T Cell Expansion Method
20200172864 ยท 2020-06-04
Assignee
Inventors
Cpc classification
A61K35/17
HUMAN NECESSITIES
International classification
Abstract
The invention relates to the expansion of T cells and particularly, although not exclusively, to the expansion of gamma delta T cells, and the optimization of medium, serum and cytokine combinations for large-scale ex vivo expansion of gamma delta T cells for clinical use.
Claims
1. A method for generating or expanding gamma delta T cells, the method comprising culturing peripheral blood mononuclear cells (PBMCs) in the presence of IL2 and IL21.
2. A method for generating or expanding gamma delta T cells, the method comprising culturing PBMCs in the presence of 112 and IL18.
3. A method for generating or expanding gamma delta T cells, the method comprising culturing PBMCs in the presence of IL15.
4. The method according to claim 3, wherein the PBMCs are cultured in the presence of IL15 and IL21.
5. The method according to claim 4, wherein the PBMCs are cultured in the presence of IL15, IL 21 and IL18.
6. A method for generating or expanding gamma delta T cells, the method comprising culturing PBMCs in the presence of IL21.
7. The method according to claim 6 wherein the PBMCs are cultured in the presence of IL21 and IL2 and/or IL15.
8. The method according to any one of the preceding claims wherein the gamma delta T cells are V2 T cells.
9. The method according to claim 8, wherein the gamma delta T cells are V9V2 T cells.
10. The method according to any one of the preceding claims, wherein the method comprises culturing the PBMCs in culture medium supplemented with serum.
11. The method according to claim 10 wherein the culture medium is supplemented with 10% serum.
12. The method according to any one of the preceding claims the PBMCs are cultured in OpTimizer T cell media.
13. The method according to claim 10 or claim 11 wherein the serum is human AB serum or defined FBS.
14. The method according to any one of claims any one of the preceding claims, wherein the method generates a population of gamma delta T cells that is at least 60% gamma delta T cells, preferably at least 70% gamma delta T cells.
15. The method according to any one of the preceding claims, wherein the gamma delta T cells exhibit antigen presentation and effector phenotypes.
16. A gamma delta T cell generated using a method according to any one of the preceding claims.
17. The gamma delta T cell according to claim 16 for use in medicine.
18. The gamma delta T cell according to claim 16 for use in a method of adoptive T cell therapy.
19. A gamma delta T cell that expresses a higher level of at least one marker selected from HLA-ABC, HLA-DR, CD80, CD83, CD86, CD40 and ICAM-1 than a gamma delta T cell that has been generated in the presence of IL2 alone.
20. A gamma delta T cell that expresses a higher level of at least one marker selected from CCR5, CCR6, CCR7, CD27 and NKG2D than a gamma delta T cell that has been generated in the presence of IL2 alone.
21. A cell culture comprising gamma delta T cells, media, and IL2 and IL21; IL15; IL15 and IL21; IL2 and IL18; IL15, IL18 and IL21. IL2 and IL7; IL2 and IL15; IL2, IL18 and IL21; IL15 and IL7; or IL15 and IL18.
22. The cell culture according to claim 21, further comprising serum, preferably 10% serum.
23. A method for generating or expanding a population of antigen-specific T cells, comprising stimulating T cells by culture in the presence of gamma delta T cells generated/expanded according to the method of any one of claims 1 to 15 presenting a peptide of the antigen.
24. An antigen-specific T cell according generated using the method according to claim 23.
25. The antigen-specific T cell according to claim 24 for use in medicine.
26. The antigen-specific T cell according to claim 25 for use in a method of adoptive T cell therapy.
27. A method of treating or preventing a disease or disorder in a subject, comprising: (a) isolating PBMCs from a subject; (b) generating or expanding a population of gamma delta T cells according to the method of any one of claims 1 to 15, and; (c) administering the gamma delta T cells to a subject.
28. A method of treating or preventing a disease or disorder in a subject, comprising: (a) isolating PBMCs from a subject; (b) generating or expanding a population of gamma delta T cells according to the method of any one of claims 1 to 15; (c) generating or expanding a population of antigen-specific T cells by a method comprising stimulating T cells by culture in the presence of gamma delta T cells generated/expanded according to (b) presenting a peptide of the antigen; and (d) administering the antigen-specific T cells to a subject.
Description
BRIEF DESCRIPTION OF THE FIGURES
[0241] Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures in which:
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EXAMPLES
Example 1: Materials and Methods
[0250] Ethics Approval and Participant Consent
[0251] All healthy donors and nasopharyngeal cancer patients are confirmed to have given written informed consent to a tissue and blood procurement study allowing ex vivo experimentation, which is approved by the National Cancer Centre Singapore's Office of Regulatory Affairs, Institutional Review Board (IRB).
[0252] Antibodies
[0253] The following monoclonal human antibodies (MoAbs) were used: (1) TCR [FITC-conjugated, clone Immu360; Beckman Coulter, Indianapolis, USA]; (2) TCR [PE-conjugated, clone 11F2; BD Bioscience, New Jersey, USA]; (3) CD3 (Pacific Blue-conjugated, clone UCHT1, mouse IgG1; BD Pharminen, New Jersey, USA); (4) HLA-ABC (APC-Cy7-conjugated, clone W6/32, mouse IgG2a; Biolegend); (5) HLA-DR (FITC-conjugated, clone L243, mouse IgG2a; BD Bioscience); (6) CD40 (PE-Cy7-conjugated, clone 5C3, mouse IgG1; BD Pharminen); (7) CD80 (PE-Cy7-conjugated, clone L307.4, mouse IgG1; BD Pharminen); (8) CD83 (PE-Cy7-conjugated, clone HB15e, mouse IgG1; BD Pharminen); (9) CD86 (PE-Cy7-conjugated, clone 2331/FUN-1, mouse IgG1; BD Pharminen); (10) CD54 (ICAM-1) [APC-conjugated, clone HA58, mouse IgG1; BD Pharminen); (11) ICOSL (FITC-conjugated, clone MIH12, mouse IgG1; Miltenyi Biotec GmbH, Bergisch Gladbach, Germany); (12) CD1d (APC-conjugated, clone CD1d42, mouse IgG1; BD Pharminen); (13) CCR5 (CD195) [APC-Cy7-conjugated, clone 2D7/CCR5, mouse IgG2a; BD Pharminen]; (14) CCR6 (CD196) [FITC-conjugated, clone G034E3, mouse IgG2b; Biolegend]; (15) CCR7 (CD197) [APC-conjugated, clone G043H7, mouse IgG2a; Biolegend]; (16) CD27 (FITC-conjugated, clone