Anti-SARS-CoV-2 multi-domain Darpinâ® molecules as highly potent therapeutics

ABSTRACT

Background: Globally accessible preventive and therapeutic drugs against SARS-CoV-2 are urgently needed. Here, we report the generation and characterization of the first anti-SARS-CoV-2 DARPin® molecules with therapeutic potential. DARPin® molecules are an emerging class of novel therapeutics based on naturally occurring ankyrin repeat motifs which can be rapidly produced in bacteria in large quantities. Methods: From a naïve library of 1012 DARPin molecules 380 molecules were selected to target the SARS-CoV-2 spike protein. Extensive biophysical and biochemical characterization, pseudovirus and infectious virus neutralizationassays as well as cryo-EM analysis, resulted in 11 highly distinct single domain DARPin molecules which were used for the assembly of highly potent multidomain DARPin molecules. The protective efficacy of multi-domain DARPin molecules was studied in COVID-19 hamster models. Results: From the 11 single domain DARPin molecules a range of multi-domain DARPin molecules were assembled which were grouped into multi-paratopic DARPin molecules neutralizing the receptor binding domain (RBD) and multimode DARPin molecules targeting simultaneously the RBD, the S1 N-terminaldomain (NTD) and/or the S2 domain. Multi-domain DARPin molecules binding three spike protein domains simultaneously demonstrated increased binding affinity, virus neutralization potency and the potential to prevent viral escape via mutations. Cryo-EM analysis further supported the structural understanding of the multi-domain DARPin molecules and molecular modelling proved that simultaneous binding of individual DARPin domains to various spike protein domains is feasible. Two additional DARPin domains binding human serum albumin were incorporated in the DARPin molecules, conferring an expected half-life of about 3 weeks in humans. A multi-paratopic RBD-neutralizing DARPin molecule and a multi-mode DARPin molecule were found to potently block SARS-CoV-2 infection with IC50 values in the single-digit ng/mL range. Multi-paratopic DARPin molecules proved prophylactic and therapeutic efficacy in hamster SARS-CoV-2 infection models. Conclusion: The anti-SARS-CoV-2 multi-domain DARPin molecule, ensovibep or MP0420, which entered clinical phase I in November 2020, displayed very high antiviral potency, rapid and high production capacity due to bacterial fermentation and demonstrated prophylactic and therapeutic activity in hamster SARS-CoV-2 infection models.