiments performed using the indicated ovarian cancer cell lines FIG. 6(A) IGROV-1, FIG. 6(B) SKOV-3, FIG. 6(C) Kuramochi and FIG. 6(D) TOV-21G; myeloid leukemic cell lines FIG. 6(E) U937 and FIG. 6(F) KG-1 and breast cancer cell lines FIG. 6(G)MDA-MB-231, FIG. 6(H) MDA-MB-468 and FIG. 6(I) BT-20.

(7) FIGS. 7A-O illustrate cytokine production by method 1 and method 2-expanded γδ T-cells. γδ T-cells were expanded using method 1 or 2 and then co-cultivated with bisphosphonate-pulsed or unpulsed tumor cells as described in FIG. 6. Supernatants were then harvested after 24 h of co-culture and analysed for interferon-γ FIGS. 7(A-I) and interleukin-2 FIGS. 7(J-O) by ELISA. Interferon (IFN)-γ production is shown for the following ovarian cancer cell lines FIG. 7(A) Kuramochi, FIG. 7(B) IGROV-1, FIG. 7(C) SKOV-3, FIG. 7(D) TOV-21G; breast cancer cell lines FIG. 7(E) MDA-MB-468; FIG. 7(F) MDA-MB-231; FIG. 7(G) BT-20; myeloid leukemic cell lines FIG. 7(H) U937, FIG. 7(I) KG-1. Interleukin-2 production is shown for co-cultivation experiments undertaken with FIG. 7(J) Kuramochi, FIG. 7(K) U937, FIG. 7(L) KG-1, FIG. 7(M) MDA-MB-231, FIG. 7(N) MDA-MB-468 and FIG. 7(O) BT-20 tumor cells. Data show mean±SEM from 3-5 independent replicate experiments.

(8) FIGS. 8A-C show the results of immunophenotypic analysis of method 1 and method 2-expanded Vγ9Vδ2 T-cells. FIG. 8(A) Method 2 expanded cells express a distinct immunophenotype with higher levels of memory (CD45RO, CD27) and homing receptors (CCR7, CXCR4, cutaneous leukocyte antigen (CLA) and E-selectin binding receptors (detected using E-selectin-IgG fusion protein—FIG. 8B). FIG. 8(C) Relative (rel.) to method, 1, the proportion of naïve (CD45RA.sup.+ CCR7.sup.+) and central memory (CD45.sup.− CD27.sup.+) cells was higher in method 2-expanded cells. NS—not significant; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

(9) FIG. 9A shows an evaluation of cell number of γδ T-cells present in cultures obtained using method 1 and the method of the invention in a different basic medium (RPMI+10% human AB serum). FIG. 9B shows the percentage of γδ T-cells present in cultures obtained using method 1 and the method of the invention in a different basic medium (RPMI+10% human AB serum).

(10) FIG. 10 shows the in-vivo therapeutic activity of intravenously administered expanded Vγ9Vδ2 T-cells obtained using method 1 and the method of the invention, against an established burden of malignant disease (U937 leukemia) in SCID Beige mice.

(11) FIGS. 11A-B show the in-vivo therapeutic activity of intravenously administered expanded Vγ9Vδ2 T-cells obtained using the method of the invention against an established burden of malignant disease (U937 leukemia) in SCID Beige mice where FIG. 11(A) shows tumor burden, indicated by bioluminescence; and FIG. 11(B) shows the weight of mice, providing an indication of toxicity of the treatment.

(12) FIGS. 12A-C show the in-vivo therapeutic activity of intravenously administered expanded Vγ9Vδ2 T-cells obtained using method 2 against an established burden of malignant disease (MDA-MB-231 triple negative breast cancer, implanted in the mammary fat pad of SCID Beige mice). FIG. 12(A) Tumor burden, indicated by bioluminescence. FIG. 12(B) Survival of mice. FIG. 12(C) Weight of mice, providing an indication of toxicity of the treatment.

