Fig. 4. An affinity maturation strategy enhances the binding and neutralization properties of synthetic nanobodies.

a Affinity maturation workflow. b Two representative sections of position-wise post- minus pre-affinity maturation amino acid percent point change profile. White values indicate the original amino acid, yellow values indicate the beneficial mutation. Empty positions indicate amino acids not detected in either the pre- or post-selection libraries. c ELISA assay of nanobody variants. d SARS-CoV-2 S pseudotyped lentivirus neutralization assay of nanobodies on HEK293T expressing ACE2 and TMPRSS2. For c and d, the data shown are two technical replicates, bar height: mean, circle: the value of each replicate. e Scatter plot of ELISA assay absorbance versus pseudotyped lentivirus neutralization as percent infection inhibited. Nanobody concentration for both assays was 50 nM. Values are the mean of two technical replicates. Numbers on linear fitting lines were r² values for data within each family. f Dose–response curve for neutralization of pseudotyped lentiviral infection by nanobodies. Markers: mean of three technical replicates, error bars: standard deviation. g IC50 calculated from data in f, as mean ± standard deviation. Source data for a, c, d, e, and f are provided in the Source Data file.