Designed ankyrin repeat proteins (DARPins) are small antibody-mimicking proteins with high stability and target specificity. Balakrishnan and Rajan et al. found comparable antitumor CAR T cell efficacy targeting EGFR either through DARPins or antibody fragments. Three DARPins for EGFR, EpCAM, and HER2 were linked to form a trispecific CAR construct; functionality depended on the spacer (hinge and linker) format and relative positioning of each domain. Trispecific or a mixture of monospecific DARPin-CAR T cells delayed growth of Raji lymphoma expressing multiple antigens, and the cells that escaped were primarily antigen-loss variants.
Contributed by Alex Najibi
PURPOSE: The outgrowth of antigen negative variants is a significant challenge for adoptive therapy with T cells that target a single specificity. Chimeric antigen receptors (CARs) are typically designed with one or two scFvs that impart antigen specificity fused to activation and costimulation domains of T cell signaling molecules. We designed and evaluated the function of CARs with up to three specificities for overcoming tumor escape using Designed Ankyrin Repeat Proteins (DARPins) rather than scFvs for tumor recognition. EXPERIMENTAL DESIGN: A monospecific CAR was designed with a DARPin binder (E01) specific for EGFR and compared to a CAR designed using an anti-EGFR scFv. CAR constructs in which DARPins specific for EGFR, EpCAM and HER2 were linked together in a single CAR were then designed and optimized to achieve multispecific tumor recognition. The efficacy of CAR-T cells bearing a multispecific DARPin CAR for treating tumors with heterogeneous antigen expression was evaluated in vivo. RESULTS: The monospecific anti-EGFR E01 DARPin conferred potent tumor regression against EGFR(+) targets that was comparable to an anti-EGFR scFv CAR. Linking three separate DARPins in tandem was feasible and in an optimized format generated a single tumor recognition domain that targeted a mixture of heterogeneous tumor cells, each expressing a single antigen, and displayed synergistic activity when tumor cells expressed more than one target antigen. CONCLUSIONS: DARPins can serve as high-affinity recognition motifs for CAR design, and their robust architecture enable linking of multiple binders against different antigens to achieve functional synergy and reduce antigen escape.