An ultrapotent synthetic nanobody neutralizes SARS-CoV-2 by stabilizing inactive Spike

Abstract

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus enters host cells via an interaction between its Spike protein and the host cell receptor angiotensin-converting enzyme 2 (ACE2). By screening a yeast surface-displayed library of synthetic nanobody sequences, we developed nanobodies that disrupt the interaction between Spike and ACE2. Cryo-electron microscopy (cryo-EM) revealed that one nanobody, Nb6, binds Spike in a fully inactive conformation with its receptor binding domains locked into their inaccessible down state, incapable of binding ACE2. Affinity maturation and structure-guided design of multivalency yielded a trivalent nanobody, mNb6-tri, with femtomolar affinity for Spike and picomolar neutralization of SARS-CoV-2 infection. mNb6-tri retains function after aerosolization, lyophilization, and heat treatment, which enables aerosol-mediated delivery of this potent neutralizer directly to the airway epithelia.

Fig. 1. Discovery of two distinct classes of anti-Spike nanobodies.

(A) Selection strategy for identification of anti-Spike nanobodies that disrupt Spike-ACE2 interactions using magnetic bead selections (MACS) or fluorescence-activated cell sorting (FACS). (B) Flow cytometry of yeast displaying Nb6 (a class I nanobody) or Nb3 (a class II nanobody). Nb6 binds Spike^S2P-Alexa 647 and the RBD (RBD-Alexa 647). Nb6 binding to Spike^S2P is completely disrupted by an excess (1.4 μM) of ACE2-Fc. Nb3 binds Spike^S2P but not the RBD. Nb3 binding to Spike^S2P is partially decreased by ACE2-Fc. (C) SPR of Nb6 and Nb3 binding to either Spike^S2P or RBD. Red traces are raw data, and global kinetic fits are shown in black. Nb3 shows no binding to RBD. (D) SPR experiments with immobilized Spike^S2P show that class I and class II nanobodies can bind Spike^S2P simultaneously. By contrast, two class I nanobodies or class II nanobodies do not bind simultaneously. (E) Nanobody inhibition of 1 nM Spike^S2P-Alexa 647 binding to ACE2-expressing HEK293T cells. n = 3 (ACE2, Nb3) or n = 5 (Nb6, Nb11) biological replicates. All error bars represent SEM.

Fig. 2. Cryo-EM structures of Nb6 and Nb11 bound to Spike.

(A) Cryo-EM maps of the Spike^S2P-Nb6 complex in either closed (left) or open (right) Spike^S2P conformation. (B) Cryo-EM maps of the Spike^S2P-Nb11 complex in either closed (left) or open (right) Spike^S2P conformation. The top views show RBD up or down states. (C) Nb6 straddles the interface of two down-state RBDs, with CDR3 reaching over to an adjacent RBD. (D) Nb11 binds a single RBD in the down state (displayed) or similarly in the up state. No cross-RBD contacts are made by Nb11 in either RBD up or down state. (E) Comparison of RBD epitopes engaged by ACE2 (purple), Nb6 (red), or Nb11 (green). Both Nb11 and Nb6 directly compete with ACE2 binding.

Fig. 3. Multivalency improves nanobody affinity and inhibitory efficacy.

(A) SPR of Nb6 and multivalent variants. Red traces show raw data, and black lines show global kinetic fit for Nb6 and independent fits for association and dissociation phases for Nb6-bi and Nb6-tri. (B) Dissociation phase SPR traces for Nb6-tri after variable association times ranging from 4 to 520 s. Curves were normalized to maximal signal at the beginning of the dissociation phase. Percent fast-phase dissociation is plotted as a function of association time (right) with a single exponential fit. n = 3 independent biological replicates. (C) Inhibition of pseudotyped lentivirus infection of ACE2-expressing HEK293T cells. n = 3 biological replicates for all but Nb11-tri (n = 2). (D) Inhibition of live SARS-CoV-2 virus. Representative biological replicate with n = 3 (right) or n = 4 (left) technical replicates per concentration. n = 3 biological replicates for all but Nb3 and Nb3-tri (n = 2). All error bars represent SEM.

Fig. 4. Affinity maturation of Nb6 yields a picomolar SARS-CoV-2 neutralizing molecule.

(A) SPR of mNb6 and mNb6-tri binding to immobilized Spike^S2P. Red traces show raw data, and black lines show global kinetic fit. No dissociation was observed for mNb6-tri over 10 min. (B) mNb6 and mNb6-tri inhibit SARS-CoV-2 infection of VeroE6 cells in a plaque assay. Representative biological replicate with n = 4 technical replicates per concentration. n = 3 biological replicates for all samples. All error bars represent SEM. (C) Comparison of closed Spike^S2P bound to mNb6 and Nb6. Rotational axis for RBD movement is highlighted. (D) Comparison of RBD engagement by Nb6 and mNb6. One RBD was used to align both structures (RBD align), demonstrating changes in Nb6 and mNb6 position and the adjacent RBD. (E) CDR1 of Nb6 and mNb6 binding to the RBD. As compared to I27 in Nb6, Y27 of mNb6 hydrogen bonds to Y453 and optimizes π-π and π-cation interactions with the RBD. N, Asp; R, Arg. (F) CDR3 of Nb6 and mNb6 binding to the RBD demonstrating a large conformational rearrangement of the entire loop in mNb6. A, Ala; L, Leu; F, Phe. (G) Comparison of mNb6 complementarity-determining regions in either the cryo-EM structure of the Spike^S2P-mNb6 complex or an x-ray crystal structure of mNb6 alone.

Fig. 5. mNb6 and mNb6-tri retain activity after aerosolization, lyophilization, and heat treatment.

(A) Size exclusion chromatography of nanobodies after lyophilization or aerosolization. (B) Summary table of SPR kinetics data and affinities for aerosolized or lyophilized mNb6 and mNb6-tri. (C) Inhibition of SARS-CoV-2 infection of VeroE6 cells by mNb6-tri after aerosolization, lyophilization, or heat treatment at 50°C for 1 hour. Representative biological replicate with n = 2. Technical replicates are n = 3 per concentration.