Shark nanobodies with potent SARS-CoV-2 neutralizing activity and broad sarbecovirus reactivity

Abstract

Despite rapid and ongoing vaccine and therapeutic development, SARS-CoV-2 continues to evolve and evade, presenting a need for next-generation diverse therapeutic modalities. Here we show that nurse sharks immunized with SARS-CoV-2 recombinant receptor binding domain (RBD), RBD-ferritin (RFN), or spike protein ferritin nanoparticle (SpFN) immunogens elicit a set of new antigen receptor antibody (IgNAR) molecules that target two non-overlapping conserved epitopes on the spike RBD. Representative shark antibody variable NAR-Fc chimeras (ShAbs) targeting either of the two epitopes mediate cell-effector functions, with high affinity to all SARS-CoV-2 viral variants of concern, including the divergent Omicron strains. The ShAbs potently cross-neutralize SARS-CoV-2 WA-1, Alpha, Beta, Delta, Omicron BA.1 and BA.5, and SARS-CoV-1 pseudoviruses, and confer protection against SARS-CoV-2 challenge in the K18-hACE2 transgenic mouse model. Structural definition of the RBD-ShAb01-ShAb02 complex enabled design and production of multi-specific nanobodies with enhanced neutralization capacity, and picomolar affinity to divergent sarbecovirus clade 1a, 1b and 2 RBD molecules. These shark nanobodies represent potent immunotherapeutics both for current use, and future sarbecovirus pandemic preparation.

Isolation and characterization of single domain antibodies from banded houndshark (Triakis scyllium) targeting SARS-CoV-2 spike RBD protein

“…To combat the pandemic, researchers have sought to develop antibodies against the SARS-CoV-2 receptor binding domain (RBD) for use in viral therapeutics and diagnostics [ 2 , 3 ], including single-domain antibodies (sdAbs). SdAbs are antibody fragments of heavy chain-only antibodies derived from camelid IgG2 and IgG3 [ [4] , [5] , [6] , [7] , [8] , [9] , [10] ], or cartilaginous fish immunoglobulin new antigen receptor (IgNAR) [ [11] , [12] , [13] , [14] , [15] , [16] ]. SdAbs obtained from camelids or cartilaginous fishes are known as nanobodies or VNARs, respectively.…”

“…SdAbs possess several advantageous properties that make them attractive for use in therapeutics and diagnostics, including small size, high physicochemical stability, solubility, specificity for cryptic epitopes, low immunogenicity, ease of protein engineering, more efficient cell penetration, and low production cost [ 17 , 18 ]. Because of these advantages, some researchers have attempted to develop multivalent [ 6 , 14 , 16 ] or nasal drugs [ 7 ] using SARS-CoV-2 RBD-specific sdAbs.…”

“…A primary reason medical and biotechnological researchers prefer nanobodies is the relatively limited availability of animal models for discovering VNARs [ 19 ]. Although various IgNARs have been discovered in numerous cartilaginous fish species [ 20 ], the six reported SARS-CoV-2 specific VNARs were derived exclusively from the nurse shark (Ginglymostoma cirratum) [ 11 , 12 , 16 ], white-spotted bamboo shark (Chiloscyllium plagiosum) [ 13 , 14 ], and a synthetic library [ 15 ]. Nurse and white-spotted bamboo sharks are suitable animal models for immunization; however, these species have not been found in the coastal areas of Korea [ 21 ].…”

Revolutionizing antiviral therapy with nanobodies: Generation and prospects

“…However, recent research has shown that other animals also have similar strategies to diversify their antibodies. For example, sharks produce a type of antibody called IgNAR (Variable domain of the immunoglobulin new antigen receptor) that is also small and stable like nanobodies [11] . Additionally, some rodents have been found to produce single-domain antibodies similar to nanobodies [12] .…”

“…The production of nanobodies or similar antibodies in different animals involves a complex process of genetic rearrangement and selection. In camels and llamas, for example, the genes encoding the heavy chain of their antibodies undergo a unique process called VHH gene rearrangement that leads to the production of nanobodies [13] In sharks, IgNAR antibodies are produced by a different mechanism involving gene conversion and somatic hypermutation [11] . Similarly, rodents use a process called VHH gene conversion to produce single-domain antibodies [13] .…”