Hinging on Success: Leveraging the Power of CAR T-Cell Therapy through In-Silico Modeling of Hinge Length and Epitope Location

Abstract Genetic engineering of T-cells using chimeric antigen receptors (CARs) has shown promise in the treatment relapsed/refractory B-cell leukemia. However, a significant percentage patients will eventually relapse, often due to emergence tumor cells expressing low levels target antigen. Previous research demonstrated that hinge domain CAR plays crucial role regulating cytotoxic capabilities these cells. In this study, we assessed potential model optimal length for given single-chain variable fragment (scFv) relation location targeted epitope. We designed an array hinges originating from CD8α and incorporated into CARs targeting CD19, CD22, CD33, CD20 antigens. found epitope is critical determining hinge; narrow range short more effective against membrane-distal epitopes while long are membrane-proximal epitopes. To build in-silico model, used AlphaFold2 (AF2), reducing multiple sequence alignment (MSA) clusters introduce stochasticity increase conformational diversity structural predictions proteins. discovered with predicted intermembrane distance 15nm between ligand were particularly antigen-low leukemic Our findings suggest can predict responsiveness specific epitope, enabling rapid optimization by adjusting length. The development modeling strategy facilitate design optimized wide novel targets. Intramural Research Program National Cancer Institute (Bethesda, MD)