Engineered Multivalent Nanobodies Potently and Broadly Neutralize SARS‐CoV‐2 Variants

Abstract

The COVID-19 pandemic continues to be a severe threat to human health, especially due to current and emerging SARS-CoV-2 variants with potential to escape humoral immunity developed after vaccination or infection. The development of broadly neutralizing antibodies that engage evolutionarily conserved epitopes on coronavirus spike proteins represents a promising strategy to improve therapy and prophylaxis against SARS-CoV-2 and variants thereof. Herein, a facile multivalent engineering approach is employed to achieve large synergistic improvements in the neutralizing activity of a SARS-CoV-2 cross-reactive nanobody (VHH-72) initially generated against SARS-CoV. This synergy is epitope specific and is not observed for a second high-affinity nanobody against a non-conserved epitope in the receptor-binding domain. Importantly, a hexavalent VHH-72 nanobody retains binding to spike proteins from multiple highly transmissible SARS-CoV-2 variants (B.1.1.7 and B.1.351) and potently neutralizes them. Multivalent VHH-72 nanobodies also display drug-like biophysical properties, including high stability, high solubility, and low levels of non-specific binding. The unique neutralizing and biophysical properties of VHH-72 multivalent nanobodies make them attractive as therapeutics against SARS-CoV-2 variants.

Deep mutational engineering of broadly-neutralizing nanobodies accommodating SARS-CoV-1 and 2 antigenic drift

“…The therapeutic potential of VHH72 resides in its ability to neutralize SARS-CoV-2 in spite of its moderate affinity by recognizing an uncommon conserved class IV epitope. 41 The marked difference in affinity of the parent molecule for the antigens of the two viruses is probably explained by the presence of three substitutions found in the contact zones with VHH72 (residues A372, S373 and P384 in SARS-CoV-2 epitope are threonine, phenylalanine and alanine residues, respectively, in the SARS-CoV-1 epitope). On this basis, we first sought to improve the affinity of VHH72 by searching all individual favorable mutations conferring enhanced binding to SARS-CoV-2 RBD.…”

Deep Mutational Engineering of broadly-neutralizing and picomolar affinity nanobodies to accommodate SARS-CoV-1 & 2 antigenic polymorphism

“…The therapeutic potential of VHH72 resides in its ability to neutralize SARS-CoV-2 in spite of its moderate affinity by recognizing an uncommon conserved class IV epitope 39 . The marked difference in affinity of the parent molecule for the antigens of the two viruses is probably explained by the presence of three substitutions found in the contact zones with VHH72 (residues A372, S373 and P384 in SARS-CoV-2 epitope are respectively threonine, phenylalanine and alanine residues in the SARS-CoV-1 epitope).…”

Bovine-derived antibodies and camelid-derived nanobodies as biotherapeutic weapons against SARS-CoV-2 and its variants: A review article

“…bipNbs have conferred protection against SARS-CoV-2 variants, significantly reduced disease progression, and increased survival rates [ 113 ]. Because of their potent and broad neutralizing actions, outstanding biophysical characteristics, stability, solubility, and long extended half-lives, multivalent VHH-72 Fc fusion proteins are promising therapeutic candidates [ 114 ]. It was successfully attached to spike proteins in alpha and beta SARS-CoV-2 variants.…”

“…Many recent reports documented the promising antiviral efficacy of nanobodies and their potential as biotherapeutics against SARS-CoV-2 are being investigated [ 54 , 58 , 87 , 92 , 112 , [122] , [123] , [124] , [125] , [126] , [127] ]. Nanobodies are currently engineered into different multivalent forms, fused to Fc domains, and their affinity matured to increase neutralization potency [ 58 , 112 , 114 , [125] , [126] , [127] ]. Bivalent Fc–VHH variants, recently recovered in both immune and pre-immune nanobody libraries, had a 10-fold higher neutralizing activity than humanized nanobodies from a synthetic library [ 122 ].…”