Arsenal of nanobodies shows broad-spectrum neutralization against SARS-CoV-2 variants of concern in vitro and in vivo in hamster models

Abstract

Nanobodies offer several potential advantages over mAbs for the control of SARS-CoV-2. Their ability to access cryptic epitopes conserved across SARS-CoV-2 variants of concern (VoCs) and feasibility to engineer modular, multimeric designs, make these antibody fragments ideal candidates for developing broad-spectrum therapeutics against current and continually emerging SARS-CoV-2 VoCs. Here we describe a diverse collection of 37 anti-SARS-CoV-2 spike glycoprotein nanobodies extensively characterized as both monovalent and IgG Fc-fused bivalent modalities. The nanobodies were collectively shown to have high intrinsic affinity; high thermal, thermodynamic and aerosolization stability; broad subunit/domain specificity and cross-reactivity across existing VoCs; wide-ranging epitopic and mechanistic diversity and high and broad in vitro neutralization potencies. A select set of Fc-fused nanobodies showed high neutralization efficacies in hamster models of SARS-CoV-2 infection, reducing viral burden by up to six orders of magnitude to below detectable levels. In vivo protection was demonstrated with anti-RBD and previously unreported anti-NTD and anti-S2 nanobodies. This collection of nanobodies provides a potential therapeutic toolbox from which various cocktails or multi-paratopic formats could be built to combat multiple SARS-CoV-2 variants.

Nanobodies against SARS-CoV-2 reduced virus load in the brain of challenged mice and neutralized Wuhan, Delta and Omicron Variants

“…So far, several Nbs have been developed with promising diagnostic and therapeutic applications [20,38]. Both monomeric Nbs and engineered molecules showed strong neutralizing activity to the different VOCs that have emerged, including the Omicron variants [39][40][41][42][43][44][45][46]. The intense production of new recombinant Nbs highlights the relevance of this technological platform, which is expected to reach clinical use for SARS-CoV-2 and other viral diseases in the near future [47].…”

Dendritic Cell‐Mediated Cross‐Priming by a Bispecific Neutralizing Antibody Boosts Cytotoxic T Cell Responses and Protects Mice against SARS‐CoV‐2

“…RBD-targeted V HH s have shown effective in neutralizing SARS-CoV-2 infection in vitro and in protecting animals from SARS-CoV-2 challenge, [23,25,27,28,54] making them a valuable alternative for the development of multivalent and multiparatopic agents (targeting multiple viral epitopes) with improved neutralization efficiency and decreased vulnerability to escape mutations. [29,55,56] Here, we used the high-affinity biparatopic V HH -tandem, TN, previously described to potently neutralize SARS-CoV-2 strain Wuhan-Hu-1 and prevent the emergence of escape mutants. [23] To maximize its neutralizing efficacy instead of generating a conventional Ig-like Fc-fusion, [24,26,56] we generated an anti-RBD hexavalent nAb, TN T , made by fusing TN to the human collagen XVIII homo-trimerization domain, an effective scaffold for the generation of trimeric antibodies for clinical use.…”

“…[29,55,56] Here, we used the high-affinity biparatopic V HH -tandem, TN, previously described to potently neutralize SARS-CoV-2 strain Wuhan-Hu-1 and prevent the emergence of escape mutants. [23] To maximize its neutralizing efficacy instead of generating a conventional Ig-like Fc-fusion, [24,26,56] we generated an anti-RBD hexavalent nAb, TN T , made by fusing TN to the human collagen XVIII homo-trimerization domain, an effective scaffold for the generation of trimeric antibodies for clinical use. [44] TN T demonstrated a complete, potent blocking of the RBD-hACE2-interaction and a strong similar binding to beta, gamma, kappa, and delta variants RBDs.…”

Tuning the immune response: sulfated archaeal glycolipid archaeosomes as an effective vaccine adjuvant for induction of humoral and cell-mediated immunity towards the SARS-CoV-2 Omicron variant of concern

“…Purified proteins were analyzed by SDS-PAGE using NuPAGE 4-12% Bis-Tris gels (Invitrogen) followed by Coomassie Blue staining or western blotting. Primary antibodies for western blotting were from Cell Signaling (E7M5X) or produced in-house (V H H S2A4) (41). The absence of endotoxin contamination was verified using Endosafe cartridge-based Limulus amebocyte lysate tests (Charles River Laboratories, Charleston, SC, USA).…”

“…The binding analysis was carried out in two steps, first using indirect capture of the S-protein trimer via the human resistin trimerization domain immobilized onto the SPR surface, followed by flowing the ACE2 over top to generate the binding sensorgrams. The capture surface was generated using an antiresistin single-domain (VHH) antibody fused to a human IgG1 Fc (41). This antibody was diluted to 10 µg/mL in 10 mM sodium acetate immobilization buffer pH 4.5 (Cytiva, Marlborough, MA) and immobilized to approximately 2000 RUs using the Immobilization Wizard for NHS/EDC amine coupling within the Biacore T200 Control instrument software (v2.0.1).…”