Llamas, camels and COVID-19
For SARS-CoV-2 to infect humans, the viral Spike (S) protein must interact with and bind to the angiotensin-converting enzyme 2 (ACE2) receptor on the surface of human cells. The S protein achieves this via the receptor binding domains (RBDs), and thus blocking the RBD using antibodies is just one therapeutic avenue that is being explored for the treatment of SARS-CoV-2.
Back in May, Technology Networks covered the work of scientists exploring the potential utility of nanobodies against SARS-Cov-2. Nanobodies, also known as "single-domain antibodies" are extremely small antibodies that are found in llamas and camels.
Compared to conventional antibodies, nanobodies are advantageous for therapeutic use for several reasons: They can be constructed into multivalent forms, nebulized (a desirable property as it simplifies drug delivery) and possess higher thermal chemostability.
Several research teams have now demonstrated that nanobodies are able to inhibit the binding of the S protein to the ACE2 receptor, neutralizing SARS-CoV-2.
The fact that nanobodies are derived from animals raises some logistical challenges, namely; how can their production be scaled-up in a manner that is safe but also efficient in the context of a global pandemic?
Synthetic biology steps up to SARS-CoV-2
Scientists have turned to synthetic versions of nanobodies – known as sybodies – as a potential alternative. Sybodies are designed to mimic the natural shape and diversity of nanobodies, and due to their small size can target epitopes that are tricky for traditional antibodies to bind to. In 2018, the laboratory of Professor Markus Seeger at the University of Zurich published a protocol which provides researchers with the tools to rapidly select sybodies against specific membrane protein targets of interest and creating libraries of sybodies.1
A group of researchers led by Christian Löw at EMBL Hamburg have utilized this platform to search for existing sybodies that could block SARS-CoV-2. Their findings are published in Nature Communications.2 Discussing their rationale in the paper the research team say, "This methodology is an attempt to minimize the overwhelming impact that SARS-CoV-2 is having on the healthcare systems and to prepare for future pandemics. Thus, the R&D community can act in a prompter way for the development of efficient medication."
A spotlight on Sb23
The team used the SARS-CoV-2 S protein as a lure to analyze which sybodies would bind to the RBD. "Six of our analyzed sybodies (Sb12, Sb23, Sb42, Sb76, Sb95, and Sb100) bound RBD with affinities of 24.2, 10.6, 5.0, 58.1, 43.9, and 38.7 nM, respectively," they describe in the paper.
Using thermal shift assays, Low and colleagues highlighted Sb23 as having a higher affinity to the RBD of SARS-CoV-2 compared to the other sybodies tested. "In particular the interaction of Sb23 with RBD increases the melting temperature of RBD by almost ten degrees, which is considerably more than for other binders," the scientists comment in the publication. As such, Sb23 became the sybody of focus for the study.
To further understand the mechanism of Sb23's interaction with the S protein RBD, Low and colleagues collaborated with other scientists, bringing together a team of scientists highly-skilled in an array of techniques. Dmitri Svergun's group at EMBL Hamburg conducted small-angle X-ray scattering studies, whereas Martin Hällberg at CSSB and the Karolinska Institute conducted cryo-EM work to take a closer look at the structure of the S protein when bound to Sb23.
The collective results are interesting. The S RBDs are known to switch between an "up" and "down" position; in the former, they essentially stick out, and in the latter, they are curled and hidden from the human immune system. Encouragingly, Low, Svergun and Hällberg found that Sb23 binds to the RBDs in both conformations, blocking the site where the human ACE2 would typically bind.
They continue, "We can at this stage only speculate if the binding of Sb23 itself induces a conformational change that results in the “2-up” conformation or if Sb23 promotes a conformational stabilization of the “2-up” in the spike’s dynamical landscape. Clearly, this conformation makes epitopes accessible for the development of therapeutic binders in the central cavity of the spike, this includes the lower portion of the RBD and possibly also the central helical region. Hence, the use of Sb23-bound spike protein preparations may be an excellent avenue to develop novel binders in these regions," the authors note in the paper.
But what about neutralization capabilities? Löw and colleagues worked with Ben Murrell at the Karolinsta Institute to assess this. The scientists performed a neutralization assay utilizing lentiviral particles that were modified to carry the S protein on their surface. "Thirty-six sybodies were screened for neutralization, identifying 11 capable of neutralizing SARS-CoV-2 at an IC50 < 20 µg/ml," the authors write. Sb23 was found to be the most potent neutralizer, with an IC50 of 0.6 µg/ml.
What is IC50?
IC50 represents half of the maximum concentration of a molecule required to inhibit a specific biological function.
The collaborative spirit
Impressively, the research team selected the candidate sybodies and completed their analyses in a matter of weeks, which Low attributes to the spirit of the scientists involved: “The collaborative spirit has been enormous in these times, and everybody was motivated to contribute,” he commented in a press release. Samuel Pazicky – a co-author on the paper – took to Twitter to discuss the study, saying, "It was amazing to play a part in this project. Good science can progress fast! Only if non-COVID projects could move with that speed!"
The data gathered in this study warrants further exploration of Sb23's potential therapeutic value against SARS-CoV-2. But, we know that the drug development and testing process is thorough and it is long. Sb23 is merely at the beginning of this process, but Low and team conclude their publication by nodding to the potential of synthetic libraries to accelerate timelines: "Synthetic libraries are an alternative approach to rapid drug development, quickly generating highly specific binders with neutralization potential."
1. Zimmermann I, Egloff P, Hutter CA, et al. Synthetic single domain antibodies for the conformational trapping of membrane proteins. eLife. 2018;7:e34317. doi:10.7554/eLife.34317.
2. Custódio TF, Das H, Sheward DJ, et al. Selection, biophysical and structural analysis of synthetic nanobodies that effectively neutralize SARS-CoV-2. Nature Communications. 2020;11(1):5588. doi:10.1038/s41467-020-19204-y.