M-T271, mouse IgG1; BD Pharminen); (17) NKG2D (CD314) [APC-conjugated, clone 1D11, mouse IgG1; BD Pharminen]; (18) PD-1 (CD279) [APC-conjugated, clone MIH4, mouse IgG1; eBioscience, San Diego, USA); (19) CLTA-4 (PE-Cy7-conjugated, clone 14D3, mouse IgG2a; eBioscience); (20) Fas ligand (FASL/CD178) [APC-conjugated, clone NOK-1, mouse IgG1; BD Pharminen]; (21) IFN- (PE-conjugated, clone 4S.B3, mouse IgG1; Biolegend, San Diego, USA); (22) TNF- (APC-Cy7-conjugated, clone Mab11, mouse IgG1; Biolegend); (23) IL-10 (PE-Cy7-conjugated, clone JES3-9D7, mouse IgG1; Biolegend); (24) IL-17A (APC-conjugated, clone BL168, mouse IgG1K; Biolegend); (25) TIM3 (CD366, PE-conjugated, clone F38-2E2 Mouse IgG IgG1, eBioscience); (26) LAG3 (CD223, PE-Cy7-conjugated, clone 3DS223H, mouse IgG1, eBioscience; (27) MICA (PE-conjugated, clone #159227, mouse IgG2b; R&D Systems); (28) MICB (APC-conjugated, clone #236511, mouse IgG2b; R&D Systems); (29) BTN3A1 (CD277) [APC-conjugated, clone BL168, mouse IgG1; Novus Biologicals, Colorado, USA); (30) NKG2D blocking antibody (purified, clone 1D11, mouse IgG1; Biolegend); (31) TCR blocking antibody (purified, clone B1, mouse IgG1; Biolegend). Cells were washed twice with DPBS and resuspended in cold staining buffer (HBSS containing 2% heat-inactivated FBS) for 10 min blocking on ice. Then, they were stained with the relevant MoAbs for 30 min on ice, washed twice with staining buffer and acquired on the same day on a BD Canto II flow cytometer (Becton Dickinson, Franklin Lakes, N.J.). Data were analyzed using the Pro CellQuest software. T cells were first gated using the forward and side scatter dot plots, and the cell population highly expressing TCR and CD3 was further analyzed for other phenotypic markers or intracellular cytokines.
[0254] Synthetic Peptides
[0255] HLA-restricted immunodominant peptides derived from NY-ESO-1 and EBV Prombix pooled peptides were purchased from Proimmune (Oxford, UK). Purities were 279% as indicated by reverse-phase high performance liquid chromatography and mass spectrometry. MACS GMP PepTivator EBV LMP2A consisted of lyophilized overlapping oligopeptides (mainly 15-mer), covering the sequence of the LMP2A protein of Epstein-Barr virus strain B95-8 [Swiss-Prot Acc. no. P13285] (total purity of 290% as determined by RP-HPLC; Miltenyi).
[0256] Tumor Cell Lines
[0257] C666-1, Hep3B, DLD-1 and K562 (all except C666-1 were purchased from American Type Culture Collection [ATCC], Manassas, Va.; C666-1 was a gift) were maintained at 37 C., 5% CO.sub.2 in DMEM medium supplemented with 10% defined FBS, 100 units/ml penicillin, 100 units/ml streptomycin and 100 units/ml L-glutamine (all from Life Technologies). C666-1, Hep3B, DLD-1 and K562 tumour lines were derived from nasopharyngeal carcinoma, hepatocellular carcinoma, colorectal carcinoma and myelogenous leukemia, respectively. All tumour cell lines were tested regularly and found to be negative for Mycop/asma infection (Mycoplasma Detection Kit; American Type Culture Collection).
[0258] Isolation of Peripheral Blood Mononuclear Cells from Fresh Whole Blood
[0259] Peripheral blood mononuclear cells (PBMCs) were prepared from 100 ml of fresh whole blood from healthy volunteers. PBMCs were first separated on Ficoll lymphoprep (Nycomed Pharma, Oslo, Norway; 400 g, 30 min, brake off) and washed twice with HBSS (400 g, 5 min, with brake). Then the PBMCs were resuspended in 90% heat-inactivated defined fetal bovine serum (FBS) and 10% DMSO, and frozen to 80 C. with a controlled-rated freezer. After that, they were transferred to 150 C. liquid nitrogen until ready for use to generate gamma-delta () T cells, dendritic cells (DCs) or nave CD4.sup.+ and CD8.sup.+ T cells.
[0260] Gamma-Delta T Cell Preparation
[0261] Cryopreserved PBMCs were rapidly thawed in 37 C. water bath and washed twice with HBSS (400 g, 8 min, with brake) before use. For cell culture media and serum optimization experiments, a total of 110.sup.7 healthy donor PBMCs were seeded into a T25 flasks and cultured for a total of 10 days in either OpTimizer T cell medium (Gibco; supplemented with 1 Optimizer T cell supplement and 100 units/ml HEPES) or Click's medium (Irvine Scientific; supplemented with 100 units/ml HEPES) with different percentages of human AB serum (i.e. 2% or 5%) or 10% heat-inactivated defined fetal bovine serum (FBS). Zoledronic acid (5 M) was added to the PBMCs on Day 1 and 3 to activate gamma-delta () T cells, while human recombinant interleukin (IL)-2 (200 IU/ml; clinical grade, Proleukin) was added to assist in T cell proliferation following zoledronic acid activation. For cytokine optimization experiments, PBMCs were cultured for 10 days in Optimizer T cell media supplemented with 1 Optimizer T cell supplement, 100 units/ml HEPES and 10% heat-inactivated defined FBS. Zoledronic acid (5 M) was added on Day 1 and 3 together with different combinations of human recombinant cytokines (i.e. IL-2 at 200 IU/ml, IL-7 at 10 ng/ml, IL-15 at 10 ng/ml, IL-18 at 10 ng/ml and IL-21 at 30 ng/ml; all except IL-2 were GMP grade and purchased from CellGenix). At the end of Day 10, unpurified T cells were harvested for evaluation of purity, cell number and phenotypic analysis. In some experiments, T cells were purified with magnetic bead separation following manufacturer's instructions (Miltenyi) and used in tumour cell cytotoxic assays and nave CD4.sup.+ and CD8.sup.+ T cell cocultures.
[0262] Monocyte-Derived Dendritic Cell Preparation
[0263] Cryopreserved PBMCs were rapidly thawed in 37 C. waterbath, washed twice with HBSS (400 g, 8 min, with brake), resuspended in RPMI medium supplemented with 10% heat-inactivated defined FBS, and seeded at 110.sup.6 cells/ml in 6-well plates (Corning). After 4 hours of incubation at 37 C., 5% CO.sub.2, nonadherent representing lymphocytes were removed by gentle washing and adherent representing monocytes were cultured for a total of 7 days in RPMI medium containing 10% heat-inactivated defined FBS, 500 IU/ml human recombinant granulocyte macrophage colony-stimulating factor (GM-CSF; GMP grade, GellGro) and 250 IU/ml IL-4 (GMP grade, GellGro). On Day 5, fresh GM-CSF and IL-4 were added. On Day 7, these dendritic cells (DCs) were >95% pure (as judged by the absence of CD3- or CD19-expressing lymphocytes and expression of CD1a). The cells had an immature phenotype characterized by absence of CD83; low levels of CD86; and moderate levels of HLA-DR, HLA-ABC, and CD40. They showed a typical dendritic cell appearance by light microscopy.
