Sybodies targeting the SARS-CoV-2 receptor-binding domain

Abstract

The COVID-19 pandemic, caused by the novel coronavirus SARS-CoV-2, has resulted in a global health and economic crisis of unprecedented scale. The high transmissibility of SARS-CoV-2, combined with a lack of population immunity and prevalence of severe clinical outcomes, urges the rapid development of effective therapeutic countermeasures. Here, we report the generation of synthetic nanobodies, known as sybodies, against the receptor-binding domain (RBD) of SARS-CoV-2. In an expeditious process taking only twelve working days, sybodies were selected entirely in vitro from three large combinatorial libraries, using ribosome and phage display. We obtained six strongly enriched sybody pools against the isolated RBD and identified 63 unique anti-RBD sybodies which also interact in the context of the full-length SARS-CoV-2 spike protein. It is anticipated that compact binders such as these sybodies could feasibly be developed into an inhalable drug that can be used as a convenient prophylaxis against COVID-19. Moreover, generation of polyvalent antivirals, via fusion of anti-RBD sybodies to additional small binders recognizing secondary epitopes, could enhance the therapeutic potential and guard against escape mutants. We present full sequence information and detailed protocols for the identified sybodies, as a freely accessible resource. This report will be updated as we further characterize the identified sybodies, in terms of affinities, scaled-up purification yields, and their potential to neutralize SARS-CoV-2 infections.

Versatile, Multivalent Nanobody Cocktails Efficiently Neutralize SARS-CoV-2

“…The binding mode of Nb20 to the RBD is distinct from other previously reported SARS-CoV-2 neutralizing Nbs, which generally recognize similar epitopes in the RBD external loop region (33,34,35 ) ( Fig S6). The extensive hydrophobic and polar interactions (Fig 3b-c) between the RBD and Nb20 stem from the remarkable shape complementarity (Fig 3d) between all the CDRs and the external RBD loop, leading to ultrahigh-affinity (~ 10 pM).…”

“…In contrast, higher mass species (from early elution volumes) corresponding to the trimeric complexes composed of Nb21, RBD, and one of the Nbs (34,36,93,105, and 95) were evident (Fig 2b). Moreover, Nb105 competed with Nb34 and Nb95, which did not compete for RBD interaction, suggesting the presence of two distinct and non-overlapping epitopes.…”

An ultra-potent synthetic nanobody neutralizes SARS-CoV-2 by locking Spike into an inactive conformation

“…Thus, when mNb6-tri engages with Spike, it prevents ACE2 binding by both directly occluding the binding site and by locking the RBDs into an inactive conformation. Although a multitude of other monoclonal and single-domain antibodies against SARS-CoV-2 Spike have been discovered to date, there are few if any molecules as potent and stable as mNb6-tri (33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43).…”

A high-affinity RBD-targeting nanobody improves fusion partner’s potency against SARS-CoV-2

“…Examples of nanobody drugs include caplacizumab (32) for the treatment of acquired thrombotic thrombocytopenic purpura, and ozoralizumab and vobarilizumab that are in the clinical trials for rheumatoid arthritis (29,33). Recently, several groups have independently reported neutralizing nanobodies (22,(34)(35)(36)(37)(38)(39) or single-chain VH antibodies (40) against SARS-CoV-2 with variable potencies.…”