(13) FIGS. 13A-H show the results of the use of various purification methods, where FIG. 13(A) illustrates how Vγ9Vδ2 T-cells were purified from freshly isolated PBMC by negative selection using a CD19 and αβ T-cell microbead isolation kit; FIG. 13(B) shows the results of attempts to expand these cells; FIG. 13(C) shows the % cell type obtained in experiments in which γδ T-cells were expanded from PBMC using the method of the invention prior to subsequent depletion of CD19 and αβ T-cells by negative selection; FIG. 13(D) shows the results of flow cytometry analysis of these cells following depletion of contaminating CD19 and αβ T-cells; FIG. 13(E) shows the results of a 24 hour cytotoxicity test of the cells (5:1 effector:target ratio) against MDA-MB-231 (231), MDA-MB-468 (468) or BT20 triple negative tumor cells or FIG. 13(F) against U937 or KG-1 myeloid leukemic cells; FIG. 13(G) illustrates cytokine concentration in supernatants that had been harvested from treated breast cancer co-cultures and FIG. 13(H) illustrates cytokine concentration in supernatants that had been harvested from treated leukemia co-cultures (n=2).

(14) FIGS. 14A-C show that the flow cytometry results of genetically engineered γδ T-cells obtained using the method of the invention, using a technique in which viral vector was pre-loaded onto a RetroNectin coated solid phase FIG. 14(B), or by addition of viral supernatant to cells FIG. 14(C), as compared to untransduced controls FIG. 14(A).

(15) FIGS. 15A-B show the results of in-vitro cytoxicity assays against tumor cells (FIG. 15(A) U937 cells and FIG. 15(B) KG1 cells) when treated with the chemotherapeutic agent, cytarabine, at various concentrations for 24 hours preceding the addition of γδ T-cells, including some obtained using the method of the invention (M2).

(16) FIGS. 16A-B are a set of graphs showing the results of in-vivo tests in which γδ T-cells of the invention are administered in combination with cyatarabine and IL-2, as compared to the use of cyatarabine and IL-2 alone where FIG. 16(A) shows the tumor burden as indicated by bioluminescence from malignant cells on data 4, 11, 19 and 26 after administration and FIG. 16(B) shows the weight of the mice over the period of the test. In each case, the cyatarbine was injected as a single dose 24 hours before infusion of γδ T-cells.

COMPARATIVE EXAMPLE A

(17) In previous studies, the applicants have shown that healthy donors have 19,916±29,887 (mean±SD, n=21) circulating γδ T-cells. By comparison, patients with newly diagnosed EOC had 14,240±15,215 γδ cells/ml blood (mean±SD, n=13; not statistically significant (NS))[16].

(18) To enrich these cells, peripheral blood mononuclear cells (PBMC) were activated with ZA and cultured in AB serum-containing RPMI 1640 medium, supplemented with IL-2/IL-15. Specifically, PBMC isolated from normal (healthy) donors (n=21 separate donors) and from patients with EOC (n=13 separate donors) were cultured with ZA (1 μg/ml day 1 only), IL-2 (100 U/ml) and IL-15 (10 ng/ml). Cytokines and medium were added daily.

(19) The percentage number of γδ T-cells and the absolute number of γδ T-cells per 20 ml blood sample was evaluated at initiation of the culture period and after 15 days. The results are shown in FIGS. 2(A) and 2(B) respectively. This “research grade” method resulted in an average expansion of γδ T-cells by 97-fold (EOC patients) or 172-fold (healthy donors; NS) (FIG. 2B).

(20) Expression of the expected Vγ9 and Vδ2 T-cell receptor subunits was determined by flow cytometry and the results are shown in FIG. 2(C). As is clear, expanded γδ T-cells from patients and healthy donors expressed the Vγ9Vδ2 T-cell receptor.

