We report the engineering and selection of two synthetic proteins - FSR16m and FSR22 - for possible treatment of SARS-CoV-2 infection. FSR16m and FSR22 are trimeric proteins composed of DARPin SR16m or SR22 fused with a T4 foldon and exhibit broad spectrum neutralization of SARS-Cov-2 strains. The IC 50 values of FSR16m against authentic B.1.351, B.1.617.2 and BA.1.1 variants are 3.4 ng/mL, 2.2 ng/mL and 7.4 ng/mL, respectively, comparable to currently used therapeutic antibodies. Despite the use of the spike protein from a now historical wild-type virus for design, FSR16m and FSR22 both exhibit increased neutralization against newly-emerged variants of concern (39- to 296-fold) in pseudovirus assays. Cryo-EM structures revealed that these DARPins recognize a region of the receptor binding domain (RBD, residues 455-456, 486-489) overlapping a critical portion of the ACE2-binding surface. K18-hACE2 transgenic mice inoculated with a B.1.617.2 variant and receiving intranasally-administered FSR16m were protected as judged by less weight loss and 10-100-fold reductions in viral burden in the upper and lower respiratory tracts. The strong and broad neutralization potency make FSR16m and FSR22 promising candidates for prevention and treatment of infection by current and potential future strains of SARS-CoV-2.

Potent and pan-neutralization of SARS-CoV-2 variants of concern by DARPins