BACKGROUND TTFields has the ability to induce immunogenic cell death (ICD). As immunotherapy and TTFields have different mechanisms of action (MOA), combining these therapies is a rational approach. Contrarily, TTFields may interfere with immune functions critical for effective T cell function. MATERIAL AND METHODS We cultured T cells from healthy donors’ peripheral blood or from viably dissociated glioblastoma samples under normal or TTFields conditions, with or without superantigen-stimulation. In order to assess T cell responses we used eight-color flow cytometry by monitoring select pivotal antitumoral functions: proliferation (CFSE), IFNγ secretion, cytotoxic degranulation (CD107a), activation/exhaustion (PD1) and viability. Evaluation of direct cytotoxicity was done by using chimeric antigen receptor (CAR) T cells. RESULTS TTFields did not change T cell activation rates for all evaluated functions with the exception of reduced proliferation - in line with TTFields’ MOA. TTFields substantially reduced the viability of activated proliferating T cells, moderately affected activated nonproliferating T cells and had almost no effect on the viability of non-activated cells. Polyfunctionality analysis of T-cells, associated with effective antitumoral responses, demonstrated that under TTFields, the activated non-proliferating T cells retained polyfunctional capabilities. PD1-expressing TILs, a subset containing most of the tumor antigen-specific TILs, exhibited unaltered viability and functionality under TTFields. CAR T-cells, which utilize the same killing machinery as unmodified T cells, exhibited unaltered cytotoxic capability under TTFields. Immunohistochemical evaluation of GBM samples before TTFields treatment and after recurrence showed that some patients had accommodated large increases in their CD8 and CD4 counts. RNA-Seq performed on GBM samples from 6 standardly-treated and 6 TTFields-treated patients before treatment and after recurrence. The data shows differential increases in TTFields-treated patients to controls, in the expression of immune genes associated with favorable prognosis (e.g. t-bet, NKG2D, ICOS-L, CD70) and concurrent decreases in genes associated with poor prognosis (e.g. IL4, TSLP, various complement genes). CONCLUSION The preclinical data showed that all antitumoral T cell functions examined, but proliferation, were unhindered by TTFields. The clinical data showed that TTFields may shift treated tumors to a state more conducive of antitumoral immune responses. Our findings support the further preclinical and clinical investigation into combining TTFields with immunotherapy.

Background: Tumor Treating Fields (TTFields) are low-intensity electric fields that target proliferating cells by hindering mitotic spindle formation and charged organelle translocation. TTFields are FDA-approved for treatment of newly diagnosed and recurrent glioblastoma. Combining TTFields with immunotherapy is a rational approach due to their different mechanisms of action (MOA) and to TTFields' ability to induce immunogenic cell death (ICD). Conversely, TTFields may interfere with immune functions critical for effective T cell responses. Here, we evaluated the effects of TTFields on select pivotal anti-tumoral T cell functions. Methods: T cells from healthy donors' peripheral blood or from viably dissociated fresh glioblastoma samples were cultured under normal or TTFields conditions, with or without superantigen stimulation. Multiparametric flow cytometry (8 colors) was used to assess T cell responses by monitoring select pivotal anti-tumoral functions: proliferation (CFSE dilution), IFNγ secretion (intracellular staining), cytotoxic degranulation (CD107a surface expression) and activation/exhaustion (PD1 expression). T cell viability under TTfields was assessed in a dedicated assay. Direct cytotoxicity under TTFields was evaluated using a chimeric antigen receptor (CAR) T cell-based assay. Results: The viability of superantigen-stimulated T cells that attempted to proliferate decreased under TTFields, in line with TTFields' MOA. Small or no reductions in viability were found in activated T cells that did not attempt to proliferate and in unstimulated T cells. The functionality of peripheral-blood T cells and tumor-infiltrating T cells (TILs) stimulated by superantigen under TTFields was unhindered: T cells exhibited comparable PD1 upregulation, IFNγ secretion and CD107a surface-expression as controls. T cell polyfunctionality was previously reported to be associated with effective anti-tumoral responses. A polyfunctionality analysis conducted on our data demonstrated that T cells that had lost the capacity to proliferate under TTFields conditions, retained all other polyfunctional combinations. PD1+ TILs, a subset containing most of the tumor antigen-specific T cells, exhibited unaltered viability and functionality when cultured under TTFields. Lastly, the cytotoxic capability of CAR T-cells (which utilize the same killing machinery as normal T cells) was unaffected by TTFields. Conclusions: All anti-tumoral T cell functions examined, with the exception of proliferation, were unhindered by TTFields. Our findings, together with the plausible synergism of TTFields with systemic anti-tumoral immune responses, warrant the further preclinical and clinical investigation into the combination of these therapeutic approaches. Citation Format: Gil Diamant, Hadar Simchony, Tamar Shiloach, Anat Globerson-levin, Zelig Eshhar, Rachel Grossman, Zvi Ram, Ilan Volovitz. Evaluating the compatibility of tumor treating electric fields with key anti-tumoral T cell functions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 621.