(21) Pooled and representative immunophenotypic data of γδ T-cells, expanded ex-vivo for 15 days from healthy donors and women with newly diagnosed EOC (donor number indicated in brackets) was also obtained and the results are shown in FIGS. 2(D) and 2(E) respectively. There was a predominance of γδ T-cells, with small numbers of contaminating γδ T-cells and natural killer (CD16+56.sup.+, CD3.sup.−) cells. Expanded cells predominantly exhibit an effector and effector memory phenotype, which is similar in patients and healthy volunteers. We subsequently found that addition of IL-15 made no significant difference to the yield of cells obtained and this was omitted from subsequent expansion runs (data not shown).

(22) To adapt manufacture of γδ T-cell products for clinical use, we tested commercially available GMP media for their ability to support the expansion of these cells using ZA+IL-2. The method as described above was repeated using clinical grade serum-free medium. PBMC were cultured in RPMI+10% human AB serum or two commercially available GMP grade media, with or without 10% human AB serum. In each case, ZA (1 μg/ml) was added to activate γδ T-cells, which were then expanded by addition of IL-2 (100 U/ml). The results are shown in FIGS. 3A-C. These show that the TexMACS medium in particular enables the expansion of these cells under serum-free conditions in “method 1”.

(23) Cytotoxicity assays were established in triplicate at a 5:1 effector:target ratio in 96 well plates and the results are shown in FIGS. 4A-F. Where indicated, tumor cells were pulsed for 24 h with the indicated concentration of zoledronic (ZA) or pamidronic acid (PA), prior to addition of γδ T-cells. Residual tumor cell viability was measured after overnight co-culture with Vγ9Vδ2 T-cells by MTT assay for FIG. 4(A) IGROV-1; FIG. 4(B) KOC7C; FIG. 4(C) PEO1; FIG. 4(D) PEA; FIG. 4(E) SKOV-3; FIG. 4(F) TOV-21G. The results show that Vγ9Vδ2 T-cells expanded using method 1 exhibited broad and NBP-enhanced anti-tumor activity against a range of ovarian and other tumor cell lines.

EXAMPLE 1

Expansion of T-cells in Accordance with the Invention

(24) Next, we modified method 1 such that transforming growth factor (TGF)-β was added together with IL-2 at all times. This approach is referred to hereafter as method 2.

(25) In a variation of the method of Example A above, blood was collected from healthy donors or patients, in a tube with citrate anticoagulant. Using Ficoll-Paque (GE), PBMCs were isolated according to previously published methodology [17].

(26) Isolated PBMC cells were then reconstituted in GMP TexMACS Media (Miltenyi) at 3×10.sup.6 cell/mL. To the reconstituted cells, 1 μg/mL Zoledronic Acid (Zometa, Novartis) was added as an activator, together with 100 U/mL IL-2 and 5 ng/mL TGF-β. The cells were incubated at 37° C. in air containing 5% carbon dioxide.

(27) On day 3, cells were fed with 100 U/mL IL-2 and 5 ng/mL TGF-β. Thereafter, on days 4, 7, 9, 11, 13, 15, cells were counted by trypan exclusion using a hemocytometer. If the number of T-cells was less than 1×10.sup.6 cells/mL, a further 100 U/mL IL-2 and 5 ng/mL TGF-β were added. If the number of T-cells was between 1×10.sup.6 and 2×10.sup.6 cells/mL, an equivalent volume of TexMACS medium was added together with 100 U/mL IL-2 and 5 ng/mL TGF-β. If the number of T-cells was greater than 2×10.sup.6 cells/nL, double the volume of TexMACS media was added together with 100 U/mL IL-2 and 5 ng/mL TGF-β.

(28) After 15 days, the cells were analyzed by flow cytometry with a pan γδ antibody to confirm the enrichment of γδ T-cells in these cultures. The results are shown in FIGS. 5A-B. These show that the modified method achieves enrichment FIG. 5(A) and improved expansion FIG. 5(B) of Vγ9Vδ2 T-cells (mean±SEM, n=13 independent replicates).

