The trispecific DARPin ensovibep inhibits diverse SARS-CoV-2 variants

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with potential resistance to existing drugs emphasizes the need for new therapeutic modalities with broad variant activity. Here we show that ensovibep, a trispecific DARPin (designed ankyrin repeat protein) clinical candidate, can engage the three units of the spike protein trimer of SARS-CoV-2 and inhibit ACE2 binding with high potency, as revealed by cryo-electron microscopy analysis. The cooperative binding together with the complementarity of the three DARPin modules enable ensovibep to inhibit frequent SARS-CoV-2 variants, including Omicron sublineages BA.1 and BA.2. In Roborovski dwarf hamsters infected with SARS-CoV-2, ensovibep reduced fatality similarly to a standard-of-care monoclonal antibody (mAb) cocktail. When used as a single agent in viral passaging experiments in vitro, ensovibep reduced the emergence of escape mutations in a similar fashion to the same mAb cocktail. These results support further clinical evaluation of ensovibep as a broad variant alternative to existing targeted therapies for Coronavirus Disease 2019 (COVID-19)

Modelling of flow conditions in the microfluidic chip.

<p>(<b>a</b>) represents the velocity field at half the height of the inlet channel. (<b>b</b>) represents the velocity field 15 um above the culture plane (ACP). (<b>c</b>) shows velocity profiles at x₀ along the z-axis.</p

Staining of hESC colonies following perfusion culture.

<p>Representative images of the feeder cells and hESC colonies in the culture device after 2 days of continuous perfusion culture. Each row shows the phase contrast images (<b>a</b>, <b>e</b>) of the feeder-attached hESC colonies and the corresponding results from DAPI (<b>b</b>, <b>f</b>,) and pluripotency marker staining for Oct-4 (<b>c</b>), Tra-1-81 (<b>d</b>) and SSEA-3 (<b>g</b>). All images were taken with a 20× objective, scale bar is 200 µm.</p

Monitoring a hESC colony in the microfabricated culture device during the course of an experiment.

<p>The same colony is shown after (a) 1 day static culture, (b) 1 day perfused culture and (c) 2 days perfused culture. The columns show, from left to right, the raw phase contrast image taken with a 4× objective, an overlay of the automated detection using the image processing algorithm, and the detected area. The scale bars are 500 µm.</p

Design of the microfabricated culture device.

<p>(<b>a</b>) Exploded view showing all parts of the modular microfabricated culture device. (<b>b</b>) Schematic representation of a longitudinal section of the interconnect assembly, showing compression of the PDMS chip around the inlet/outlet ports (dashed rectangle), by the interconnect. (<b>c</b>) Top view of the microfluidic chip with dashed lines showing the footprints of the lid and interconnect bosses. (<b>d</b>) Cross-sectional view showing the two PDMS layers of the microfluidic chip. The lower ‘spacer’ layer elevates the flow equalisation barriers of the top layer and thus reduces the hydrodynamic shear exposure for the cells.</p

Schematic representation of the continuous media perfusion setup.

<p>A syringe pump is used to pump media through gas-permeable tubing to adjust gaseous tension levels before entering the culture device.</p

Quantification of hESC colony size.

<p>Due to the time required to image larger culture areas, only the central area of the control dish, which contained the majority of colonies, was imaged. The numbers of distinct colonies shrinks as nearby colonies grow into each other.</p

Photograph of the assembled modular culture device with the re-sealable lid attached.

<p>Photograph of the assembled modular culture device with the re-sealable lid attached.</p

Co-cultured hESCs in microfabricated culture device and control dish.

<p>Representative phase contrast images of iMEF feeder cells and individual hESC colonies cultured in the microfabricated culture device (<b>a-c</b>) and in the control dishes (<b>d-f</b>). The same two colonies are shown at each of three time points; after 1 day of static culture (a, d), after 1 day of the perfused culture (b, e), and at the end of the 2 days of perfused culture (c, f). All images were taken with a 4× objective, scale bar is 500 µm.</p

High-throughput single-cell ChIP-seq identifies heterogeneity of chromatin states in breast cancer

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