The coronavirus disease 2019 (COVID-19) pandemic, brought on by the rapid outbreak of severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2), has drastically affected the healthcare system and economy worldwide. Several COVID-19 vaccines have received emergency use authorization (EUA) from global regulatory bodies, akin to the U.S. Food and Drug Administration (FDA). Because of the continual emergence of SARS-CoV-2 variants owing to genomic mutations, the efficacy of the available vaccines against some strains of the virus has been reduced.

Study: A humanized nanobody phage display library yields potent binders of SARS CoV-2 spike. Image Credit: Huen Structure Bio/Shutterstock

Background

There’s an urgent need for novel, cost-effective, high-throughput, and adaptable pipelines to discover effective therapeutics against COVID-19 and other similar viruses that may potentially cause pandemics.

Mechanistically, the spike protein of SARS-CoV-2 is answerable for host invasion. This protein detects and binds to the host’s angiotensin-converting enzyme 2 (ACE2) and promotes the fusion of membranes to cause host infection. SARS-CoV-2 genomic mutations, akin to N501, have been related to increased transmissibility and virulence in comparison with the unique SARS-CoV-2 strain that emerged in Wuhan. Moreover, some variants can escape immune responses elicited after COVID-19 vaccination or natural infection.

Camelidae and a few shark species contain nanobodies known to be a novel class of heavy chain-only antibodies. Nanobodies (VHHs) and shark variable latest antigen receptors (VNARs) are smaller by one order of magnitude than full-length IgG counterparts. The predominant advantage of utilizing these nanobodies as antiviral biologics is straightforward bulk production at a multi-kilogram scale in a prokaryotic system. Moreover, nanobodies have an extended shelf life and better tissue permeability.

Nanobodies are administered via different routes, for instance, orally (e.g., V565) or by inhalation (e.g., ALX-0171). A recent PLoS ONE study detected neutralizing nanobodies against SARS-CoV-2 from nanobody phage display libraries and via in vitro and ex vivo high-throughput screenings.

Key findings

Synthetic humanized nanobody libraries were developed by combining randomized complementarity-determining regions (CDRs) framework to diversify antigen recognition. Among the many ten enriched RBD binders, RBD-1-2G(NCATS-BL8125) and RBD-2-1F were probably the most potent exhibiting an IC50 of 490 and 470 nM against SARS-CoV-2 pseudotyped particles, respectively.

The event of bi- or tri-valent formats of the RBD-1-2G domains enabled an enhanced affinity and neutralization capability against each pseudotyped particles in addition to the SARS-CoV-2 virus. Further, two distinct binding modes were noted for 4 nanobodies along side a stable SARS-CoV-2 S-protein ectodomain. The epitope for ‘Group 1’ nanobodies was found to overlap with the receptor binding motif (RBM), while the N-terminal domain of the adjoining monomer was targeted by the ‘Group 2’ binders. The latter did not inhibit ACE binding and didn’t overlap with the RBM.

It was also observed that 3 copies of RBD-1-2G could bind to the identical spike trimmer. Pseudotyped particles containing the N501Y mutation were neutralized by the RBD-1-2G nanobody. The viral loads in WA1 and B.1.1.7 infections were reduced by RBD-1-2G-Fc treatment, and molecular dynamic simulations make clear nanobodies that certain epitopes solely focussed on the spike-ACE2 interface. It was noted that one among the explanations for the affinity of RBD-1-2G to B.1.1.7 RBD could possibly be the presence of the tyrosine in position 501.

To find out the effect of the mutation on the nanobody, the B.1.1.7/Alpha (N501Y) was used, which enhanced the RBD binding to the ACE2 receptor. RBD-1-2G could bind to each the Alpha (N501Y) and WT RBD but to not latest mutant lineages, which could possibly be brought on by the E484 mutations. Binding was also absent with the Delta and Lambda variants, which shouldn’t have the E484 mutation.

Future perspectives

Nanobodys’ effectiveness as therapeutics are mainly driven by aspects akin to protein solubility, potential immunogenicity, and binding affinity. Several latest approaches have been reported concerning developing effective therapeutics against the SARS-CoV-2 spike domain. Previous research showed that direct immunization of llamas produced effective nanobodies that neutralize pseudotyped particles within the low nanomolar range. In the long run, the technology to supply llama hybridomas needs to be developed. 

In a recent study, anti-SARS-CoV-2 binders were generated using ribosome-display of synthetic llama libraries by performing an affinity maturation step. Subsequently, a mutagenized library was created by combining the sequences. In the same vein, such an affinity maturation process could possibly be helpful for the RBD-1-2G binder to help in restoring binding to novel SARS-CoV-2 mutants.

Rapid screening of artificial humanized nanobody libraries to detect compatible binders could possibly be useful for countering the immunogenic defenses possessed by viral pathogens. The invention method described on this study was capable of generate high-affinity llama binders in a brief span of fewer than two weeks. In the long run, the library could possibly be utilized in the treatment of other diseases besides COVID-19 and act as a reserve for quick neutralizing nanobodies discovery.