(29) Additionally, the T-cells were immunophenotypically characterised and subjected to functional tests. The relative ability of the T-cells obtained using method 1 above, or the present method of the invention to mediate cytotoxic destruction of tumor cells was evaluated. After expansion of γδ T-cells for 2 weeks using either method 1 or 2, cytotoxicity assays were established in triplicate at a 5:1 effector:target ratio in 96 well plates. Where indicated, tumor cells were pulsed for 24 h with the indicated concentration of zoledronic (ZA), alendronic acid (AA) or pamidronic acid (PA), prior to addition of γδ T-cells. Residual tumor cell viability was measured after overnight co-culture with Vγ9Vδ2 T-cells by MTT or luciferase assay. The results are shown in FIGS. 6A-I. FIG. 6(A) IGROV-1, FIG. 6(B) SKOV-3, FIG. 6(C) Kuramochi and FIG. 6(D) TOV-21G; myeloid leukemic cell lines FIG. 6(E) U937 and FIG. 6(F) KG-1 and breast cancer cell lines: FIG. 6(G) MDA-MB-231, FIG. 6(H) MDA-MB-468 and FIG. 6(I) BT-20.

(30) Activation of γδ T-cells when co-cultivated with tumor cells was assessed by measurement of release of IL-2 and IFN-γ. Ability of these expanded γδ T-cells to control an established burden of malignant disease was also assessed in SCID Beige mice with an established burden of U937 myeloid leukemia.

(31) The original rationale for inclusion of TGF-β in the culture process was to try to improve expression of homing receptors such as CXCR4 on these cells. Completely unexpectedly however, addition of TGF-β resulted in substantially enhanced yields of Vγ9Vδ2 T-cells as shown in FIGS. 5A-B.

(32) Method 2-expanded cell products also demonstrated equivalent or enhanced anti-tumor activity against EOC (IGROV-1, SKOV-3, Kuramochi, TOV-21G), breast cancer (MDA-MB-231) and myeloid leukemic cells (U937), even in the absence of NBP exposure (FIGS. 6A-I; FIG. 10). However, anti-tumor activity was consistently enhanced by prior NBP sensitization (FIGS. 6A-I).

(33) After expansion of γδ T-cells for 2 weeks using either method 1 or 2, co-cultures were established in triplicate at a 5:1 effector:target ratio in 96 well plates. Where indicated, tumor cells were pulsed for 24 h with the indicated concentration (μg/ml) of zoledronic (ZA) or pamidronic acid (PA), prior to addition of γδ T-cells. After a further 24 hours, supernatants were harvested and analysed for Interferon-γ or Interleukin-2 by ELISA. The results are shown in FIGS. 7A-O. Interferon (IFN)-γ production is shown for the following tumor cell monolayers: ovarian cancer cell lines FIG. 7(A) Kuramochi, FIG. 7(B) IGROV-1, FIG. 7(C) SKOV-3, FIG. 7(D) TOV-21G; breast cancer cell lines FIG. 7(E) MDA-MB-468; FIG. 7(F) MDA-MB-231; FIG. 7(G) BT-20; myeloid leukemic cell lines FIG. 7(H) U937, FIG. 7(I) KG-1. In addition, interleukin-2 production is shown for co-cultivation experiments undertaken with FIG. 7(J) Kuramochi, FIG. 7(K) U937, FIG. 7(L) KG-1, FIG. 7(M) MDA-MB-231, FIG. 7(N) MDA-MB-468 and FIG. 7(O) BT-20 tumor cells.

(34) When compared to cells that had been expanded using method 1, method 2-expanded cells produced significantly higher levels of IFN-γ when engaging tumor cell targets. This effect was most pronounced when transformed cells had been pulsed with very low concentrations of NBP agents (FIGS. 7A-G). Method 2-expanded cells also produced IL-2 under these conditions, a finding that was not observed using method 1-expanded cells (FIGS. 7H-K).

(35) Finally, the phenotype of method 1 and method 2 cells was investigated using conventional methods and the results are illustrated in FIGS. 8A-C. Method 2-expanded cells were found to express a distinctive phenotype, with high levels of homing receptors (CXCR4, CLA, E-selectin binding activity) and memory markers (CD27, CD45RO). In addition, the proportion of naïve (CD45RA.sup.+ and CCR7.sup.+) and central memory (CD45.sup.− and CD27.sup.+) cells was higher in method 2 expanded cells as compared to method 1 expanded cells. Thus these cells are distinguishable from cells produced using other expansion protocols.

