Impact of scFv structure in chimeric antigen receptor on receptor expression efficiency and antigen recognition properties

Gene-modifying T cells expressing chimeric antigen receptor (CAR) with an extracellular domain consisting of single chain variable fragment (scFv) and an intracellular domain with a T cell activation motif, are promising cancer immuno-medicines that can exert long term potent antitumor activity. However, CAR-T cells have a high risk of causing fatal side effects. Thus, more effective and safer CAR-T cells are urgently needed. Although antigen specificity and reactivity of CAR-T cells are defined by CAR expression level and affinity, information on optimizing the scFv structure that defines CAR avidity is lacking. Here, we investigated the impacts of scFv substitution and structural modification in CAR on receptor expression and antigen recognition properties. Four CARs with distinct scFvs targeting the same antigen were unexpectedly separated into a CAR expressed on T cells and bound to the antigen, CARs that did not show antigen-binding because of cell surface aggregation, and a rarely expressed CAR. Among the scFv structural modifications of CARs, changes in the Fv order and linker did not noticeably affect CAR expression or antigen-binding. In contrast, complementarity-determining region (CDR)-grafting to the stable framework region in Fv dramatically improved the surface expression level of non-producible CAR. These results revealed that CAR expression efficiency and stability on T cells are influenced by the Fv structure. Therefore, stabilization of the Fv structure by CDR-grafting may be an effective means for expressing scFvs, which have excellent antigen specificity and appropriate affinity but low structural stability, as a CAR on T cells.