[0264] Nave T Cell Isolation and CFSE-Labeling
[0265] Nave CD4.sup.+ and CD8.sup.+ T cells were derived from the nonadherent lymphocyte population after 4 hours of plastic adhesion as described in the DC preparation. The nave CD4.sup.+ and CD8.sup.+ T cells were isolated using magnetic bead separation kit (Miltenyi) following manufacturer's instructions. Then, they were labeled with cell membrane CFSE (carboxyfluorescein diacetate succinimidyl ester) dye (final concentration of 5 M; Molecular Probes) for 20 min at 37 C. and excess CFSE was adsorbed by adding an equal volume of RPMI medium containing 10% heat-inactivated defined FBS with further 5 min incubation. After that, they were washed once with HBSS (400 g, 8 min, with brake) and used for 2-week cocultures with peptide-pulsed T cells or DCs.
[0266] Coculturing CFSE-Labeled Nave T Cells with Peptide-Pulsed T Cells or DCs
[0267] Day 10 purified T cells or Day 7 DCs were pulsed with Epstein-Barr virus (EBV) or NY-ESO-1 Prombix peptides (10 g/ml; Proimmune) for 2 hours and activated overnight with lipopolysaccharides (LPS) [100 ng/ml; Invivogen]. T cells or DCs were harvested the next day and washed twice with HBSS (400 g, 5 min, with brake) before coculturing with CFSE-labeled nave CD4.sup.+ and CD8.sup.+ T cells (ratio of 10 nave T cells to 1 T cell or DC). After a week of coculture, viable CD4.sup.+ and CD8.sup.+ T cells were restimulated with fresh Day 10 T cells or Day 7 DCs that had been pulsed with relevant peptides and activated with LPS as described above. As controls, unpulsed T cells or DCs were cocultured with CFSE-labeled nave CD4.sup.+ and CD8.sup.+ T cells as described above. At the end of 2 weeks, CD4.sup.+ and CD8.sup.+ T cells were assessed for their proliferation as visualized by the dilution of CSFE staining on flow cytometry. These CD4.sup.+ and CD8.sup.+ T cells were also evaluated for their phenotypes and antigen-specificities with flow cytometry and pentamer staining, respectively.
[0268] Large-Scale Coculturing of Peripheral Blood Lymphocytes Cells with EVB-LMP2 Peptide-Pulsed T Cells or DCs
[0269] To mimic the large-scale procedure we would perform in the clinic, we obtained 300 ml of whole blood from healthy volunteers to isolate sufficient PBMCs, PBLs and monocytes for generating T cells, responder T cells and DC, respectively. One-third of the PBMCs were used for generating T cells, while the rest were plated to obtain monocytes and PBLs. The T cells and DCs were generated as described earlier in the methods and materials. For the coculture, Day 10 purified T cells or Day 7 DCs were pulsed with MACS GMP PepTivator EBV LMP2A (a pool of mainly 15mer overlapping oligopeptides covering the sequence of EBV LMP2A protein; final concentration of 0.6 nmol or 1 g of each peptide per ml; Miltenyi) for 2 hours and activated overnight with lipopolysaccharides (LPS) [100 ng/ml; Invivogen] for T cells, or with proinflammatory cytokines (prostaglandin 2A, TNF-, IL-1 and IL-6; all from Cellgro) for DCs overnight. The next day, T cells or DCs were harvested, washed twice with HBSS (400 g, 5 min, with brake) before coculturing with PBLs at a ratio of 10 nave PBLs to 1 T cell or DC). After a week of coculture, viable PBLs were restimulated with fresh Day 10 T cells or Day 7 DCs that had been pulsed with relevant peptides and activated with LPS or proinflammatory cytokines as described above. During the coculture, IL-7 and IL-15 were added at 10 ng/ml each on Day 2 and every 3 days thereafter to support T cell growth. At the end of 2 weeks, PBLs were assessed for exhaustion and activation markers, and IFN- secretion in response to PepTivator EBV LMP2A peptide pool.
[0270] Phenotypic Analysis
[0271] For phenotype studies, cells were resuspended in cold staining buffer (HBSS containing 2% heat-inactivated defined FBS) for 10 min blocking at 4 C. Then, the cells were incubated with the relevant MoAbs for 30 min at 4 C. Following that, the cells were washed twice (500 g, 5 min, with brake) with staining buffer and analyzed immediately with BD Canto II flow cytometer (Becton Dickinson). Data were analyzed using Data were analyzed using the Pro CellQuest software. For T cell analysis, the relevant cells were first gated using the forward and side scatter dot plots and the cell population that highly expressing both T cell receptor (TCR) and CD3 was further analyzed for HLA-ABC, HLA-DR, CD40, CD80, CD83, CD86, ICAM-1, CCR5, CCR6, CCR7, NKG2D, PD-1, CTLA-4, TIM-3, LAG-3 toll-like receptor (TLR)-1, TLR-2, TLR-3, TLR-4, TLR-5, TLR-7 and TLR-9. For CD4.sup.+ and CD8.sup.+ T cell analysis, the relevant cell population that highly expressing al TCR, CD3 and CD4/CD8 was further analyzed for effector, effector memory, central memory, exhaustion (PD-1, CTLA-4, TIM-3, LAG-3) and FOXP3 regulatory T cell markers. For DC analysis, relevant cell population that highly expressing CD11c and HLA-DR was further analyzed for CD40, CD80, CD83, CD86 and ICAM-1. For tumour cell line analysis, relevant cell population was gated with forward and side scatter dot plots and further analyzed for MICA, MICB and BTN3A1 expressions.
[0272] Intracellular Cytokine Staining
[0273] T cells were stimulated with phorbol myristate acetate (PMA) [50 g/ml] and ionomycin (100 g/ml) [both from Sigma-Aldrich] to evaluate their cytokine profile. After the 1.sup.st hour of the total 5 hour incubation, T cells were pelleted by centrifugation (500g, 5 min with brake) and GolgiStop containing brefeldin A (1000 dilution according to manufacturer's instructions; BD Pharmingen) was added to the cells for the remainder of the incubation period. After that, the cells were harvested and stained for FITC-conjugated anti- TCR and Pacific Blue-conjugated anti-CD3 for 30 min at 4 C. This is followed by fix-permeabilization treatment (BD) for 30 min at 4 C. to stain intracellularly for IFN-, TNF-, and IL-17. Then, the cells were washed twice with staining buffer (HBSS containing 2% heat-inactivated FBS) and interrogated on the same day with BD Canto II flow cytometer (Becton Dickinson). The data was analyzed with Pro CellQuest software. For IL-10 intracellular staining, GolgiStop containing brefeldin A was added in the 1.sup.st hour of the total 12 hour incubation, stained and analyzed as described above. T cells that were positive for intracellular IFN-, TNF-, IL-10 or IL-17 were expressed as a percentage of the gated TCR.sup.+ CD3.sup.+ T cells. T cells not stimulated with PMA and ionomycin were evaluated the same way to account for background cytokine secretions.