EXAMPLE 2

Alternative Cell Expansion Process

(36) The methodology of Example 1 above was repeated using a different basic medium, specifically RPMI+human AB serum. In particular, PBMC (3×10.sup.6 cells/ml) were cultured in RPMI+10% human AB serum containing zoledronic acid (1 μg/ml)+IL-2 (100 U/ml; method 1) or zoledronic acid (1 μg/ml)+IL-2 (100 U/ml)+TGF-β (5 ng/ml; method 2). Cell number was evaluated on day 15 and the results are shown in FIG. 9A. The percentage of γδ T-cells present in each culture was evaluated on the day of initiation of the cultures (day 1) and after a further 14 days (day 15) and the results are shown in FIG. 9B.

(37) As before, it is clear that the addition of TGF-β has enhanced cell expansion.

EXAMPLE 3

In-Vivo Therapeutic Activity

(38) In addition, the in-vivo therapeutic activity of expanded Vγ9Vδ2 T-cells against an established burden of malignant disease were compared. Twenty SCID Beige mice were inoculated with 1×10.sup.6 firefly luciferase-expressing U937 leukemic cells by tail vein injection and were then divided into 4 groups of 5 mice each. After 4 days, mice were treated as follows: Group 1 is a control group that received PBS alone. Group 2 received pamidronic acid (200 μg IV) alone. Group 3 received pamidronic acid (200 μg IV on day 4) followed by 20×10.sup.6 (day 5) and 10×10.sup.6 (day 6) Vγ9Vδ2 T-cells that had been expanded using method 1 (IV). Group 4 received pamidronic acid (200 μg IV on day 4) followed by 20×10.sup.6 (day 5) and 10×10.sup.6 (day 6) Vγ9Vδ2 T-cells that had been expanded using method 2 (administered IV). Leukemic burden was monitored thereafter by serial bioluminescence imaging.

(39) The results are shown in FIG. 10. It is clear that the efficacy of the cells obtained by method 2 of the invention is significantly greater in this assay.

EXAMPLE 4

In-Vivo Activity of Cells of the Invention in Conjunction with IL-2

(40) In a separate experiment, the in-vivo therapeutic activity of intravenously administered expanded Vγ9Vδ2 T-cells obtained using the method of the invention (M2) against an established burden of malignant disease (U937 leukemia) in SCID Beige mice was measured. Mice were divided into 4 groups of 5 mice and each received 1 million U937 cells IV on day 1. Thereafter, one group received treatment that may be summarised as follows:

(41) TABLE-US-00001 Group Treatment 1 PBS (control) 2 Zoledronic acid + IL-2 3 M2 + IL-2 4 M2 + IL-2 + Zoledronic acid

(42) Where administered, 20 μg Zoledronic acid was administered intravenously 24 hours after treatment with U937 cells. Mice receiving M2 cells were given 2 treatments of 15 million γδ T-cells intravenously, one day later. Those receiving IL-2 were given 10,000 U of IL-2 by the intraperitoneal (IP) route at the same time as M2 administration. On the following 2 days, mice received 10,000 U IL-2 IP. A control group received phosphate-buffered saline (PBS) alone.

(43) Bioluminescence from the malignant cells was measured on days 7, 15, 21 and 28 as an indicator of tumor burden. The results are shown in FIG. 11A. The results show that Vγ9Vδ2 T-cells obtained using the method of the invention significantly reduce tumor burden, in particular when administered with an activator.

(44) Mice were weighed over the course of the treatment to provide an indication of the toxicity of the treatment. The results, shown in FIG. 11B, indicate that that there is no significant toxicity associated with the treatment.

