Specific loading of oncolytic VSV on CAR enhances CAR-T cell signaling and antitumor activity.

Oncolytic viruses (OVs) have been shown to increase the efficacy of chimeric antigen receptor (CAR) T cells in treating solid tumors. However, their combined effect has been limited by the unbalanced distribution of two agents in tumor tissue and viral infection-mediated CAR-T cell exhaustion. Here, we designed a CAR moiety by inserting the CR2 and CR3 domains (CR2/3-CAR) of low-density lipoprotein receptor, which is the viral receptor of oncolytic vesicular stomatitis virus (VSV) mutant (VSVΔ51), enabling specific loading of VSVΔ51 onto CAR-T cells. The anchored VSVΔ51 could be released from CAR-T cells and efficiently delivered to tumor tissue. Further investigation revealed that the cross-connection between viral envelope proteins and CR2/3-CAR moieties facilitated forming antigen-free CAR clusters and antigen-induced CAR synapse, triggered CAR signaling transduction, and directly pre-activated the CAR-T cells. Consequently, this approach potently enhanced the proliferation, metabolic fitness, and immunological activities of CAR-T cells, and subsequently enhanced the OV/CAR-T synergetic cytotoxicity, revealing an effective strategy for treating solid tumors.