Fig. 2: Laboratory-matured nanobodies bind to RBD and spike proteins with high affinity.
From: Neutralizing nanobodies bind SARS-CoV-2 spike RBD and block interaction with ACE2
a, Maturation by mutagenesis of CDR3 region of H11 resulted in H11-D4 and H11-H4. The five changes from the parent are shown in bold. b, SPR sensorgram showing that H11-H4 bound to RBD (immobilized as RBD-Fc on the chip) with 5 nM affinity. A repeat experiment is shown in Extended Data Fig. 1c and H11-D4 data are provided in Extended Data Fig. 1d. c, RBD was bound by ACE2 (immobilized as ACE2-Fc on the chip). When RBD was pre-mixed with H11-H4, there was no binding, indicating that H11-H4 and ACE2 compete for binding to RBD. Similar results were observed using spike protein instead of RBD. The antibody E08R (anti-Caspr2 Fab) was used as a negative control. Data for H11-D4 are provided in Extended Data Fig. 1e. d, RBD was bound by CR3022 (immobilized as CR3022-Fc on the chip). When RBD was pre-mixed with H11-H4, binding occurred with similar on and off rates, indicating that H11-H4 and CR3022 recognize different epitopes on RBD. The response for the RBD H11-H4 mixture was larger, consistent with an H11-H4–RBD complex binding to CR3022. Antibody E08R was again used as a negative control. The spike protein shows binding to CR3022 in the presence or absence of H11-H4. Data for H11-D4 are provided in Extended Data Fig. 1f. e, ITC measurements show a KD of 12 ± 1.5 nM and a 1:1 ratio for H11-H4 and RBD association. Replicates and data for H11-D4 are provided in Extended Data Fig. 2a. f, ITC measurements show a KD of 44 ± 3 nM and a 1:1 ratio for association between spike protein and H11-H4. Replicates and data for H11-D4 are provided in Extended Data Fig. 2b.
