Yeast display for generating SARS-CoV-2 RBD-specific nanobodies

Yeast display for generating SARS-CoV-2 RBD-specific nanobodies

In a recent study published in iScience Researchers have developed biparatopic nanobodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

Studie: Biparatopische Nanokörper, die auf die Rezeptorbindungsdomäne abzielen, neutralisieren effizient SARS-CoV-2. Bildnachweis: Juan Gaertner/Shutterstock

background

Despite the rapid development of effective and safe vaccines against SARS-CoV-2, questions remain regarding their long-term effectiveness, justifying the continued search for new therapeutic strategies. Recombinant protein biologics such as monoclonal antibodies (mAbs) offer potential for the prophylaxis and treatment of infected individuals.

In addition, nanobodies, antibodies containing a single variable domain, are native to the camelid family. The small size, high stability and simple architecture of nanobodies are advantageous over conventional mAbs. These enable improved penetration into tissue and tend to bind to small epitopes with high affinity. In addition, nanobodies can be covalently linked to improve functionality and typically have higher yields and lower production costs.

The study and results

In the present study, researchers isolated nanobodies against the receptor binding domain (RBD) of the SARS-CoV-2 spike using a synthetic yeast display library. RBDs were prepared in two formats - 1) RBD monomer with an AVI tag for biotinylation and 2) dimeric RBD-Fc, in which the RBD was fused to the fragment crystallizable (Fc) domain of mouse IgG1.

The RBD probes were validated by transient transfection of 293T cells with human angiotensin-converting enzyme 2 (hACE2) and stained with tetramerized RBD monomers. The authors found ACE2-dependent binding of RBD probes to 293T cells, confirming their functional integrity. These tetramers were used to generate RBD-specific neutralizing nanobodies from a yeast display library.

The selection involved two consecutive magnetic enrichment steps, followed by fluorescence-activated cell sorting (FACS)-based enrichment, resulting in a library with approximately 72% RBD-binding yeast clones. Next, the library was co-stained with RBD tetramers of SARS-CoV-1 and SARS-CoV-2, yielding distinct populations.

One population (main population) bound exclusively SARS-CoV-2 RBD tetramer, while the other (minor) population bound RBD tetramer of both viruses. It is likely that the cross-reactive SARS-CoV-1/2 clones may have a conserved RBD epitope, which represents an important target. Next, the clones were sorted by single cells and the top ten clones with the brightest RBD tetramer staining were sequenced.

Mammalian expression vectors were cloned with the DNA of RBD-specific nanobodies and the nanobodies were purified. The authors tested whether the purified nanobodies inhibited ACE2-RBD interaction using a surrogate virus neutralization assay (sVNT) and found four nanobody clones (A11, B1, C8, and G8) that inhibited binding. Notably, only the G8 nanobody was cross-reactive with SARS-CoV-1/2.

The RBD binding capacity of the nanobody clones was evaluated using surface plasmon resonance (SPR). The four nanobodies bound SARS-CoV-2 RBD with moderate affinity, while only G8 bound the SARS-CoV-1 RBD, albeit with reduced affinity. Further SPR-based experiments revealed two different binding modes - one involved binding to a common epitope (for A11, B1 and C8 constructs) and the other to a different epitope (G8) that was more conserved within the SARS-CoV-1 RBD.

Although the nanobodies had moderate affinities, they were unlikely to strongly neutralize infection compared to the multiple high-affinity mAbs used. Therefore, the researchers created 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 included 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 via a glycine-serine (GS) linker (biparatopic constructs). The 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 both nanobodies improved their neutralizing activity. Notably, the monomeric biparatopic constructs significantly enhanced neutralization and increased with linker length. Fc dimerization of biparatopic constructs resulted in potent neutralizing activity; However, they were less effective than their monomer counterparts.

Furthermore, the biparatopic nanobodies were tested using a multiplex RBD variant array to evaluate their ability to overcome virus leakage. Binding of nanobodies to RBDs of SARS-CoV-2 variants of concern (VOCs) and RBD-ACE2 inhibition were measured. The G8-Fc construct bound with high potency to all variants tested, but the B1-Fc nanobody showed reduced binding to beta and gamma variant RBDs as well as those containing E484D, E484K, Q493K, and S494P substitutions.

Nevertheless, 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 in a bead-based sVNT using 20 different RBDs, including those from SARS-CoV-1, bat and pangolin coronaviruses, and the Omicron BA.1 and BA.2 variants.

The results were similar to those of the multiplex array. However, the nanobody constructs were unable to neutralize the Omicron variants. Finally, the mice were treated separately with B1-Fc, G8-Fc and BP10-Fc constructs and infected with SARS-CoV-2 after 24 hours. Treatment with G8-Fc moderately reduced viral load in the lungs. In contrast, mice treated with B1-Fc or BP10-Fc were completely 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 apparently improve their neutralizing effectiveness due to the cross-linking of different spike proteins. Interestingly, dimerization of the biparatopic constructs failed to enhance neutralization compared to their monomeric counterparts.

Reference:

• Pymm, P. et al. (2022) „Biparatopische Nanokörper, die auf die Rezeptorbindungsdomäne abzielen, neutralisieren effizient SARS-CoV-2“, iScience, S. 105259. doi: 10.1016/j.isci.2022.105259. https://www.sciencedirect.com/science/article/pii/S2589004222015310