Biparatopic sybodies neutralize SARS‐CoV‐2 variants of concern and mitigate drug resistance

Abstract

The ongoing COVID-19 pandemic represents an unprecedented global health crisis. Here, we report the identification of a synthetic nanobody (sybody) pair, Sb#15 and Sb#68, that can bind simultaneously to the SARS-CoV-2 spike RBD and efficiently neutralize pseudotyped and live viruses by interfering with ACE2 interaction. Cryo-EM confirms that Sb#15 and Sb#68 engage two spatially discrete epitopes, influencing rational design of bispecific and tri-bispecific fusion constructs that exhibit up to 100and 1,000-fold increase in neutralization potency, respectively. Cryo-EM of the sybody-spike complex additionally reveals a novel up-out RBD conformation. While resistant viruses emerge rapidly in the presence of single binders, no escape variants are observed in the presence of the bispecific sybody. The multivalent bispecific constructs further increase the neutralization potency against globally circulating SARS-CoV-2 variants of concern. Our study illustrates the power of multivalency and biparatopic nanobody fusions for the potential development of therapeutic strategies that mitigate the emergence of new SARS-CoV-2 escape mutants.

Structural Characterization of a Neutralizing Nanobody With Broad Activity Against SARS-CoV-2 Variants

“…The IC 50 for the Beta (K417N/E484K/N501Y), Gamma (K417T/E484K/N501Y), and Delta (L452R/T478K) increased to 2.5, 3, and 4-fold, respectively ( Figure 2A ). Interestingly, despite having the highest number of mutations (Walter et al, 2022 ; Figure 2B ), the Omicron strain remained sensitive to DL28 (IC 50 = 0.66 μg mL −1 ; Figure 2A ).…”

“…The heat stability of nanobodies opens the possibility of using them as inhaling drugs for respiratory diseases (Muyldermans, 2013 ) [and indeed potentially for SARS-CoV-2 as demonstrated in hamsters Nambulli et al, 2021 ] and offers convenience in storage and transport. In the past months, dozens of neutralizing nanobodies against SARS-CoV-2 have been reported (Chi et al, 2020 ; Custódio et al, 2020 ; Esparza et al, 2020 ; Hanke et al, 2020 ; Huo et al, 2020a ; Schoof et al, 2020 ; Xiang et al, 2020 ; Koenig et al, 2021 ; Li et al, 2021a ; Pymm et al, 2021 ; Walter et al, 2022 ).…”

Isolation, characterization, and structure-based engineering of a neutralizing nanobody against SARS-CoV-2

“…To outcompete ACE2-RBD engagement, therefore, may require high concentrations of antibodies in the case of the monomeric DL4. A survey of the literature found this to be a general trend: most monomeric nanobodies (20 out of 24) show an IC 50 value that is at least 5-fold higher than the K D value [ 37 , [54] , [55] , [56] ], and 8 of them report a >20 fold difference [ 44 , 46 , 54 , [57] , [58] , [59] , [60] ].…”

A Bispecific Antibody Targeting RBD and S2 Potently Neutralizes SARS-CoV-2 Omicron and Other Variants of Concern

“…Therefore, it is still a top priority to develop highly potent and broadly monospecific or multispecific neutralizing mAbs targeting SARS-CoV-2 heavily mutated variants, such as the Omicron variant [21].…”

“…The substantial mutations of the Omicron variant make it completely or partially resistant to neutralization by most potent monoclonal antibodies against other VOCs and result in significantly reduced efficacy of existing COVID-19 vaccines[17-20]. Therefore, it is still a top priority to develop highly potent and broadly monospecific or multispecific neutralizing mAbs targeting SARS-CoV-2 heavily mutated variants, such as the Omicron variant[21].…”