[0274] Pentamer Staining
[0275] CD4.sup.+ and CD8.sup.+ T cells that had been stimulated with peptide-pulsed T cells or DCs for 2 weeks were evaluated for their antigen-specificities with pentamer staining. 110.sup.6 T cells per group were washed once with staining buffer (HBSS with 2% FCS) and stained with a phycoerythrin (PE)-conjugated HLA-A*1101-restricted EBV LMP2 pentamer (abbreviated p-EBV LMP2.sub.369-377; ProImmune) or HLA-A*2401-restricted NY-ESO-1 pentamer (abbreviated p-NY-ESO-1.sub.69-377; ProImmune) for 20 min at 370C. T cells were then counterstained with anti-CD8-APC or anti-CD4-APC-Cy7 for 30 min at 4 C. Following that, the cells were washed twice with staining buffer and analyzed by flow cytometry, gating on CD4.sup.+ or CD8.sup.+ cells. T cells that were double positive for CD4/CD8 and pentamer were expressed as a percentage of the total number of CD4.sup.+/CD8.sup.+ T cells gated.
[0276] Tumor Cytotoxic Assay
[0277] DELFIA EuTDA Cytotoxicity assay was used to evaluate tumour cell lysis by T cells. Briefly, T cells were seeded in 96-well V bottom plates in graded numbers (i.e. 110.sup.5, 510.sup.4, 2.510.sup.4 per well). Then, tumor cells (i.e. C666-1, Hep3B, DLD-1 and K562) were added to the T cells at 510.sup.3 cells per well. The cells were cocultured for a total of 2 hours at 37 C., 5% CO.sup.2 before the supernatants were analyzed for lysis of labeled tumor cell targets according to the manufacturer's protocol. All assays were performed in triplicate. The measured fluorescence signal was correlated directly with the amount of lysed cells and the results were expressed as % tumor cell lysis by T cells.
[0278] Cytokine and Chemokine Array Analysis
[0279] T cells were cocultured with different tumor lines (i.e. C666-1, Hep3B, DLD-1 and K562) at a ratio of 20 effector T cells (110.sup.5) to 1 tumor cells (510.sup.3) in 96-well V bottom plate for 24 hours at 37 C., 5% CO.sub.2. Then, the coculture supernatants were collected and evaluated for granzymes A and B, perforin, granulysin, IFN-, IL-17, IL-8, Eotaxin, IP-10, MIG, GRO A, MIP-3A, I-TAC, MCP-1, RANTES, MIP-1A, MIP-1B and ENA-78 with Biolegend Legendplex cytometric bead array (Biolegend) and BD Canto II flow cytometer according to the manufacturer's protocol. As negative controls, supernatants from tumor cells or T cells alone were evaluated. All assays were performed in duplicate. The data was presented as g/ml or ng/ml.
[0280] Statistical Analysis
[0281] Means for different experimental groups were analyzed from 3 to 6 independent experiments (i.e. DCs from 3 to 6 different individuals). The analysis of significance was carried out using unpaired Student's t-tests or one-way ANOVA. A significance level of 0.05 or less was considered statistically significant. Analyses were conducted in GraphPad Prism.
Example 2: Results and Discussion
[0282] Optimizer T Cell Medium is Superior than Click's Medium in Generating a Higher Yield and Purity of Peripheral Blood Derived-92 T Cells.
[0283] Click's and Optimizer T cell media are two widely used clinical grade serum-free defined media for expanding large numbers of tumor-infiltrating T cells (TILs) or activated tumor-specific CD4.sup.+ and CD8.sup.+ T cells. However, no clinical trials and preclinical studies had explored the use of these media in generating T cells from peripheral blood. Serum derived from bovine or human provides a good source of nutrients for rapidly expanding CD4.sup.+ and CD8.sup.+ T cells. Autologous serum from cancer patients is not an ideal source as it could contain high levels of inhibitory cytokines such as IL-10, IL-6 or transforming growth factor (TGF)- to suppress the proliferation and function of T cells. A suitable alternative is the pooled normal human AB serum which is free from T cell inhibitory cytokines and infectious agents. Defined FBS is also available for clinical use. As opposed to normal FBS, defined FBS is certified free from bovine-related infectious agents and other contaminants that could adversely affect T cell generation. We have successfully used defined FBS to generate large numbers of cytotoxic T lymphocytes (CTLs) specific to Epstein-Barr virus (EBV) in a Phase I/II trial of nasopharyngeal carcinoma (NPC) [21].
[0284] In this study, we evaluated different media and serum combinations for culturing peripheral blood-derived 92 T cells. We compared Click's and Optimizer T cell medium supplemented with 2% or 5% pooled human AB serum, or 10% defined FBS. Using 10 million cryopreserved PBMCs as the starting population, we determined the yield and percentage purity of the generated T cells after 10 days of culture. In summary, we found that Optimizer T cell medium was superior than Click's medium in supporting T cell expansion whether serum-free or serum-supplemented (
[0285] Recombinant Human IL-21 Further Enhanced the Purity and Yield of Ex Vivo Generated 92 T Cells.
[0286] IL-2 and IL-15 are widely used cytokines for ex vivo expansion of CD4.sup.+ and CD8.sup.+ T cells. IL-2 is commonly used for generating T cells in the clinics (22, 23), while IL-15 is known for inducing the proliferation of memory CD4.sup.+ and CD8.sup.+ T cells (24). IL-7 is required for the homeostatic maintenance and proliferation of nave CD4.sup.+ and CD8.sup.+ T cell (22, 23). IL-18 has been shown to elicit a stronger IFN- response from T cells (25), while IL-21 could enhance the cytotoxic activity of ex vivo generated 92 T cells (26). All these cytokines are available in GMP grade for clinical use. The synergistic effects of these cytokines on the generation of V9V2 T cells have not been explored in detail. Thus, we analyzed the effect of these recombinant human cytokines on the yield and purity of the generated 92 T cells from a starting population of 10 million cryopreserved PBMCs. We observed that IL-15 alone or in combination of IL-7, IL-18 or IL-21 was more superior in expanding T cells than IL-2 alone or in combinations with the above mentioned cytokines (
[0287] Ex Vivo Generated 92 T Cells Exhibited Desirable Antigen Presentation and Effector Phenotypic Markers and were Highly Proinflammatory Upon Activation.
