Fig. 8: Generation and functional characterization of homotrimeric tcTRP9x₃-VHH₃ constructs.

a Each construct (sequences and locations of inserted VHH domains provided in Supplementary Table S1) corresponds to a fusion at their N- or C-termini with a VHH domain previously shown to bind to the SAR—CoV2 spike protein receptor binding domain²³. Eight constructs were tested, corresponding to various linkers that fuse the VHH domain to the N- or C-terminal ends of each subunit, in either case generating a trimeric array of VHH domains arranged on one side of the tcTRP9x₃ scaffold. The ability of each construct to bind the SARS CoV2 spike receptor-binding domain (RBD) was measured using two approaches (Biolayer interferometry and ELISA); the ability of the same constructs to inhibit cell infection was measured using a pseudoviral neutralization assay. b Purification of individual constructs from E. coli. Yields ranged from ~15 to ~ 80 mg/L. c The relative ability of each construct to bind to the SARS-Cov2 receptor-binding domain (RBD) was measured using an ELISA analysis, leading to EC₅₀ values (Supplementary Table S2) ranging from a low of 0.4 nM (construct 681) to 12 nM (construct 680). All binding experiments were conducted in triplicate using independent aliquots of each protein. Data shown as mean and s.d. for n = 3 measurements. d The relative ability of each construct to bind to the SARS-Cov2 receptor-binding domain (RBD) was measured using Biolayer interferometry (BLI). The rate of association and dissociation are calculated from concentration-dependent responses. The rate of association (k_a) is plotted on the Y-axis as inverse molar second (1/MS), and the rate of dissociation (k_dis) is plotted on the X-axis as inverse second. Corresponding equilibrium binding constants (K_D) are plotted on a diagonal axis as nano molar concentrations. See also Supplementary Fig. S5. e The relative ability of each construct to achieve 50% inhibition of infection of 293T-Ace2 cells using a pseudoviral neutralization assay with SARS-CoV-2 spike pseudotyped lentiviruses was measured. The IC₅₀ values (Supplementary Table S2) ranged from 0.78 nM (construct 681) to 5.5 nM (construct 680). All experiments were conducted in triplicate using independent aliquots of each protein. Data shown as the mean and standard deviation for n = 3 measurements.