The development of effective radioligand therapeutics (RLTs) is frequently hampered by the lack of high-quality targeting agents that selectively deliver radioactive payloads to the site of disease while sparing healthy tissues. Antibodies can have high affinity and specificity to tumor targets, but their large size results in limited tumor penetration and long systemic half-life is frequently causing haematological toxicities. Alternatively, targeting agents with low molecular weight such as small molecules and peptides often suffer from limited affinity and specificity to the tumor target, resulting in off-target effects and limited tumor retention. DARPins (Designed Ankyrin Repeat Proteins) developed by Molecular Partners combine small size (15 kDa) and ideal binding properties. Due to their rigid-body target binding mode DARPins combine very high affinity and specificity and unless engineered accordingly, DARPins have very short systemic half-lives. Thanks to a simple and robust architecture, DARPins can be efficiently coupled with radioactive payloads, even at elevated temperatures; and they can tolerate sequence-engineering approaches, which are not compatible with other protein scaffolds. To establish the DARPin platform for RLT, we have used DARPin candidates against different tumor targets. We have previously shown that increasing affinity to the tumor target correlates with elevated tumor uptake and long tumor residence in preclinical mouse models. We now also show that DARPins exhibit a homogeneous and deep tumor penetration in vivo that is highly superior to antibody benchmarks. Globular proteins below 60 kDa in size are typically cleared from the bloodstream via the renal pathway. This generally results in a strong kidney accumulation of small sized, protein-based targeting agents and their coupled residualizing radionuclides, leading to dose-limiting kidney toxicities. To overcome this limitation, we have undertaken an extensive engineering approach of the DARPin scaffold. Our results show that sequence engineering strongly reduces kidney uptake of DARPins without affecting their tumor uptake. This effect was confirmed with independent DARPin candidates suggesting a general applicability of the approach. Combined with other orthogonal strategies, we are able obtain favourable tumor to kidney ratios in preclinical mouse models. These results show that our proprietary optimized DARPin platform offers an attractive solution to the limitations of protein-based targeting agents for RLT applications. Together with the fact that high-affinity DARPins can be generated against a large variety of tumor targets, we conclude that our platform provides a powerful basis for the development of next-generation RLTs. Several DARPin-RLT programs in indications with high unmet medical need are currently in development.

Citation Format: Andreas Bosshart, Stephan Wullschleger, Martin Behe, Alain Blanc, Stefan Imobersteg, Alexandra Neculcea, Jacqueline Blunschi, Liridon Abduli, Sarah Schütz, Julia Wolter, Christian Reichen, Amelie Croset, Alessandra Villa, Christian Lizak, Anne Goubier, Roger Schibli, Daniel Steiner. DARPins as powerful targeting agents for radioligand therapeutics. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5037.