[0288] Next, we investigated the effect of different cytokine combinations in influencing the phenotypes and cytokine profiles of the generated 92 T cells. In
[0289] We further evaluated the cytokine profile of these ex vivo generated T cells by activating them with PMA and ionomycin. After 5 hours of stimulation, we performed intracellular cytokine staining to determine the % of T cells expressing IFN-, TNF-, IL-17 and IL-10. We found that a high % of T cells could be activated by PMA and ionomycin to produce proinflammatory IFN- only (54.8212.09% to 66.424.68%) or TNF- only (54.111.6% to 66.98.3%) regardless of their cytokine culture condition (Table 1; columns 1 and 2). A smaller % of T cells were also capable of producing both IFN- and TNF- upon activation (Table 1; 17.75.6% to 48.69.4%, column 3). Notably, very small % of these ex vivo generated T cells produced IL-17 (00% to 1.080.72%) and IL-10 (00.02 to 0.510.28%), suggesting that they preferentially elicit proinflammatory T helper (Th)-1 and cytotoxic T cell (CTL) responses. This finding was important as both IL-17 and IL-10 have been implicated in assisting tumor progression, thus low expression of these cytokines from T cells was preferred for generating a strong anti-tumor response. The following groups were selected for further functional analysisi.e. IL-2 alone, IL-2+IL-21, IL-15 and IL-15+IL-21. We selected the IL-2 alone group because all the published clinical trials so far had used IL-2 alone for T cell expansion. This group served as the baseline response for comparison of all the functional analysis in our study. The other three groups (IL-15 alone, IL-2+IL-15 and IL-15+IL-21) were selected because they consistently gave one of the highest yield and % purity of T cells compared to IL-2 alone and other groups (see
TABLE-US-00001 TABLE 1 Percentage of gamma-delta T cells producing IFN-, TNF-, IL-17 and IL-10 following PMA and Ionomycin stimulation % gamma-delta T cells producing cytokines following PMA and ionomycin stimulation Cytokine condition IFN- TNF- IFN- + TNF- IL-17A IL-10 IL-2 only 54.82 12.09 63.39 11.4 17.7 5.6 0 0 0.03 0.04 IL-2 + IL-7 56.91 12.13 63.88 10.3 14.8 3.3 0 0 0.05 0.07 IL-2 + IL-15 65.62 6.47 64.04 9.7 32 8.9 0 0 0.02 0.06 IL-2 + IL-18 62.79 12.57 66.90 8.3 48.6 9.4 4.11 2.4 0.12 0.1 IL-2 + IL-21 66.01 5.09 64.07 11.9 21.8 7.3 0.74 0.17 0.33 0.15 IL-2 + IL-18 + IL-21 62.89 7.70 62.58 10.7 26 5.8 0 0 0.06 0.07 IL-15 only 66.42 4.68 61.70 8.1 41.7 4.6 0 0 0.02 0.01 IL-15 + IL-7 55.78 14.30 61.90 7.4 41.4 6.3 0 0 0 0.02 IL-15 + IL-18 55.46 5.76 57.23 10.1 18.5 4.8 0.61 1.40 0.02 0.01 IL-15 + IL-21 61.53 6.72 56.69 6.5 35.9 3.1 1.08 0.72 0.51 0.28 IL-15 + IL-18 + IL-21 62.70 4.66 54.10 11.6 25.1 5.4 0 0 0.06 0.01
[0290] Ex Vivo Generated 92 T Cells were Highly Efficient in Killing Broad Range of Tumor Cells Via NKG2D Ligand Recognition and Displayed Differential Cytokine and Chemokine Profiles.
[0291] To determine if the ex vivo generated 92 T cells could directly recognize and lyse tumor cells, we cocultured the purified T cells with different tumor types at varying effector to tumor target ratios (i.e. 20 to 1, 10 to 1 and 5 to 1) as described in the Materials and Methods section. We chose four cell lines derived from different tumor typesC666-1 (nasopharyngeal carcinoma), Hep3B (hepatocellular carcinoma), DLD-1 (colorectal carcinoma) and K562 (myeloid leukemia) for analysisand determined their MICA, MICB and BTN3A1 expressions which were ligands for direct tumor cytolysis by T cells via their NKG2D and T cell receptors (TCRs). We determined that all the tumor lines expressed MICA, MICB and BTN3A1, with K562 line expressing the highest levels of these three ligands amongst the four tumor cell lines (
[0292] In
[0293] We further evaluated the mode of direct tumor cytolysis of T cells by measuring their secreted granzymes A and B, perforin, granulysin and IFN- following 24 hours incubation with the above tumor cell lines. All the T cells were able to produce granzymes A and B, granulysin, perforin and IFN- (
[0294] Next, we used cluster analysis to evaluate the chemokine profiles of T cells in response to live tumor cells (
[0295] Ex Vivo Generated 92 T Cells were More Efficient than Monocyte-Derived Dendritic Cells in Stimulating the Proliferation of Nave CD4.sup.+ and CD8.sup.+ T Cells.
[0296] We investigated whether ex vivo generated 92 T cells could act as antigen-presenting cells to stimulate the proliferation of nave CD4.sup.+ and CD8.sup.+ T cells in cocultures. To test this, we pulsed the T cells with MHC Class I-restricted peptides derived from either EBV or NY-ESO-1 (a tumor-associated antigen) and cocultured with CFSE-labeled nave CD4.sup.+ and CD8.sup.+ T cells for two weeks. At the end of two weeks, we observed that that peptide-pulsed T cells, regardless EBV or NY-ESO-1 derived, stimulated a more robust proliferation of nave CD4.sup.+ and CD8.sup.+ T cells than Day 7 classical monocyte-derived dendritic cells pulsed with the same peptides (
[0297] Lower % of CD4.sup.+ and CD8.sup.+ T Cells Expressed PD-1, CTLA-4, TIM3 and LAG3 Exhaustion Phenotypes Following Stimulation with 7962 T Cells Pulsed with EBV-LMP2A Pooled Peptides Compared to DCs Pulsed with the Same Peptide Pool.
