CD44 antagonizing oHSV promotes the formation of tertiary lymphoid structures and improves anti-tumor immunity

Oncolytic HSV (oHSV) therapy is currently approved for melanoma (in USA and EU) and recurrent brain tumors (conditional approval in Japan). To understand opportunities to improve therapeutic index, we analyzed transcriptomic changes in patients with recurrent brain tumors before and after treatment with CAN-3110. Gene set pathway analysis showed a significant enrichment of glycosaminoglycan (GAG) synthesis and extracellular matrix (ECM) interactions in patients after oHSV treatment. CD44 is a cell surface receptor that engages with receptors and also with ECM to orchestrate cell-ECM interactions. To evaluate the significance of the multiple CD44 regulated ECM interactions during virotherapy, we created an oHSV that encodes for the secreted extracellular CD44 (Ox44) which functions as a decoy receptor to inhibit cell-ECM interactions. Treatment with Ox44 also destabilized SLC7A11 (xCT) inducing oxidative stress and inhibiting antiviral STING signaling in infected tumor cells. Interestingly the ensuing increased viral burst and immunogenic cell death amplified innate immune activation in immune cells. This correlated with the induction of inflammatory cytokines like interferon (IFN) and IL-1 and the formation of tertiary lymphoid structures (TLS) in intracranial tumors. Single cell sequencing uncovered that Ox44-treated tumors had increased antigen presentation and increased T cell diversity and anti-tumor efficacy. To our knowledge, this is the first report to show that CD44 presents a barrier to both oncolytic virus replication as well as anti-tumor immunity.