Triple knockdown of <i>CD11a</i> , <i>CD49d</i> , and <i>PSGL1</i> in T cells reduces CAR-T cell toxicity but preserves activity against solid tumors in mice

Chimeric antigen receptor (CAR)-T cell therapies have revolutionized the landscape of cancer treatment, in particular in the context of hematologic malignancies. However, for solid tumors that lack tumor-specific antigens, CAR-T cells can infiltrate and attack nonmalignant tissues expressing the CAR target antigen, leading to on-target, off-tumor toxicity. Severe on-target, off-tumor toxicities have been observed in clinical trials of CAR-T therapy for solid tumors, highlighting the need to address this issue. Here, we demonstrated that targeting the cell adhesion and migration molecules lymphocyte function-associated antigen 1 (LFA-1; CD11a/CD18) and very late activation antigen 4 (VLA-4; CD49d/CD29) with blocking antibodies reduced the on-target, off-tumor toxicity of CAR-T cells in mice. To translate this observation into improved CAR-T cell therapy, we either knocked out both <i>CD11a</i> and <i>CD49d</i> or knocked down <i>CD11a</i> and <i>CD49d</i> along with <i>PSGL1</i>, another cell adhesion molecule, in CAR-T cells. We found that these modified CAR-T cells exhibited reduced on-target, off-tumor toxicity in vivo without affecting CAR-T cell efficacy. Furthermore, we showed that this approach promoted T cell memory formation and decreased tonic signaling. On the basis of these data, we engineered a human version of these low-toxicity CAR-T cells and further validated the feasibility of this approach in vitro and in vivo. Together, these results provide a potential solution to address the clinical challenge of on-target, off-tumor toxicity in CAR-T therapy.