[0298] To further evaluate the phenotype and cytokine profile of the T cells stimulated by antigen-pulsed 92 T cells, we cocultured PBLs with 92 T cells pulsed with pooled overlapping peptides of EBV-LMP2A. By comparing to monocyte-derived DCs pulsed with the same peptide pool, we observed that peptide-pulsed T cells stimulated an overall higher, though not significant, number of CD3.sup.+ T lymphocytes (
[0299] Discussion
[0300] Compelling evidence from clinical and preclinical studies now shows that T cells play an important role in tumor surveillance through active surveying and elimination of transformed cells in the body. T cells exhibit unique antigen specificities compared to CD4.sup.+ and CD8.sup.+ T cells that recognize tumor-derived peptides presented by professional antigen-presenting cells such as DCs; T cells show diverse antigen specificity towards phosphoantigens (e.g. IPP), self-derived stress-induced ligands on tumor cells (e.g. MICA, MICB, ULBP and HSP) and lipids. They also recognize protein antigens via their TCR (reviewed in 1). Recently, they have been shown to display antigen-presentation and the ability to activate CD4.sup.+ and CD8.sup.+ T cells (39, 40). Promising results have been observed in B cell leukemia, prostate and renal cell carcinoma patients whereby some of them achieved partial remission and stable diseases after V9V2 T cell treatment (18-20). Thus, these findings strongly support the rationale of V9V2 T cell-based tumor immunotherapy.
[0301] As the procedures for ex vivo generation of V9V2 T cell are highly variable and difficult to compare in many clinical trials even within a given clinical setting or disease, there is a strong need to define a set of robust criteria for producing V9V2 T cells that are highly immunogenic and effective for the clinic. We were the first to describe and define a set of important culture parameters for large-scale clinical production of V9V2 T cells that could highly influence the quality of their phenotype, cytokine profile and anti-tumor functions.
[0302] First, we evaluated two clinical grade media (i.e. Click's and Optimizer T cell media) in combination with different serum (i.e. pooled human AB serum and defined FBS) for culturing V9V2 T cells.
[0303] We chose these two cell culture media because they have been used extensively for culturing CD4.sup.+ and CD8.sup.+ T cells in the clinics, in particular, Optimizer T cell medium could be used serum-free. However, no study have evaluated the effect of these culture media on V9V2 T cells. We chose to supplement the cell culture with pooled human AB serum (2% and 5%) or defined FBS (10%) because both types of sera are already in clinical use and are compatible for producing CD4.sup.+ and CD8.sup.+ T cells. In our Phase I/II clinical trial in NPC, we had successfully used Click's media supplemented with 10% defined FBS for large-scale production of EBV-specific CD8.sup.+ CTLs. Thus, this positive experience led us to select Click's medium and defined FBS as two cell culture components to be evaluated in this study. When used serum-free, we found that Optimizer T cell medium and not Click's medium was able to support V9V2 T cell growth. The yield and % purity of V9V2 T cells generated from PBMCs were significantly increased when pooled human AB serum or defined FBS was added. We also determined that 10% defined FBS was superior to 2% and 5% pooled human AB serum in supporting the rapid proliferation of V9V2 T cells. Hence, we chose Optimizer T cell media supplemented with 10% defined FBS as the optimal medium for further evaluation.
[0304] IL-2, IL-15, IL-7, IL-18 and IL-21 are well-studied cytokines for CD4.sup.+ and CD8.sup.+ T cell growth and functions. Amongst these cytokines, only IL-2 is used widely in the clinics for T cell proliferation. We evaluated the above cytokines either individually or in combinations on T cell growth. It was noted that IL-18 and IL-21 were not known to support CD4.sup.+ and CD8.sup.+ T cell growth, therefore we did not assessed them individually in this study. Similar to reported studies, IL-2 alone was able to induce strong proliferation of V9V2 T cells. V9V2 T cell yield and % purity were increased when IL-2 was used in combination with IL-7 or IL-21. On the other hand, the addition of IL-15 or IL-18 to IL-2 adversely reduced the growth of V9V2 T cells. The use of IL-15 alone or in combinations with IL-7 and IL-21 also supported a stronger V9V2 T cell proliferation. Similarly, the addition of IL-2 or IL-18 led to reduced V9V2 T cell yield and purity. Notably, IL-21 synergized with IL-2 and IL-15 to significantly enhance the yield and % purity of V9V2 T cells. The contrasting effects of IL-18 and IL-21 on V9V2 T cell proliferation were outside the scope of this study. As IL-21 is known to support CD4.sup.+ T cell differentiation, we speculated that V9V2 T cells might share similar properties as CD4.sup.+ T cell and hence IL-21 exerted a beneficial effect on their growth.
[0305] We found that all the V9V2 T cells generated exhibited both antigen presentation and effector phenotypes. This is important as it suggested that these V9V2 T cells have the ability to perform both antigen-presentation and direct tumor cytolysis functions. We found that the V9V2 T cells generated, regardless of cytokine combinations, were highly capable of producing IFN- and TNF-. This finding is important as IFN- and TNF- exert important anti-tumor functions and are required for activating DCs, CD4.sup.+ and CD8.sup.+ T cells. Thus, this suggested that the generated V9V2 T cells are highly capable of activating these immune cells after administration.
[0306] Interestingly, we observed that the use of IL-15 (alone or in combination with IL-7 or IL-21) in the cell culture assisted in generating a higher percentage of V9V2 T cells that produced IFN- and TNF- simultaneously upon PMA and ionomycin activation. Thus, the use of IL-15 not only improved the yield and purity of V9V2 T cells, it also helped to enhance their proinflammatory cytokine secretions. Encouragingly, very low % of V9V2 T cells produce IL-17 and IL-10, indicating that they would not actively support tumor and T regulatory cell growth.
[0307] Similar to CD8.sup.+ CTLs, the ex vivo generated V9V2 T cells used granzymes A and B, perforin and granulysin to target and lyse tumor cells. We also noted that the V9V2 T cells reacted most strongly against C666-1 NPC line which actively expressed EBV-related antigen, indicating that V9V2 T cell-based immunotherapy might be particularly useful against viral-related cancers. In addition, we also found that the V9V2 T cells generated were superior to monocyte-derived classical Day 7 DCs in simulating the proliferation of nave CD4.sup.+ and CD8.sup.+ T cells. In our large-scale study, we further showed that peptide-pulsed T cells (whether generated with IL-2+IL-21 or IL-15+IL-21) were more efficient than monocyte-derived DCs in stimulating more antigen-specific IFN- secreting CD8.sup.+ and CD4.sup.+ T cells, as well as CD8.sup.+ and CD4.sup.+ T cells that were less exhausted in phenotype and fewer Tregs.
