CD3scFv-Engineered AAV Capsid Drives Superior In Vitro and In Vivo CAR-T Cell Generation and Leads to Complete Eradication of CD19+ Tumors.
TL;DR
It is demonstrated that site-selective CD3scFv insertion into a single AAV capsid protein provides a robust, versatile platform for improved T cell transduction and effective in vivo CAR-T cell generation, offering a promising off-the-shelf approach for treating B cell malignancies.
Abstract
Adeno-associated viruses (AAV) are emerging vectors for in vivo delivery of chimeric antigen receptor (CAR) therapies. Natural AAV serotypes were previously considered unsuitable due to their poor transduction of non-activated human T cells. To overcome this limitation, we inserted an anti-CD3 single-chain variable fragment (CD3scFv) into one of the three AAV capsid proteins (VP1, VP2, or VP3) to specifically target and activate human T cells. This strategy preserved normal vector production yields, while enabling specific T cell targeting. Using AAV6 as a representative serotype, our lead candidate AVO-100 achieved more than 200-fold higher transduction efficiency of primary human T cells compared to unmodified counterpart. In vitro, AVO-100-mediated CD19-CAR delivery resulted in efficient CAR expression and killing of CD19+ Raji lymphoma cells in co-culture assays. In vivo, a single intravenous dose of AVO-100 at 2×108 vector genomes (vg) per subject using NSG mice bearing Raji xenografts, prompted complete tumor eradication and significantly prolonged survival, whereas control untreated animals succumbed to a progressive disease. These results demonstrate that site-selective CD3scFv insertion into a single AAV capsid protein provides a robust, versatile platform for improved T cell transduction and effective in vivo CAR-T cell generation, offering a promising off-the-shelf approach for treating B cell malignancies.
