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As there are currently no clinically validated vaccines and limited therapeutic treatments for SARS-CoV-2, the development of new therapies is a worldwide effort, with neutralizing antibodies as a central component. However, traditional antibody production suffers from long development times and costly production.
As an alternative to human antibodies, nanobodies (single domain antibodies) offer a promising approach, conferring the advantages of smaller size, greater stability and superior simplicity in manufacturing. Indeed, the selection of specific attachments from a synthetic nanobody library can be achieved in a time frame of as little as two to three weeks.
Traditionally, the isolation of nanobodies from immunized camelids can take three to four months, while the synthetic nanobody (sb) approach requires no immunization step and requires only a fraction of the purified antigen for the selection process, accelerating so the development process. Synthetic libraries produce large and diverse ligand repertoires that can facilitate rapid development of nanobody-based drugs.
The SARS-CoV-2 trimeric spike protein is the major antigenic determinant of the host immune response and one of the major drug targets for COVID-19. Each S1 subunit comprises a receptor binding domain (RBD) that can transition from an exposed “up” conformation to a “down” conformation, where the latter is inaccessible for angiotensin converting enzyme 2 (ACE2) binding ).
The ability of SARS-CoV-2 to infect cells is based on the interactions between the viral spike protein (magenta) and the ACE2 protein (blue) found on the surface of human cells. These interactions can be disrupted by (black) sybodies – synthetic miniantibodies similar to those produced by camels and llamas. Image courtesy of Rayne Zaayman-Gallant / EMBL.
First, the researchers selected synthetic RBD antibody sequences of SARS-CoV-2 from three sybody (sb) libraries, yielding 85 unique sb sequences derived from a wide variety of phylogenetic sources. Sybody 23 (sb23) showed the highest RBD binding affinity during affinity analysis.
The team then selected 36 sb candidates and conducted a neutralization test with pseudo-typed lentiviral particles with the SARS-CoV-2 spike protein. Eleven shielded sybodies were capable of neutralizing SARS-CoV-2, with sb23 exhibiting the most potent neutralization efficiency.
The neutralization efficiency of Sb23 was substantially increased by fusing it with an antibody-derived Fc domain. Results from a bio-layer interferometry (BLI) test and a competition test demonstrated that sb23 competes with ACE2 for the same overlapping binding sites on the SARS-CoV-2 RBD.
Next, to gain insight into the structural basis of sb23’s neutralization activity against SARS-CoV-2, the researchers collected small-angle X-ray scatter data on the RBD-sb23 complex. Importantly, they found that sb23 binds alongside the ACE2 binding site in a lateral fashion.
They then determined the cryo-electron microscopy structure of the sb23-bound spike protein. In these models, sb23 binds to the inner edge of the ACE2 interaction interface in the “up” and “down” spike conformations. This demonstrates the strength of the small high affinity and strongly neutralizing SARS-CoV-2 spike ligands, as they are not sterically occluded in the same way as larger traditional antibodies.
Furthermore, the data show that sb23 hampers ACE2 binding in both “1-up” and “2-up” confirmations. The “2-up” conformation of the spike protein confers additional avidity with sb23 by blocking two binding sites for ACE2. According to the authors, the unique binding of sb23 in the “2-up” conformation may lead to the development of new therapeutic bonds, including those that bind to the lower portion of the RBD and possibly to the central helical region.
Finally, the researchers suggested that combining sb23 with other non-overlapping sybodies could potentially increase overall avidity towards the spike protein. These bispecific chimeric proteins could be advantageous in the development of novel COVID-19 therapeutic agents.
Senior author Christian Löw, PhD, group leader at the European Molecular Biology Laboratory (EMBL), emphasized the collaborative nature of the work in this paper.
“The spirit of collaboration has been enormous these days and everyone was motivated to contribute,” Löw said. “Getting the results so quickly was only possible because the methodologies we used were already established for other research projects not related to SARS-CoV-2. The development of these tools would have required much more time and resources.”
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