Engineering Sonogenetic EchoBack-CAR T cells
Chimeric antigen receptor (CAR) T cell therapy marks a significant advancement in oncology, offering the promise of durable remissions in hematological malignancies. However, solid tumors present formidable challenges, including on-target off-tumor toxicity, exhaustion, and limited persistence of therapeutic T cells. Here, we engineered sonogenetic EchoBack-CAR T cells using a highly heat-sensitive promoter, evolved through a sort-seq platform, and integrated it with a positive feedback loop from CAR signaling pathways that enables the conversion of tumor engagement into CAR production, resulting in long-lasting CAR expression upon remote stimulation by focused ultrasound (FUS). EchoBack-CAR T cells with a high-affinity single-chain variable fragment (scFv) targeting disialoganglioside GD2 (EchoBack-hGD2CAR) exhibited potent cytotoxicity and persistence in an in vitro glioblastoma spheroid model. In both subcutaneous and orthotopic glioblastoma models in mice, EchoBack-hGD2CAR T cells achieved robust and safe tumor suppression without off-tumor toxicity. Through single-cell RNA sequencing, we uncovered a distinctive transcriptomic landscape that underscores the enhanced cytotoxicity and reduced exhaustion of EchoBack-CAR T cells after chronic antigen stimulation compared to standard CAR T cells that constitutively express CAR. The EchoBack-CAR can also be generalized and extended to treat prostate cancer by targeting prostate-specific membrane antigen (EchoBack-PSMACAR), demonstrating a remarkable tumor suppression but minimal off-tumor toxicity to healthy tissues. Collectively, these findings not only highlight the potential of EchoBack-CAR T cells to promote solid tumor immunotherapy via ultrasound remote control but also present a modular approach adaptable to various cancer targets, poised to mitigate the limitations of current CAR T cell therapies. Overall design: EchoBack CAR-T cells and standard CAR-T cells were rechalleneged with glioblastoma cancer cells daily at a E:T ratio of 1:2 for 10 days to achieve chronic antigen stimulation in vitro. EchoBack and standard CAR-T cells were isolated by FACS and analyzed using scRNA-seq