Abstract B27: A DARPin-based toolbox to understand and treat RAS-addicted cancers
Authors: Kapp Jonas N. , Schaefer Jonas V. , Verdurmen Wouter , Nagy Gabriela , Degen Ralph , Ernst Patrick , Scholl Claudia , Plückthun Andreas 
Published: June 2021

Abstract
The repeating failure of small molecules as specific inhibitors of KRAS has drawn the attention to macromolecular structures, which can recognize their target with high affinity and specificity. Designed Ankyrin Repeat Proteins (DARPins) are multipurpose alternative affinity reagents that have proved to recognize targets with exceptional specificities and selectivity that often surpass those of antibodies. Due to their additional outstanding ability to act intracellularly within living cells and their recognition of structural rather than linear epitopes, DARPins have enabled a multitude of more advanced projects. In-house selections against GTP- or GDP-loaded KRAS were performed, and several hundred different DARPins could be identified. In subsequent validations, we not only analyzed their affinities, but also focused on the essential features of cross-reactivities, the recognition of various epitopes and their biologic functionalities. A number of candidates directly interfere with the RAS-Raf interaction and SOS-mediated nucleotide exchange by binding to the identical epitope on RAS as proved by crystal structures. One of these lead candidates with an affinity of 10 nM was used to elucidate whether anti-KRAS DARPins can mediate a biologic effect in model systems of human cancers. For this purpose, we chose cell lines categorized as KRAS dependent to generate stable cell lines expressing a highly active anti-KRAS DARPin under an inducible promotor, and it revealed potent antitumor activity, reducing the proliferation, colony formation and anchorage-independent growth. We could furthermore show that the observed effect resulted from reduced signaling of KRAS through its downstream MEK-ERK and PI3K-AKT pathways and the induction of apoptosis. Importantly, this anti-KRAS DARPin was shown to have no effects in the immortalized cell line HEK293T. In addition to traditional knockdown approaches, this model could be used to assess RAS dependency of human cancers. Combined with various suitable intracellular delivery techniques currently under development in our laboratory, the potential of this model will be further investigated for its potential in the treatment of solid tumors in a mouse model. In our broad effort many additional DARPins were identified that recognize different, nonoverlapping epitopes and most likely will interfere with other essential functions, such as the nanoclustering of RAS. This toolbox also allows the development of various biosensors in living cells. This large set of binders has the potential not just to help the community to gain detailed insights into the various functions and of RAS, but also might highlight novel vulnerabilities and innovative ways to finally make these key players druggable.
Citation Format: Jonas N. Kapp, Jonas V. Schaefer, Wouter Verdurmen, Gabriela Nagy, Ralph Degen, Patrick Ernst, Claudia Scholl, Andreas Plückthun. A DARPin-based toolbox to understand and treat RAS-addicted cancers [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B27.