EXAMPLE 5

In-Vivo Therapeutic Effect Against Breast Cancer

(45) In this experiment, 20 SCID Beige mice having an established burden of malignant disease in the form of MDA-MB-231 triple negative breast cancer, implanted in the mammary fat pad of the mice, were used. Again, mice were divided into four groups for treatment. Mice were treated as follows: Group 1 is a control group that received PBS alone. Group 2 received 20 μg Zoledronic acid intravenously. Group 3 received 20×10.sup.6 (day 2) and 10×10.sup.6 (day 3) Vγ9Vδ2 T-cells that had been expanded using method 2 intravenously. Group 4 received 20 μg Zoledronic acid intravenously on day 1 followed by 20×10.sup.6 (day 2) and 10×10.sup.6 (day 2) Vγ9Vδ2 T-cells that had been expanded using method 2.

(46) The resultant tumor burden as measured by bioluminesence was measured over a period of 28 days. The results are shown in FIG. 12. In this case, the cells obtained using the method of the invention produced a significant reduction in tumor burden (FIG. 12A) accompanied by prolonged survival (FIG. 12B).

(47) Mice were weighed over the course of the treatment to provide an indication of the toxicity of the treatment. The results, shown in FIG. 12C, indicate that that there is no significant toxicity associated with the treatment.

EXAMPLE 6

Purification of Expanded γδ T-Cells

(48) In a first experiment, Vγ9Vδ2 T-cells were purified from freshly isolated PBMC by negative selection using a CD19 and/or a αβ T-cell microbead isolation kit. Where both kits were used, residual contaminating CD19 and αβ T-cells were <0.1% as shown in FIG. 13(A).

(49) The purified cells were subjected to expansion using method 2 as described in Example 1. However, these cells were not able to expand as illustrated in FIG. 13(B). Thus it appears that the starting material must comprise PBMCs.

(50) In other experiments, γδ T-cells were expanded from PBMCs using method 2 for 15 days. At this point, flow cytometry analysis demonstrated that significant numbers of αβ T-cells remain, accompanied by small numbers of CD19.sup.+ cells (n=4) (FIG. 13(C)).

(51) The resultant product was then depleted of CD19 and αβ T-cells by negative selection, as described above in relation to FIG. 13(A). Two representative flow cytometric analyses are shown in FIG. 13(D) to indicate the efficiency of the depletion process.

(52) Following purification by negative selection using the MACS beads (Miltenyi), method 2-expanded γδ T-cells were tested in a 24 hour cytotoxicity assay (5:1 effector:target ratio) against MDA-MB-231, MDA-MB-468 or BT20 triple negative tumor cells or U937 or KG-1 myeloid leukemic cells using methodology similar to that described in Example 1. Cells were tested alone, or in combination with zoledronic acid. There was a negative control and a control with activator alone. Tumor cell viability was measured by luciferase assay and/or MTT assay (n=2). The results are shown in FIG. 13(E) and FIG. 13(F) respectively. As is clear, the combination of T cells and activator produced a significant reduction in tumor cell viability.

(53) Supernatants were harvested from these breast cancer and leukemia co-cultures, after 24 h, and analysed for the presence of IFN-γ and/or IL-2. The results are shown in FIGS. 13(G) and 13(H) respectively. Cytokine levels were substantially raised in the case of the combination of T-cells expanded in accordance with the invention and activator.

(54) These experiments show that method 2 expanded γδ T-cells are fully functional if purified by negative selection after expansion, but not before. This purification facilitates the safe allogeneic use of these cells since potentially hazardous B-cells (CD19.sup.+) and up T-cells have been removed.

EXAMPLE 7

Genetic Engineering of Expanded Cells

(55) To further confirm the functionality of γδ T-cells expanded in accordance with the invention, they were genetically engineered by retroviral transduction. Cells were either transduced by pre-loading viral vector onto a RetroNectin coated solid phase or by addition of viral supernatant to the expanding cells.

