Generation and optimization of off-the-shelf immunotherapeutics targeting TCR-Vβ2+ T cell malignancy

Current treatments for T cell malignancies encounter issues of disease relapse and off-target toxicity. Using T cell receptor (TCR)Vβ2 as a model, here we demonstrate the rapid generation of an off-the-shelf allogeneic chimeric antigen receptor (CAR)-T platform targeting the clone-specific TCR Vβ chain for malignant T cell killing while limiting normal cell destruction. Healthy donor T cells undergo CRISPR-induced TRAC, B2M and CIITA knockout to eliminate T cell-dependent graft-versus-host and host-versus-graft reactivity. Second generation 4-1BB/CD3zeta CAR containing high affinity humanized anti-Vβ scFv is expressed efficiently on donor T cells via both lentivirus and adeno-associated virus transduction with limited detectable pre-existing immunoreactivity. Our optimized CAR-T cells demonstrate specific and persistent killing of Vβ2+ Jurkat cells and Vβ2+ patient derived malignant T cells, in vitro and in vivo, without affecting normal T cells. In parallel, we generate humanized anti-Vβ2 antibody with enhanced antibody-dependent cellular cytotoxicity (ADCC) by Fc-engineering for NK cell ADCC therapy.