[0308] In conclusion, we had optimized different culture parameters for large-scale V9V2 T cell production. We evaluated important cell culture parameters that could highly influence the quality of V9V2 T cell phenotype, cytokine prolife, direct tumor cytolysis and antigen presentation for activating anti-tumor CD4.sup.+ and CD8.sup.+ T cells. We determined that Optimizer T cell medium supplemented with 10% defined FBS, IL-15 (10 ng/ml) and IL-21 (30 ng/ml) was optimal for generating more than 25 million V9V2 T cell with a purity of 285% from a starting population of 10 million cryopreserved PBMCs. We also determined that the V9V2 T cell generated under this culture condition exhibited desirable antigen-presentation and effector phenotypes, were highly tumor cytolytic and stimulated strong nave CD4.sup.+ and CD8.sup.+ T cell proliferation. The ex vivo generated T cells stimulated more IFN- antigen-specific CD8.sup.+ T cells as well as less exhausted T cells and fewer Tregs compared to DCs in our experimental system. T cell-based therapy could highly benefit from immune checkpoint blockage of TIM-3, LAG-3 and/or CTLA-4 to augment the anti-tumor activities of T cells, CD8+ and CD4+ T cells. This is the first study that provides important insight into the anti-tumor properties of V9V2, as well as essential preclinical data for the development of potent V9V2 T cell-based immunotherapy which is applicable to many tumor types.
REFERENCES
[0309] 1. Silva-Santos B, Serre K, Norell H. T cells in cancer. (2015) Nat Rev Immunol 15:683-91. [0310] 2. Parker C M, Groh V, Band H, et al. (1990). Evidence for extrathymic changes in the T cell receptor / repertoire. J Exp Med 1990; 171:1597-612. [0311] 3. Morita C T, Jin C, Sarikonda G, et al. (2007). Nonpeptide antigens, presentation mechanisms, and immunological memory of human V2V2 T cells: discriminating friend from foe through the recognition of prenyl pyrophosphate antigens. Immunol Rev 2007; 215:59-76. [0312] 4. Tanaka, Y., Morita, C. T., Nieves, E., et al. (2015) Natural and synthetic non-peptide antigens recognized by human T cells. Nature 375:155-158. [0313] 5. Morita, C. T. et al. (1995) Direct presentation of nonpeptide prenyl pyrophosphate antigens to human T cells. Immunity 3:495-507. [0314] 6. Thompson K, Rogers M J. (2004). Statins prevent bisphosphonate-induced -T-cell proliferation and activation in vitro. J Bone Miner Res 19:278-88. [0315] 7. Gober H J, Kistowska M, Angman L., et al. (2003). Human T cell receptor cells recognize endogenous mevalonate metabolites in tumor cells. J Exp Med 197:163-8. [0316] 8. Zheng B J, Chan K W, Im S., et al. (2001). Anti-tumor effects of human peripheral T cells in a mouse tumor model. Int J Cancer 92:421-5. [0317] 9. Guo B L, Liu Z, Aldrich W A, Lopez R D. (2005). Innate anti-breast cancer immunity of apoptosis-resistant human -T cells. Breast Cancer Res Treat 93:169-75. [0318] 10. Kenna T, Golden-Mason L, Norris S., et al. (2004). Distinct subpopulations of T cells are present in normal and tumor-bearing human liver. Clin Immunol 113:56-63. [0319] 11. Ferrarini M, Heltai S, Pupa S M., et al. (1996). Killing of laminin receptor-positive human lung cancers by tumor infiltrating lymphocytes bearing + T-cell receptors. J Natl Cancer Inst 88:436-41. [0320] 12. Kobayashi H, Tanaka Y, Yagi J., et al. (2001). / T cells provide innate immunity against renal cell carcinoma. Cancer Immunol Immunother 50:115-24. [0321] 13. Viey E, Fromont G, Escudier B., et al. (2005). Phosphostim-activated T cells kill autologous metastatic renal cell carcinoma. J Immunol 174:1338-47. [0322] 14. Kabelitz D, Wesch D, Pitters E, et al. (2004). Characterization of tumor reactivity of human V9V2 T cells in vitro and in SCID mice in vivo. J Immunol 173:6767-76. [0323] 15. Liu Z, Guo B L, Gehrs B C, et al. (2005). Ex vivo expanded human V 9V2+-T cells mediate innate antitumor activity against human prostate cancer cells in vitro. J Urol 173:1552-56. [0324] 16. Schilbach K E, Geiselhart A, Wessels J T, et al. (2000). Human T lymphocytes exert natural and IL-2-induced cytotoxicity to neuroblastoma cells. J Immunother 23:536-48. [0325] 17. Malkovska V, Cigel F K, Armstrong N, et al. (1992). Antilymphoma activity of human T cells in mice with severe combined immune deficiency. Cancer Res 52:5610-16. [0326] 18. Wilhelm M, Kunzmann V, Eckstein S, et al. (2003). T cells for immune therapy of patients with lymphoid malignancies. Blood 102:200-06. [0327] 19. Dieli F, Vermijlen D, Fulfaro F., et al. (2007). Targeting human T cells with zoledronate and interleukin-2 for immunotherapy of hormone-refractory prostate cancer. Cancer Res 67:7450-7. [0328] 20. Deniger D C, Moyes J S, Cooper U. (2014). Clinical applications of gamma delta T cells with multivalent immunity. Front Immunol 5:636. [0329] 21. Chia W K, Teo M, Wang W W, et al. (2014). Adoptive T-cell transfer and chemotherapy in the first-line treatment of metastatic and/or locally recurrent nasopharyngeal carcinoma.
