In a recent study published in iScience, researchers designed biparatopic nanobodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
Study: Biparatopic Nanobodies Targeting Receptor Binding Domain Efficiently Neutralize SARS-CoV-2. Image credit: Juan Gaertner/Shutterstock
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Despite the rapid development of effective and safe vaccines against SARS-CoV-2, questions about their long-term efficacy prevail, justifying the continued search for new therapeutic strategies. Recombinant protein biologics such as monoclonal antibodies (mAb) have potential for prophylaxis and treatment of infected individuals.
In addition, nanobodies, antibodies that contain a single variable domain, originate from the camelid family. The small size, high stability and simple architecture of nanobodies are beneficial over conventional mAbs. These allow improved tissue penetration, with a propensity for high-affinity binding to small epitopes. In addition, nanobodies are amenable to covalent bonding to enhance function and typically have higher yields and lower production costs.
The study and conclusions
In the present study, the researchers isolated nanoparticles against the SARS-CoV-2 spike receptor-binding domain (RBD) using a synthetic yeast display library. RBDs were designed in two formats: 1) monomeric RBD with an AVI tag for biotinylation and 2) dimeric RBD-Fc, in which the RBD was fused to the crystallizable fragment (Fc) domain of IgG1 of the mouse
RBD probes were validated by transiently transfecting 293T cells with human angiotensin-converting enzyme 2 (hACE2) and stained with tetramerized RBD monomers. The authors observed ACE2-dependent binding of RBD probes to 293T cells, validating their functional integrity. These tetramers were used to generate RBD-specific neutralizing nanobodies from a yeast display library.
Selection involved two successive magnetic enrichment steps, followed by fluorescence-activated cell sorting (FACS)-based enrichment, which resulted in a library with approximately 72% yeast clones that join RBD. The library was then co-stained with SARS-CoV-1 and SARS-CoV-2 RBD tetramers, which revealed distinct populations.
One (major) population bound SARS-CoV-2 RBD tetramers exclusively, while the other (minor) population bound RBD tetramers from both viruses. Probably, SARS-CoV-1/2 cross-reacting clones could have a conserved RBD epitope, which represents an important target. Single-cell clones were then sorted and the top 10 clones with the brightest RBD tetramer staining were sequenced.
Mammalian expression vectors were cloned with RBD-specific nanobody DNA and the nanobodies were purified. The authors tested whether the purified nanobodies inhibited the ACE2-RBD interaction using a surrogate virus neutralization assay (sVNT) and found four nanobody clones (A11, B1, C8, and G8) that inhibited binding. It should be noted that only nanobody G8 was cross-reactive with SARS-CoV-1/2.
The RBD binding ability of the nanobody clones was assessed by surface plasmon resonance (SPR). All four nanobodies bound the SARS-CoV-2 RBD with moderate affinities, while only G8 bound the SARS-CoV-1 RBD, albeit with reduced affinity. Additional SPR-based experiments revealed two distinct modes of binding: one involved binding to a shared epitope (for constructs A11, B1, and C8) and the other to a distinct epitope (G8) that was more conserved within the SARS-CoV-1. RBD.
Although the nanobodies exhibited moderate affinities, they were unlikely to strongly neutralize infection compared to multiple high-affinity mAbs in use. Therefore, the researchers generated a series of nanobody constructs to increase the neutralization capacity of nanobody monomers. The nanobody clones with the highest affinities (G8 and B1) were used. These improvement experiments involved three different strategies.
First, a human IgG1 Fc domain was fused to the nanobodies (B1-Fc and G8-Fc constructs). Second, B1 and G8 were covalently linked (biparatopic constructs) via a glycine-serine (GS) bond. Biparatopic (BP) constructs were generated with three different linker lengths (10, 19, and 39 amino acids). Third, dimeric biparatopic constructs were generated using human IgG1 Fc domains. A microneutralization assay was performed to test whether the constructs inhibited SARS-CoV-2 infection.
As monomers, B1 and G8 moderately inhibited infection; however, Fc dimerization of either nanobody enhanced its neutralizing activity. In particular, monomeric biparatopic constructs markedly improved neutralization, increasing with bond length. Fc dimerization of biparatopic constructs resulted in potent neutralizing activity; however, they were less effective than their monomeric counterparts.
In addition, biparatopic nanobodies were tested using a multiplex RBD variant array to assess their ability to overcome viral escape. Nanobody binding to RBDs of SARS-CoV-2 variants of concern (VOCs) and inhibition of RBD-ACE2 was measured. The G8-Fc construct bound all tested variants with high potency, but the B1-Fc nanobody showed reduced binding to the RBDs of Beta and Gamma variants and those containing E484D, E484K, Q493K, and S494P substitutions.
However, the biparatopic nanobody with a 10 amino acid linker (BP10) had a similar profile to G8-Fc. Inhibition of the RBD-ACE2 interaction was tested on a bead-based sVNT using 20 different RBDs, including those from SARS-CoV-1, bat and pangolin coronaviruses, and the Omicron variants BA.1 and BA.2.
Findings were similar to those of the multiplex array. However, the nanobody constructs did not neutralize the Omicron variants. Finally, mice were treated separately with B1-Fc, G8-Fc, and BP10-Fc constructs and challenged with SARS-CoV-2 after 24 hours. Treatment with G8-Fc moderately reduced viral loads in the lungs. In contrast, mice treated with B1-Fc or BP10-Fc were fully protected.
Conclusions
In summary, the study demonstrated the isolation of SARS-CoV-2 neutralizing nanobodies using a yeast display library and that the generation of biparatopic nanobodies could enhance their neutralizing potency, apparently due to the cross-linking of different proteins spike Interestingly, dimerization of the biparatopic constructs did not improve neutralization relative to their monomeric counterparts.