Chimeric antigen receptor T cell targeting CD44E variant in HCC holds therapeutic potential.

Chimeric antigen receptor (CAR) T cell therapy has achieved remarkable success in hematological malignancies. However, its efficacy in hepatocellular carcinoma (HCC) remains limited. One major challenge is the lack of reliable tumor antigens for CAR T cell recognition. CD44E, a unique alternative splicing (AS) variant of CD44 transmembrane protein that is highly expressed in HCC tumors compared to adjacent non-tumoral liver tissues, holds promise as a potential therapeutic target. Here, we successfully developed a CD44E-specific monoclonal antibody derived from the S431 hybridoma clone. Using this antibody, we confirmed CD44E protein expression in patient-derived HCC cell lines, organoids, and primary tumor tissues. A second-generation CAR was designed by incorporating the single-chain variable fragment (scFv) of the S431 antibody. These anti-CD44E CAR T cells exhibited specific cytotoxicity against CD44-positive HCC cells in both in vitro and in vivo models and importantly, did not induce detectable toxicity in vital organs in animal models. Our findings suggested that CD44E-targeting CAR T cell therapy might represent a promising therapeutic approach for HCC treatment.