Background Evidence indicates that diacylglycerol kinases (DGK) are promising targets for the optimization of T cell activity, for example in the setting of adoptive cell therapy (ACT). The tumor microenvironment (TME) of human renal cell carcinoma (RCC) is an immunosuppressive setting where T and NK cell functionality is blocked. DGK–α is a negative regulator of TCR signaling, functioning by metabolizing diacylglycerol to phosphatidic acid and thereby limiting the activation of MAPK/ERK1/2 signaling pathway. DGK-α is found increased in tumor-infiltrating lymphocytes (TIL) from RCC patients and also in adoptively transferred T cells after infiltrating into the TME.1 We previously reported that inhibition of DGK-α restored functionality of unresponsive CD8 T cells and NK cells from RCC-TIL. Other studies demonstrated that knockdown or pharmacologic inhibition of DGK-α and DGK-ζ alone or together increased target cell killing and cytokine production, and protected T cells from inhibitory factors in the TME.2 However, there are no inhibitors for DGK-ζ and available DGK-α inhibitors have undesired pharmacokinetic/pharmacodynamic properties and are highly toxic precluding their clinical application. Here, we present data using a novel RNA interference (RNAi) technology that can specifically target each DGK isoform.
Materials and Methods INTASYL™ compounds incorporate drug-like properties into RNAi, resulting not only in enhanced cellular uptake in the presence of serum but also eliminating the need for further transfection reagents. Toxicity of compounds applied alone or in combination was assessed by 7-AAD flow cytometry analysis and WST assay. Silencing of mRNA and protein was analyzed by RT-qPCR and SimpleWestern. Downstream signaling pathways and T cell function were analyzed to demonstrate pharmacological efficacy.
Results Two DGK-ζ compounds and one DGK-α compound were analyzed using Jurkat T cells and primary human TCR-transduced T cells. No effects were seen on cell viability for the compounds applied alone or in combination. On-target knockdown was achieved in Jurkat T cells evidenced by RT-qPCR and SimpleWestern. Silencing of mRNA and protein occurred quickly after 24h, peaked between 48h and 72h and lasted at least for 96h. Stimulation under DGK-targeting INTASYL treatment resulted in enhanced levels of phosphorylated ERK1/2 and enhanced secretion of IL-2.
Conclusions INTASYL™ self-delivering RNAi compounds represent a promising approach to target intracellular immune checkpoints such as DGKs. The good toxicity profile allows for combined application of several compounds enabling targeting of multiple checkpoints, which likely is necessary to counteract the complex and heterogeneous inhibitory influences of the TME. The technology enables the anti-tumor activity of T and NK cells for immunotherapy, and can be used in ACT and direct therapeutic applications towards the TME.
Moon EK, Wang L-C, Dolfi DV, Wilson CB, Ranganathan R, Sun J, et al. Multifactorial T-cell hypofunction that is reversible can limit the efficacy of chimeric antigen receptor-transduced human T cells in solid tumors. Clin Cancer Res 2014;20(16):4262–73.
Jung I-Y, Kim Y-Y, Yu H-S, Lee M, Kim S, Lee J. CRISPR/Cas9-mediated knockout of DGK improves antitumor activities of human T cells. Cancer Res 2018;78(16):4692–703.
Disclosure Information A.S. Herbstritt: C. Other Research Support (supplies, equipment, receipt of drugs or other in-kind support); Significant; Phio Pharmaceuticals. M. Maxwell: A. Employment (full or part-time); Significant; Phio Pharmaceuticals. D. Yan: A. Employment (full or part-time); Significant; Phio Pharmaceuticals. B. Cuiffo: A. Employment (full or part-time); Significant; Phio Pharmaceuticals. J. Cardia: A. Employment (full or part-time); Significant; Phio Pharmaceuticals. S.P. Fricker: A. Employment (full or part-time); Significant; Phio Pharmaceuticals. E. Noessner: C. Other Research Support (supplies, equipment, receipt of drugs or other in-kind support); Significant; Phio Pharmaceuticals.
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