[0330] Mol Ther 22: 132-9. [0331] 22. Toe J G, Pellegrini M, Mak T W. (2013). Promoting immunity during chronic infectionthe therapeutic potential of common gamma-chain cytokines. Mol Immunol 56:38-47. [0332] 23. Cox M A, Kahan S M, Zajac A J. (2013). Anti-viral CD8 T cells and the cytokines that they love. Virology 435:157-169. [0333] 24. Weng N P, Liu K, Catalfamo M., et al. (2002). IL-15 is a growth factor and an activator of CD8 memory T cells. Ann N Y Acad Sci. 975:46-56. [0334] 25. Li W, Kubo S, Okuda A., et al. (2010). Effect of IL-18 on expansion of gammadelta T cells stimulated by zoledronate and IL-2. J Immunother 33:287-96. [0335] 26. Thedrez A, Harty C, Morice A., et al. (2009). IL-21-mediated potentiation of antitumor cytolytic and proinflammatory responses of human V gamma 9V delta 2 T cells for adoptive immunotherapy. J Immunol. 182:3423-31. [0336] 27. Wu H H, Hwang-Verslues W W, Lee W H, et al. (2015). Targeting IL-17B-IL-17RB signaling with an anti-IL-17RB antibody blocks pancreatic cancer metastasis by silencing multiple chemokines. J Exp Med. 212:333-349. [0337] 28. Wang D Z, Wand H B, Brown J, et al. (2006). CXCL1 induced by prostaglandin E2 promotes angiogenesis in colorectal cancer. J Exp Med. 203:941-951. [0338] 29. Acharyya S, Oskarsson T, Vanharanta S, et al. (2012). A CXCL1 Paracrine Network Links Cancer Chemoresistance and Metastasis. Cell. 150:165-178. [0339] 30. Walch-Rckheim B, Mavrova R, Henning M, et al. (2015). Stromal Fibroblasts Induce CCL20 through IL6/C/EBP to Support the Recruitment of Th17 Cells during Cervical Cancer Progression. Cancer Res. 75:5248-5259. [0340] 31. Loetscher P, Seitz M, Clark-Lewis I, et al. (1996). Activation of NK cells by CC chemokines. Chemotaxis, Ca2+ mobilization, and enzyme release. J Immunol. 156:322-327. [0341] 32. Harlin H, Meng Y, Peterson A C, et al. (2009). Chemokine Expression in Melanoma Metastases Associated with CD8+ T-Cell Recruitment. Cancer Res. 69:3077-3085. [0342] 33. Molon B, Ugel S, Del Pozzo F, et al. (2011). Chemokine nitration prevents intratumoral infiltration of antigen-specific T cells. J Exp Med. 208:1949-1962. [0343] 34. Fridlender Z G, Buchlis G, Kapoor V, et al. (2010). CCL2 Blockade Augments Cancer Immunotherapy. Cancer Res. 70:109-118. [0344] 35. Castellino F and Germain R N. (2007). Chemokine-Guided CD4+ T Cell Help Enhances Generation of IL-6RahighIL-7Rahigh Prememory CD8+ T Cells. J Immunol. 178:778-787. [0345] 36. Hickman H D, Li L, Reynoso G V, et al. (2011). Chemokines control nave CD8+ T cell selection of optimal lymph node antigen presenting cells. J Exp Med. 208:2511-2524. [0346] 37. Mikucki M E, Fisher D T, Matsuzaki J, et al. (2015). Non-redundant requirement for CXCR3 signalling during tumoricidal T-cell trafficking across tumour vascular checkpoints. Nat Commun. 25:7458. [0347] 38. Sharma S, Yang S C, Hillinger S, et al. (2003). SLC/CCL21-mediated anti-tumor responses require IFN, MIG/CXCL9 and IP-10/CXCL10. Mol Cancer. 15:22. [0348] 39. Brandes M, Willimann K, Moser B. Professional antigen-presentation function by human gammadelta T Cells. (2005). Science 309:264-268. [0349] 40. Meuter S, Eber M, Moser B. (2010). Prolonged antigen survival and cytosolic export in cross-presenting human T cells. Proc Natl Acad Sci USA. 107:8730-8735.
Example 2: In Vivo Experiments in Mice
[0350] Anticancer activity of adoptively transferred T cells was analysed in vivo in experiments performed in a mice.
[0351] Experiment 1
[0352] Tumours were established by subcutaneous injection of mice with 510.sup.6 lymphoblastoid cell line cells (LCLs) on Day 0.
[0353] Mice were divided into groups of 3-4 mice, and assigned to one of four treatment groups a) to d) below: [0354] a) No treatment (mock treatment by injection of basal media) [0355] b) T cells only (designated gd)110.sup.6 cells per mouse, per treatment [0356] c) CSFE-labelled, pan nave T cells only (designated ab)110.sup.6 cells per mouse, per treatment [0357] d) Initial administration of T cells and CSFE-labelled, pan nave T cells in a 1:1 ratio (Treatment 1), followed by administration of T cells (Treatments 2 and 3)110.sup.6 cells per mouse, per treatment.
[0358] The T cells used in Experiment 1 were prepared as described in Example 1.
[0359] All treatments were administered intratumorally from Day 12, every 10 days, and blood samples were obtained prior to every treatment. A schematic representation of the procedures for Experiment 1 is shown in
[0360] At the end of the experiment the tumors and spleens were harvested and analysed (
[0361] The size and volume measurements of the tumors harvested from the mice are shown in the Table of
[0362] Administration of T cells was therefore demonstrated to have an antitumor effect.
[0363] Experiment 2
[0364] Tumours were established by subcutaneous injection of mice with 210.sup.6 LCLs on Day 0.
[0365] Mice were divided into groups of 3-4 mice, and assigned to one of five treatment groups a) to e) below: [0366] a) No treatment (mock treatment by injection of basal media), administered intratumorally [0367] b) T cells only210.sup.6 cells per mouse, per treatment, administered subcutaneously [0368] c) T cells only210.sup.6 cells per mouse, per treatment, administered intravenously [0369] d) CSFE-labelled, pan nave T cells only210.sup.6 cells per mouse, per treatment administered intratumorally [0370] e) Initial administration of T cells and CSFE-labelled, pan nave T cells in a 1:1 ratio (Treatment 1), followed by administration of T cells (Treatments 2 and 3)210 cells per mouse, per treatment, administered intratumorally.
[0371] The T cells used in Experiment 2 were prepared as described in Example 1.
[0372] Treatments were administered from Day 12, every 10 days, and blood samples were obtained prior to every treatment. A schematic representation of the procedures for Experiment 2 is shown in
[0373] At the end of the experiment the tumors and spleens were harvested and analysed. The size and volume measurements of the tumors harvested from the mice are shown in the Table of
[0374] Tumors obtained from mice which received treatment with T cells via intravenous administration (treatment group c) were smaller and had a reduced volume as compared to the tumors obtained from mice of the other treatment groups.
[0375] Intravenous administration of T cells was therefore demonstrated to have an antitumor effect.
[0376] Experiment 3
[0377] In a further experiment, tumours are established by subcutaneous injection of mice with 510.sup.5 LCLs on Day 0.
[0378] Mice are divided into groups of 3 mice, and assigned to one of five treatment groups 1) to 5) below: [0379] 1) No treatment (mock treatment by injection of basal media) [0380] 2) 1010.sup.6 T cells only+100 g/kg zoledronic acid per mouse, per treatment [0381] 3) 1010.sup.6 peripheral blood lymphocytes (PBLs)+100 g/kg zoledronic acid per mouse, per treatment [0382] 4) 1010.sup.6 cells from a coculture of T cells and PBLs+100 g/kg zoledronic acid per mouse, per treatment [0383] 5) 100 g/kg zolendronic acid per mouse, per treatment
[0384] All treatments are administered intravenously from Day 12, every 10 days, and blood samples are obtained prior to every treatment.
[0385] Zoledronic acid is obtained from Sigma Aldrich and is diluted in basal media prior to administration.
[0386] A schematic representation of the procedures for Experiment 3 is shown in
[0387] The T cells used in Experiment 3 are prepared as described in Example 1, with the following variations: [0388] (i) 10 M is added to the cultures on days 1, 3 and 5 [0389] (ii) 400 units/ml IL-2 and 60 ng/ml of IL-21 are added to the cultures on days 1, 2, 5 and 8 [0390] (iii) The T cells are isolated on day 11.
[0391] It is expected that combination treatment with T cells and zoledronic acid (e.g. treatment group 2) will display greater antitumor activity as compared to treatment with zoledronic acid alone (treatment group 5).