Rewiring STAT signaling from the cell surface with Trikine immunotherapeutics [RNAseq of IL21 Trikine]

Cytokines dimerize two receptor chains to activate Janus kinases and STAT transcription factors that regulate immune cells but have therapeutic liabilities. We engineered “Trikines” to compel cis formation of three-chain cytokine receptor complexes at the cell surface that induce bespoke STAT transcriptional signaling programs optimized for therapeutic efficacy. Designed Trikines co-activated pSTAT5 and pSTAT3 signatures distinct from any natural cytokines, by assembling trimeric combinations of Interleukin-2 (IL-2), Interleukin-10 (IL-10), and Interleukin-21 (IL-21) receptors. IL-2-based-Trikines restrain terminal differentiation of T cells, promote stemness, and enhance durability of tumor control. Unexpectedly, an IL-10-based Trikine induced immune infiltration into poorly immunogenic tumors, showing striking efficacy in small cell lung cancer and pancreatic cancer models. Trikines obviate the need for cell engineering to customize STAT signatures for immunotherapy. Overall design: Mouse T cells were pre-activated with IL-2 as described and rested in the absence of serum or cytokine overnight. Cells were then incubated with 100 nM IL-2 (GER110), IL-21(GER112), IL-1 + IL-21 (GERcombo) or 21/2-trikine(GER119) for 24 hours. Cell wee not stimulated (NS) as control. For each condition, we performed 3 technical replicates. Total RNA was extracted from 1-2.5million cells per condition using a RNeasy Micro Kit (Qiagen) followed by mRNA library preparation (poly-A enrichment) according to manufacturer instructions. Each library was sequenced on the NovaSeq X Plus PE150 resulting in approximately 12 x 106reads per sample.