Fig. 4: Divalent engineering increases affinity and neutralizing activity.

a, b Identification of two non-competing pairs, LR1/MR3 (a) and LR5/MR3 (b), for biparatopic constructs. For BLI assays, sensors coated with RBD were soaked in 200 nM of LR1 or LR5 before further soaked in LR1- or LR5-containing buffer with (magenta) or without (black) 100 nM of MR3. The MR3–RBD interaction profile was obtained in the absence of LR1 or LR5 (blue). c Neutralization assay of the biparatopic sybody LR5-MR3 with a Gly-Ser (GS) linker of 13 (blue), 19 (red), or 34 (black) amino acids as indicated. Brackets indicate IC₅₀ values in μg mL⁻¹. Data are from a representative of two independent experiments. d Neutralization assays of divalent sybodies. The original SARS-CoV-2 was used for all assays except that the D614G mutant⁴² was additionally tested for MR3-MR3 (red asterisk). Color-coding of the tested sybodies is as indicated. For MR3-MR3(34GS) against 614G, data show a representative of three independent experiments, which were performed using non-overlapping concentrations. For the rest of the samples, mean ± standard deviation are plotted (n = 3 independent experiments). Error bars are omitted where, in rare cases, available data points are less than three due to experimental design on concentration replicates. e Summary of binding kinetics and neutralizing activities of the divalent sybodies. Source data for a–d are provided as a Source data file. BLI biolayer interferometry, N.D. not determined.