An engineered viral protein activates STAT5 to prevent T cell suppression

T cell therapy efficacy can be compromised if cytokine-induced Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling is dysregulated or insufficient to sustain functionality. Here, we demonstrate that LCK kinase activity can be recruited to non-canonical protein substrates to directly activate targeted STAT proteins in T cells. STAT activation was accomplished by engineering the Herpesvirus saimiri (HVS) tyrosine kinase interacting protein (TIP) to provide a platform for the enforced recruitment of LCK to STAT proteins. We determined that a minimal region of TIP that binds to LCK could be combined with STAT binding sites derived from endogenous cytokine receptors. These constructs activated targeted STAT proteins in a cytokine-independent manner. We identified a STAT5 activator that sustained CD8+ T cell survival and cytotoxic function ex vivo in the absence of IL-2. Tumor outgrowth was reduced in vivo due to enhanced T cell persistence and functionality. Single-cell transcriptomics revealed the STAT5 activator prevented the expression of genes associated with an exhausted T cell fate. Our findings demonstrate that signaling pathways can be rewired in T cells to sustain their function in solid tumors. Overall design: B16-OVA tumors were established in BoyJ (CD45.1+) mice prior to adoptive transfer of CD8+ OT-I T cells (ACT) transduced with empty vector, a STAT5 activator (aSTAT5), a minimal construct (PepControl), and constitutively active STAT5 (STAT5CA). Tumors were collected form 3 mice per treatument group 5- or 12-days post-ACT and CD45+ tumor infiltrating leukoctyes isolated by positive selection. Isolated cells were then stained with DNA barcoded antibodies (CD45.1, CD45.2, CD90.1, CD8a TotalSeq-C antibodies and TotalSeq-C HashTag antibodies) and fluorescent antibodies (CD45, CD8a, CD8b, TCRb) prior to isolation of CD45+CD8+ T cells by FACS. Joint HTO and ADT libraries were prepared using the 10x Genomics platform.