Description

Designed Ankyrin repeat proteins (DARPins) as recognition motifs in chimeric antigen receptors

Balakrishnan A¹, Salter AI¹, Rajan A¹,

Plückthun A², Riddell SR¹

Fred Hutchinson Cancer Research Center, Seattle, USA, University of Zurich, Zurich, Switzerland

Chimeric antigen receptors (CARs) consist primarily of single chain variable fragments (scFv) linked to the T cell activation (CD3ζ) and costimulatory domains. We investigated whether alternative recognition motifs selected for ligand binding from synthetic libraries of Designed Ankyrin Repeat proteins (DARPins) could be incorporated into CARs. As proof of principle, we linked a DARPin (DARPin E01) specific for EGFR to the CAR signaling domain and compared anti-tumor function with a high affinity CAR containing the Cetuximab scfv, which recognizes an overlapping epitope.Our in vitro results show that DARPin CARs designed with flexible linkers and with short and long spacer domains exhibit specific cytotoxicity, cytokine secretion and proliferation against EGFR⁺ targets that is comparable to the Cetuximab CAR. We tested DARPins CARs in an in vivo breast cancer tumor model with EGFR⁺ MDA-MB-231 tumor cells and found enhanced anti-tumor clearance with both E01 DARPin and Cetuximab scFV CARs. We show that an EpCAM-specific DARPin chimeric costimulatatory receptor (CCR) can also be used in conjunction with a scFV CAR targeting ROR1 to provide combinatorial antigen recognition and enhance T cell function against tumor cells expressing both targets. The EpCAM-specific DARPin CCR combined with first or second generation ROR1 scFv CARs enhanced T cell cytokine secretion and proliferation. The compact stable structure of DARPins also facilitate linking of multiple DARPins targeting different antigens reducing the likelihood of antigen loss. In summary, the stable structural scaffold of DARPins and the presence of diverse binder libraries make them attractive recognition motifs to incorporate into CARs.