(56) It was clear that in order to preserve the efficient enrichment of these cells during expansion, it is preferable to pre-load viral vector onto a RetroNectin coated solid phase (FIG. 14(B)), rather than addition of viral supernatant (FIG. 14(C)). This is indicated by the greater percentage of transduced cells and the greater percentage of γδ T-cells present when gene transfer is achieved using the pre-loading method.

EXAMPLE 8

Effects of Combination of γδ T-Cells with Chemotherapeutic Agent

(57) Cytotoxicity assays were established in triplicate at a 1:1 effector:target ratio in 96 well plates containing either U937 tumor cells or KG-1 tumor cells. Where indicated, tumor cells were pulsed for 24 h with the indicated concentrations of cytarabine, prior to addition of γδ T-cells, produced either using the method of the invention (M2) or the method of the comparative example (M1) above. There were three donors for the M2 cells and two donors for the M1 cells. A control group received no cytarabine.

(58) Residual tumor cell viability was measured after overnight co-culture with Vγ9Vδ2 T-cells by luciferase assay. The results, shown in FIGS. 15A-B show that sub-lethal doses of cytarabine potentiated the anti-tumor activity of Vγ9Vδ2 T-cells expanded using method 2 against two cell models of AML (three donors for M2 cells and two donors for M1 cells)

(59) In a separate experiment, fifteen SCID Beige mice were inoculated with 1×10.sup.6 firefly luciferase-expressing U937 leukemic cells by tail vein injection and were then divided into 3 groups of 5 mice each. After 4 days, mice were treated as follows: Group 1 is a control group that received PBS alone. Group 2 received cytarabine (480 mg/Kg IV on day 4) and IL-2 (10000 IP on day 5, 6, 7 and 8). Group 3 received cytarabine (480 mg/Kg IV on day 4) followed by 20×10.sup.6 (day 5 and 6) Vγ9Vδ2 that had been expanded using method 2 (IV) and IL-2 (10000 IP at days 5, 6, 7 and 8).

(60) Leukemic burden was monitored thereafter by serial bioluminescence imaging. Bioluminescence from the malignant cells was measured on days 4, 11, 19 and 26 as an indicator of tumor burden. The results are shown in FIG. 16(A) and indicate that Vγ9Vδ2 T-cells obtained using the method of the invention reduce tumor burden most effectively when administered with cytarabine. Mice were weighed over the course of the treatment to provide an indication of the toxicity of the treatment. The results shown in FIG. 16(B), indicate that that there is no significant toxicity associated with the treatment.

REFERENCES

(61) [1] P. Vantourout et al., Nat Rev Immunol, 13 (2013) 88-100. [2] P. Vantourout et al., Sci Transl Med, 6 (2014) 231ra249. [3] M. Brandes et al., Science, 309 (2005) 264-268. [4] M. Wilhelm et al., J Transl Med, 12 (2014) 45. [5] I. Benzaid et al., Cancer Res, 71 (2011) 4562-4572. [6] J. W. Clendening et al., Proc Natl Acad Sci USA, 107 (2010) 15051-15056. [7] U. Laggner et al., Clin Immunol, 131 (2009) 367-373. [8] J. E. Dunford et al., J Med Chem, 51 (2008) 2187-2195. [9] I. Benzaid et al., Clin Cancer Res, 18 (2012) 6249-6259. [10] F. Dieli et al., Cancer Res, 67 (2007) 7450-7457. [11] J. Bennouna et al., Cancer Immunol Immunother, 57 (2008) 1599-1609. [12] H. Kobayashi et al., Anticancer Res, 30 (2010) 575-579. [13] A. J. Nicol et al., Br J Cancer, 105 (2011) 778-786. [14] Y. Gu et al., J Immunol Methods, 402 (2014) 82-87. [15] R. Casetti et al., J Immunol, 183 (2009) 3574-3577. [16] A. C. Parente-Pereira et al., J Immunol, 193 (2014) 5557-5566. [17] A. C. Parente-Pereira et al., J Biol Methods, 1 (2014) e7. [18] S. R. Mattarollo et al., Cancer Immunol. Immunother. (2007) 56:1285-1297