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Selection and Attributes of Synthetic Nanobodies that Effectively Neutralize SARS-CoV-2

COVID-19 has imposed social and economic burden across the globe. The causative agent of COVID-19, SARS-CoV-2, yields severe atypical pneumonia. The virus works by exploiting the angiotensin converting enzyme 2 (ACE2) receptor upon entering host cells, which is orchestrated by the viral spike protein through fusion. In the ectodomain of the glycosylated spike protein, it forms a trimer that consists of a core S2 subunit and a distal S1 subunit, which contains a receptor-binding domain (RBD). This domain can be exposed in an “up” or “down” confirmation, in which “down” leads to ACE2 binding. Currently, the SARS-CoV-2 spike protein is on the major antigenic targets for drug development as it determines the host immune response.

Studies have used plasma from convalescent SARS-CoV-2 patients for improvement of clinical outcomes. Human antibodies have also been isolated as well for neutralization activity against SARS-CoV-2 in vitro. Nanobodies, single-domain antibodies, may be used as an alternative therapeutic. Their small size, increased stability, and production simplicity adds great advantage to their functionality. Prior studies have demonstrated that nanobodies are capable of virus neutralization by inhibiting binding of the spike protein to ACE2 receptor. Though usually extracted from immunized camelids, development of libraries of synthetic nanobodies enable a more efficient selection for binders for intended targets.

In this study, synthetic nanobodies were targeted against the RBD of SARS-CoV-2 and were shown to compete with ACE2 binding and neutralize a SARS-CoV-2 spike pseudovirus. Sybody 23 (Sb23) exhibited precise targeting by binding to recombinant RBD in addition to the perfusion spike glycoprotein with high affinity. Sb23 also demonstrated potent neutralization activity. Using a small angle X-ray scattering model of a RBD-Sb23 complex, the synthetic nanobody binds in the vicinity of ACE2-binding site on the RBD. Cryogenic electron microscopy was utilized to reveal Sb23 bound to the spike in the ACE2-binding site on the RBD in both “up” and “down” conformation and thus effectively blocking ACE2 binding.

The synthetic nanobody library generated was intended to select highly specific binders with neutralizing activity against SARS-CoV-2 and was completed within the time frame of 2-3 weeks. Traditionally, nanobody generation may take as long as 6 weeks for Llama immunization and a total of 3-4 months for the entire process. Using the synthetic nanobody platform did not require any immunization steps and generated a varietized but large binder repertory. The larger binder repertory helps to account for naturally-occurring antibody maturation and requires only a portion of the purified antigen for the selection process. In this library, 85 unique binders were identified in one selection round. No synthetic nanobodies were found to be identical, illustrating the potential for selecting for other high-affinity binders. It was speculated whether the Sb23 binding induces a conformational change that results in the “up” conformation or rather Sb23 promotes a conformational stabilization of the “up” in the spike’s landscape.

Increasing avidity and affinity of binders may be advantageous in the development of therapeutic agents. In combination with a non-overlapping synthetic nanobody, the neutralization potential of Sb23 could increase the overall avidity towards the spike protein. Fusion of Sb23 with Sb12 also demonstrated neutralization potential and affinity to RBD in the same range as Sb23-Fc or Sb42-Fc, also illuminating the potential of heterobivalent ligands.

As the COVID-19 pandemic shows how fast a new viral strain may emerge and spread across the globe, approaches for treatment have diversified and efforts have grown. Treatments against new viral strains will be key in combating fatality rates in the future. Synthetic nano-libraries may offer an alternative to typical drug development, offering generation of highly specific binders as well as the possibility of neutralization potential.


Custódio, T.F., Das, H., Sheward, D.J. et al. Selection, biophysical and structural analysis of synthetic nanobodies that effectively neutralize SARS-CoV-2. Nat Commun 11, 5588 (2020). https://doi.org/10.1038/s41467-020-19204-y

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