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Background: Combining Tumor Treating electrical Fields (TTFields) with immunotherapy is a rational approach due to their different mechanisms of action (MOA) and to TTFields’ ability to induce immunogenic cell death (ICD). Conversely, TTFields may interfere with immune functions critical for effective T cell responses. Methods: T cells from healthy donors’ peripheral blood or from viably dissociated glioblastoma samples were cultured under normal or TTFields conditions, with or without superantigen-stimulation. Eight-color flow cytometry was used to assess T cell responses by monitoring select pivotal antitumoral functions: proliferation (CFSE), IFNγ secretion, cytotoxic degranulation (CD107a), activation/exhaustion (PD1) and viability. Direct cytotoxicity was evaluated using chimeric antigen receptor (CAR) T cells. Results: The viability of stimulated T cells that attempted to proliferate decreased under TTFields, in line with TTFields’ MOA. Small or no reductions in viability were found in activated T cells that did not attempt to proliferate and in unstimulated T cells. The functionality of stimulated peripheral-blood T cells and tumor-infiltrating T cells (TILs) under TTFields was unhindered: T cells exhibited comparable PD1 upregulation, IFNγ secretion and CD107a expression as controls. T cell polyfunctionality, associated with effective antitumoral responses, was retained under TTFields conditions. PD1-expressing TILs, a subset containing most of the tumor antigen-specific TILs, exhibited unaltered viability and functionality under TTFields. CAR T-cells, which utilize the same killing machinery as unmodified T cells, exhibited unaltered cytotoxic capability under TTFields. Immunohistochemical evaluation of GBM samples before TTFields treatment and after recurrence showed that some patients had accommodated large increases in their CD8 and CD4 counts. RNA-Seq performed on GBM samples from 6 standardly-treated and 6 TTFields-treated patients before treatment and after recurrence. The data shows differential increases in TTFields-treated patients to controls, in the expression of immune genes associated with favorable prognosis (e.g. t-bet, NKG2D, ICOS-L, CD70) and concurrent decreases in genes associated with poor prognosis (e.g. IL4, TSLP, various complement genes). Conclusions: The preclinical data showed that all antitumoral T cell functions examined, but proliferation, were unhindered by TTFields. The clinical data showed that TTFields may shift treated tumors to a state more conducive of anti-tumoral immune responses. Our findings support the further preclinical and clinical investigation into combining TTFields with immunotherapy. Citation Format: Gil Diamant, Hadar Simchony, Tamar Shiloach, Anat Globerson-levin, Lital Gasri Plotnitsky, Zelig Eshhar, Niv Pencovich, Rachel Grossman, Zvi Ram, Ilan Volovitz. Evaluating the compatibility of tumor treating electric fields with key anti-tumoral immune functions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3954.