Generation and Characterization of SARS-CoV-2 Neutralizing Nanobodies

Abstract

Single-domain antibody fragments, also called nanobodies, that target the spike glycoprotein of SARS-CoV-2 can potently neutralize the virus and prevent infection. In our first aim, we investigated whether their neutralization potency can be further increased by multimerization and generated multivalent constructs of a recently identified SARS-CoV-2 neutralizing nanobody, Ty1. We applied a novel method for the rapid production of Ty1 dimers and tetramers. First, Ty1 nanobodies were labelled on the C-terminus with a click chemistry functional group via sortase A and subsequently, dimerized or attached to a 4-arm polyethylene glycol (PEG) molecule by copper-free strain-promoted azide-alkyne cycloaddition (SPAAC). Comparison of the neutralization activity of the different constructs presented that di- and multimerization considerably increased the potency. The second aim was to explore the use of click chemistry for the generation of bispecific constructs. For this, two neutralizing nanobodies with distinct binding sites were functionalized and fused by SPAAC reaction. The obtained heterodimer showed a 25-fold increased in vitro neutralization potency compared to the monomeric variants. The therapeutic potential of the heterodimer was confirmed in vivo, where it suppressed SARS-CoV-2 in infected mice when administered at an early stage of the disease. The third aim was to use click chemistry as a screening method to identify the most potent nanobody combination. Further characterization by enzyme-linked immunosorbent assay (ELISA) and epitope mapping using competition assays showed that receptor-binding domain (RBD)-specific nanobodies were most potent and exhibited different mechanisms for viral neutralization. This thesis describes that click chemistry reaction is an efficient method to rapidly produce nanobody multimers which leads to increased avidity and enhanced neutralization potency. Characterization of nanobodies specific for the spike protein may help to identify novel neutralization mechanisms of SARS-CoV-2. Finally, bivalent nanobodies have suppressive effects in vivo, making them therapeutic candidates for viral infections.

Popular Abstract

Nanobodies as potential antiviral agents against SARS-CoV-2

SARS-CoV-2 is the causative agent of COVID-19 and is responsible for a serious global health crisis. As of April 2021, more than 134.6 million cases of COVID-19 worldwide have been reported, including 2,918,176 deaths. While vaccination of the population progresses, the numbers of infections are still high. In addition, new virus variants have emerged that show resistance to the currently available antiviral therapies. Therefore, highly potent antiviral drugs capable of neutralizing multiple SARS-CoV-2 variants are urgently needed. Camelid-derived single-domain antibody fragments, also called nanobodies, have been shown to be potent inhibitors of viral infections. Since nanobodies derive from heavy-chain-only antibodies, they are much smaller than conventional antibodies. This feature allows nanobodies to bind to regions that may be more conserved and inaccessible for conventional antibodies. Hence, nanobodies are ideal tools to identify novel mechanisms for antiviral therapeutics.

SARS-CoV-2 presents a spike glycoprotein on its surface, which has three receptor binding domains (RBD) that play a critical role in the attachment of the virus to host cells. Nanobodies binding the spike protein have been shown to prevent SARS-CoV-2 from entering host cells and reduce infectivity. To study the effects of different nanobody constructs on SARS-CoV-2 infectivity, the focus of this project was to generate and characterize nanobody monomers, dimers, and tetramers. Verification of binding sites and evaluation of neutralization potency and mechanisms showed that RBD-specific nanobodies were most potent and neutralized the virus in different ways, giving rise to new possible neutralization mechanisms for SARS-CoV-2.

For di- and multimerization of the nanobodies, a novel method of sortase A mediated functionalization combined with click chemistry was used. Sortase A is a bacterial transpeptidase that mediates the attachment of click chemistry functional groups to the nanobodies. Different dimer combinations and nanobody tetramers were obtained using click chemistry to fuse functionalized nanobodies together or to functionalize 4-arm PEG molecules. When testing the different constructs in neutralization assays, it was shown that di- and multimerization substantially improved the neutralization potency. These results highlight that increasing valency is a powerful way of enhancing the potency of nanobodies.

Finally, click chemistry was used to generate a bispecific nanobody dimer that simultaneously targets two epitopes. This nanobody heterodimer showed extremely high potency in vitro. Due to the remarkable potency, as well as the fact that bispecific constructs might be less susceptible to viral escape, the heterodimer was tested for its therapeutic potential in SARS-CoV-2 infected mice. Treatment with the heterodimer had suppressive effects on SARS-CoV-2. To further improve neutralization in vivo, bivalent constructs were formed by click chemistry which had a longer half-life in the bloodstream and thus potentially increase viral neutralization.

In conclusion, click chemistry can be used to rapidly generate bi- and multivalent nanobody constructs. In this manner avidity is increased, resulting in enhanced neutralization potency of antiviral nanobodies. Application of neutralizing nanobodies that prevent SARS-CoV-2 from entering host cells at an early stage of infection could protect individuals in high-risk groups and reduce the severity of COVID-19.

Master’s Degree Project in Molecular Biology – Medical Biology 60 credits 2021
Department of Biology, Lund University
Supervisor: Leo Hanke
McInerney’s laboratory, Department of Microbiology, Tumour and Cell Biology, Karolinska Institute

• author: Karl, Vivien
• supervisor: Leo Hanke
• organization: Degree Projects in Molecular Biology
• course: MOBN03 20202
• year: 2021
• type: H2 - Master's Degree (Two Years)
• subject: Biology and Life Sciences
• language: English
• id: 9061433
• date added to LUP: 2021-07-09 10:04:14
• date last changed: 2021-11-05 08:39:39