Nanobody Repertoires for Exposing Vulnerabilities of SARS-CoV-2

Abstract

Despite the great promise of vaccines, the COVID-19 pandemic is ongoing and future serious outbreaks are highly likely, so that multi-pronged containment strategies will be required for many years. Nanobodies are the smallest naturally occurring single domain antigen binding proteins identified to date, possessing numerous properties advantageous to their production and use. We present a large repertoire of high affinity nanobodies against SARS-CoV-2 Spike protein with excellent kinetic and viral neutralization properties, which can be strongly enhanced with oligomerization. This repertoire samples the epitope landscape of the Spike ectodomain inside and outside the receptor binding domain, recognizing a multitude of distinct epitopes and revealing multiple neutralization targets of pseudoviruses and authentic SARS-CoV-2, including in primary human airway epithelial cells. Combinatorial nanobody mixtures show highly synergistic activities, and are resistant to mutational escape and emerging viral variants of concern. These nanobodies establish an exceptional resource for superior COVID-19 prophylactics and therapeutics.

Atomistic Simulations and Deep Mutational Scanning of Protein Stability and Binding Interactions in the SARS-CoV-2 Spike Protein Complexes with Nanobodies: Molecular Determinants of Mutational Escape Mechanisms

“…The mutational sensitivity map also shed some light on structure-functional role of sites targeted by common resistant mutations (F490S, E484K, Q493K, F490L, F486S, F486L, and Y508H) that evade many individual nanobodies. 47 Indeed, we found that E484, F486, and F490 positions can be sensitive to Nb6 binding (Figure 4A-C), while variations in these sites are known to have only minor effect on ACE2 binding.…”

“…46 Another diverse repertoire of high affinity nanobodies against SARS-CoV-2 S protein that are refractory to common escape mutants and showed synergistic neutralizing activity are characterized by proximal but non-overlapping epitopes suggesting that multimeric nanobody combinations can improve potency while minimizing susceptibility to escape mutations. 47 These studies identified a group of common resistant mutations in the dynamic RBM region (F490S, E484K, Q493K, F490L, F486S, F486L, and Y508H) that evade many individual nanobodies. 47 Structural versatility of nanobody combinations that can effectively insulate the S-RBD accessible regions suggested a mechanism of resistance to mutational escape in which combining two nanobodies can markedly reduce the number of allowed substitutions to confer resistance and thereby elevate the genetic barrier for escape.…”

“…47 These studies identified a group of common resistant mutations in the dynamic RBM region (F490S, E484K, Q493K, F490L, F486S, F486L, and Y508H) that evade many individual nanobodies. 47 Structural versatility of nanobody combinations that can effectively insulate the S-RBD accessible regions suggested a mechanism of resistance to mutational escape in which combining two nanobodies can markedly reduce the number of allowed substitutions to confer resistance and thereby elevate the genetic barrier for escape. 47, 48 Using human VH-phage library and protein engineering several unique VH binders were discovered that recognized two separate epitopes within the ACE2 binding interface with nanomolar affinity.…”

“…47 Structural versatility of nanobody combinations that can effectively insulate the S-RBD accessible regions suggested a mechanism of resistance to mutational escape in which combining two nanobodies can markedly reduce the number of allowed substitutions to confer resistance and thereby elevate the genetic barrier for escape. 47, 48 Using human VH-phage library and protein engineering several unique VH binders were discovered that recognized two separate epitopes within the ACE2 binding interface with nanomolar affinity. 48 Multivalent and bi-paratopic VH constructs showed markedly increased affinity and neutralization potency to the SARS-CoV-2 virus when compared to standalone VH domain.…”

Degenerate CD8 Epitopes Mapping to Structurally Constrained Regions of the Spike Protein: A T Cell-Based Way-Out From the SARS-CoV-2 Variants Storm

“…This would obviously imply that different spike regions are simultaneously recognized by polyclonal anti-spike antibodies of different specificity, elicited by the virus (or by a vaccine) in each patient. Moreover, a variable proportion of the elicited antibodies may be directed against largely invariable spike regions, i.e., protein regions whose mutational change would severely impair the fitness of the virus (14)(15)(16)(17)(18). Thus, loss of immune-recognition of a certain spike region caused by a mutational event, should, in principle, be compensated by the persistent recognition of the virus by antibody molecules directed against spike regions that have not changed or that are intrinsically not permissive to amino acid substitutions because of structural or functional constraints.…”