Shedding light on Sybody® candidates binding onto Sars-CoV-2 spike RBD

Introduction

Unlike traditional small molecule drug products, which typically exist as a single chemical entity, biologics such as antibodies, nanobodies, and other large molecules manufactured in living systems are highly complex. This makes their characterization challenging, especially since many biologic drugs exist as multiple variants, the nature and abundance of which is heavily influenced by the manufacturing process. Amid the current coronavirus pandemic, synthetic single-domain antibodies (also known as nanobodies or Sybody) could be key to the development of COVID-19 preventive treatments given their suitability for pulmonary delivery in the form of inhaled nanobody formulations.¹

Binding affinity is arguably one of the most widely studied properties of a biologic such as a Sybody molecule. But unless it is evaluated alongside other qualities of the drug, binding affinity cannot provide sufficient information to predict clinical efficacy. Comprehensive characterization in biologic drug development should also include real-time analysis of binding kinetics through accurate measurement of association and dissociation rates for the antibody-target interaction.

With no-clog microfluidics and superior sensitivity to conventional Surface Plasmon Resonance (SPR) technologies for more accurate measurement of binding affinity and binding kinetics, here we show how the Creoptix WAVE is driving biologics development by providing insights into binding affinities and selectivity of Sybody candidates to the receptor-binding domain (RBD) region of SARS-CoV-2 Spike protein.

Materials and methods

ACE2 binding kinetics

Biotinylated RBD-vYFP was captured onto a Streptavidin PCP-STA WAVEchip (polycarboxylate quasi-planar surface; Creoptix AG) to a density of 1000 pg/mm². ACE2 was then injected at increasing concentrations ranging from 6.25 nM to 400 nM (two-fold serial dilution, 7 concentrations) in TBS buffer supplemented with 0.05% Tween-20 (TBST). ACE2 was injected for 360 s at a flow rate of 15 μl/min per channel and dissociation was set to 1200 s to allow the return to baseline. Sensorgrams were recorded at 25°C and the data analyzed on the WAVEcontrol. Data were double referenced by subtracting the signals from blank injections and from the reference channel. A Langmuir 1:1 model was used for data fitting (See Figure 1). 

ACE2 competition

Selected Sybody (500 nM in TBST) alone, ACE2 (250 nM in TBST) alone or a mixture of Sybody (500 nM) and ACE2 (250 nM) was injected for 300 s and dissociation was set each time to 1200 s to ensure the return to baseline. Sensorgrams were recorded at 25°C and the data overlaid using the WAVEcontrol after double-referencing by subtracting the signals from blank injections and from the reference channel. 

Figure 1: binding kinetics of purified ACE2 onto biotinylated spike RBD

Results

Study history

Our collaboration started by receiving 57 well-behaving and unique Sybody candidates which were created by Linkster Therapeutics AG and Prof. Markus Seeger (Institute of Medical Microbiology; University of Zurich) using the Sybody platform.² All Sybody candidates were therefore subjected to an off-rate screen on the Creoptix WAVE, which identified six strong binders to Spike RBD. In agreement with ELISA data, yet providing on- and off-rates for more kinetic details, binding constants were then determined revealing affinities within 20-180 nM range.³ Given their tight binding properties, with affinities in the same range as that of ACE2/Spike interaction, we have decided to further characterize six Sybody candidates and investigate their ability to compete for ACE2 binding onto Spike RBD.

Sybody candidates compete for ACE2 binding onto Spike RBD. Since virulence of SARS-CoV-2 requires the binding of the viral Spike RBD to the human ACE2, we have assessed the ability of selected Sybody candidates to inhibit ACE2 binding onto Spike RBD, upon their co-injection. 

A Stretavidin PCP-STA WAVEchip was used to capture the biotinylated Spike RBD, and Sybody candidates and purified ACE2 were injected either alone or in combination, at the indicated concentrations. As shown in Figure 2E, ACE2 binding to Spike RBD was almost completely abolished in presence of Sybody 16,  demonstrating an exceptionally high nhibition level of ACE2-Spike interaction. This inhibition is partial in presence of Sybody 3 or Sybody 42 (Figures 2C and 2D, respectively), and completely inefficient with Sybody 67 (Figure 2B). This GCI data is in line with previously reported ELISA results (Figure 2E), with the advantage of enabling a faster characterisation of this inhibition assay. The evidence gathered using these types of analytical assays supports the observation that Sybody candidates 3; 16 and 42 recognize a surface region on the Spike RBD that overlaps with the ACE2 binding site. 

Figure 2: Sybody candidates compete for ACE2 binding onto spike RBD

Conclusion

In short, here we show how the Creoptix WAVE has enabled the rapid characterization of candidates originating from the Sybody libraries previously published, in addition to confirming insights into how these Sybody candidates inhibit the ACE2-Spike interaction.³ Providing fast and orthogonal validation of ELISA  data, the Creoptix WAVE is ideally suited to get ahead in the race to understand the COVID-19 pandemic, by accelerating the development of therapeutics against SARS-CoV-2.

Key takeaways

Understand binding dynamics for therapeutics development and generate novel data to strengthen results and publications with the Creoptix WAVE: 

• Fast and insightful inhibition assays : characterize binding and understand competition dynamics faster. 
• Validate your ELISA results : correlate data from competition assays with confidence.

Great for:

• Development of inhaled formulations
• Epitope mapping
• Orthogonal validation of ELISA data

Acknowledgments

SARS-CoV-2 biotinylated Spike RBD protein fused to Venus YFP (RBD-vYFP) and the Sybody candidates were kindly provided by Professor Markus Seeger (IMM, Zurich) and Linkster Therapeutics AG. Purified, Histagged ectodomain of human angiotensin-converting enzyme 2 (ACE2) was generously provided by Dr. Matthieu Botte (leadXpro).

References

1. van Heeke, G. et. al. 2017. Nanobodies as inhaled biotherapeutics for lung diseases. Pharmacol Ther. 169, 47-56. doi: 10.1016/j.pharmthera.2016.06.012 
2. Zimmermann, I. et al. 2018. Synthetic single domain antibodies for the conformational trapping of membrane proteins. eLife 7:e34317. doi: 10.7554/eLife.34317
3. Walter, J.D. et al. 2020. Sybodies targeting the SARS-CoV-2 receptor-binding domain. bioRxiv doi: 10.1101/2020.04.16.045419