CD96 as a therapeutic target for CAR T cell therapy in acute myeloid leukemia.

Therapies leveraging chimeric antigen receptor (CAR) T cells for acute myeloid leukemia (AML) are limited by the scarcity of leukemia stem cell (LSC)-specific antigens. Here, we found that CD96 is expressed in 48.3% of AML patients, with higher expression on LSCs than blasts, and is absent on normal hematopoietic stem/progenitor cells (HSPCs). We developed a panel of CD96-CAR T cells using single-chain variable fragments derived from various monoclonal antibodies, incorporating distinct transmembrane and costimulatory domains. Treatment with CD96-CAR T cells confers specific anti-leukemic activity correlated with CD96 expression. Notably, CAR T cells featuring a CD28 transmembrane and costimulatory domain (CD96-28z) exhibit enhanced proliferation and cytotoxic capabilities in vitro. In vivo, CD96-28z potently eliminated AML cells and prolonged survival in mice bearing CD96-high, but not CD96-low, AML. To address CD96-low AML, we combined CD96-28z with a CD33-targeted chimeric costimulatory receptor (CCR), thereby increasing cytotoxic efficacy. Importantly, CD96-CAR T cells did not inhibit colony formation by HSPCs during manufacturing. These findings indicate that CD96 is a promising target for AML immunotherapy, and the combination of CD96-CAR and CD33-CCR may represent a potent strategy for patients with CD96-positive AML while preserving normal hematopoiesis.