Cysteine-Engineered CAR-T Cells to Counter Antigen Escape in B Cell Lymphoma

Abstract Chimeric Antigen Receptor (CAR-) T cell therapy represents a paradigm shift in immunotherapy of hematological cancers. However, selective pressure on cancer cells often leads to suppression of target antigens, eventually causing cancer relapse1,2. This so-called antigen escape renders CAR-T cells ineffective, posing a significant clinical challenge2–5. Therefore, identifying alternative targets less susceptible to antigen escape is crucial. Here, we describe a novel type of CAR-T cells utilizing cysteine-engineered antibody fragments that target altered redox states on the surface of B cell lymphoma (BCL)6. We demonstrate that cysteine-engineered CAR-T cells exhibit specific cytotoxicity in vitro against various BCL subtypes, including antigen escape models. Additionally, we show that cysteine engineering, achieved through single amino acid substitution in the state-of-the-art anti-CD19-CAR, enables co-targeting of both CD19-positive and -negative BCL. Our findings introduce a novel class of bifunctional CAR-T cells that target conventional antigens and altered redox states simultaneously, potentially reducing the risk of antigen escape. Abnormal redox states occur in several cancers, including breast and leukemia7–12, indicating a broad therapeutic scope. Competing Interest Statement The authors have declared no competing interest.