One of the leading causes of non-responding tumors with existing immunotherapies is an immunosuppressive tumor micro-environment (TME). The suppressive nature of the TME is, in part, mediated by immunoregulatory cells (e.g. MDSC cells) and cytokines/chemokines. TGFB1 has recently been shown to be a key suppressive factor responsible for facilitating “cold” or immune-excluded tumors. In these tumors, T cell activity is restricted and by reducing TGFB1 levels we aim to support anti-tumor T cell activity. We evaluated if downregulation of TGFB1 with a self-delivering RNAi (sd-rxRNA) in MDSCs (in vitro) or TME (in vivo) promotes T cell activity and increase anti-tumor activity.
sd-rxRNA is a chemically modified RNAi which incorporates drug like properties allowing for self-delivery of RNAi compounds to immune cells (including MDSC, DC, T and NK cells) in the absence of electroporation or formulation in a delivery vehicle. Tumor induced MDSCs were incubated with sd-rxRNA targeting TGFB1, its downstream target COX2 or in combination for 2 days. The cells were washed and added to a co-culture with OT-1 T cells and OVA expressing B16 melanoma. The anti-tumor activity of T-cells was monitored using a Real-Time Cell Activity assay (RTCA). TGFB1 and COX2 downregulated MDSCs had reduced immune-suppressive activity as shown by the enhanced B16-OVA tumor cell killing by CD8 OT-1 T cells in the RTCA assay while the combination of TGFB1 and COX2 MDSCs did not further improve the anti-tumor activity.
To evaluate the effect of anti-TGFB1 sd-rxRNA in vivo, we first studied the tumor distribution and TGFB1 downregulation by intratumoral injection of sd-rxRNA to immunocompetent Balbc mice bearing 4T1-RL-GFP breast cancer in a fat pad model. DY547 labeled sd-rxRNA targeting PPIB or unlabeled sd-rxRNA targeting TGFB1 was injected into tumors. Two days later, tumors were resected, and fluorescence signal was analyzed by microscopy and TGFB1 downregulation was evaluated by RT-PCR. Efficient delivery of the DY547 labeled sd-rxRNA was observed in the vast majority of cells as shown by fluorescence microscopy. In addition, fluorescence signal could be detected in tumors up to 28 days after intratumoral injection, suggesting that sd-rxRNA was stable in tumors. Downregulation of TGFB1 in resected tumors, and an increased anti-tumor activity, was seen after three injections of sd-rxRNA. A follow up study was initiated investigating the efficacy of TGFB1 sd-rxRNA in combination with local tumor radiation on anti-tumor and metastatic activities in a 4T1 orthotopic breast cancer model. Preliminary data shows promising results.
Our results suggest that sd-rxRNA compounds targeting TGFB1 can enhance the anti-tumor activity of T cells. Local injection of sd-rxRNA into tumors can effectively target TGFB1 in the TME resulting in less immunosuppression and increased anti-tumoral activity.
Citation Format: Winnie F. Tam, Dingxue Yan, Melissa Maxwell, James Cardia, Gerrit Dispersyn. Feasibility and efficacy of using self-delivering RNAi against TGFB1 to reduce TME immunosuppression [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 1963.