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)

Crystal structures of HER3 extracellular domain 4 in complex with the designed ankyrin-repeat protein D5

The members of the human epidermal growth factor receptor (HER) family are among the most intensely studied oncological targets. HER3 (ErbB3), which had long been neglected, has emerged as a key oncogene, regulating the activity of other receptors and being involved in progression and tumor escape in multiple types of cancer. Designed ankyrin-repeat proteins (DARPins) serve as antibody mimetics that have proven to be useful in the clinic, in diagnostics and in research. DARPins have previously been selected against EGFR (HER1), HER2 and HER4. In particular, their combination into bivalent binders that separate or lock receptors in their inactive conformation has proved to be a promising strategy for the design of potent anticancer therapeutics. Here, the selection of DARPins targeting extracellular domain 4 of HER3 (HER3d4) is described. One of the selected DARPins, D5, in complex with HER3d4 crystallized in two closely related crystal forms that diffracted to 2.3 and 2.0 Å resolution, respectively. The DARPin D5 epitope comprises HER3d4 residues 568-577. These residues also contribute to interactions within the tethered (inactive) and extended (active) conformations of the extracellular domain of HER3

RMSD from the reference experimental structure as a function of time along simulations at 303 K for the models of Efb-C:C3d complex.

<p>Model r1 (blue) is close to the experimental structure and stays stable throughout the simulation. The trajectory starting from model r18 (orange) converges to the correct structure after about 30 ns.</p

RMSD (for Cα atoms) from the reference experimental structure as a function of time along simulations at 303 K for the G3:HER2_IV complex.

<p>Simulations starting from model r37 or from the experimental structure explore a narrow range of conformations close to the experimental structure. The simulation starting from model r44 converges to the correct structure after about 50 ns, suggesting that, despite the remarkable structural difference from the correct structure, model r44 is within the native basin of the free energy surface. All the other trajectories do not lead to the correct state after 32 ns simulation. A simulation started from the experimental structure is also shown: the RMSD fluctuates around 1 Å indicating high rigidity of the complex.</p

Model r37 (blue) and r44 (orange) of G3:HER2_IV show similarity at the C-terminal region.

<p><b>(A)</b> Model overview <b>(B)</b> Hydrophobic interactions between F112 and a patch formed by F555 and V563 are conserved in both models. This works as an anchor that allows a pivoting to the correct pose. <b>(C)</b> The movement (indicated with an arrow) is facilitated by the additional hydrophobic contacts from I79 and F81 that slide around F555.</p

Comparison of simulations from different models of complex Efb-C:C3d.

<p><b>(A)</b> RMSD (Cα) from the respective initial structure for the highest ranking RosettaDock models of complex Efb-C:C3d as a function of simulation time; simulations were performed at 303 K; highlighted in black is the simulation starting from the experimental structure, and in blue and orange from two selected models (also shown in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1006182#pcbi.1006182.g006" target="_blank">Fig 6</a>); in grey are shown all simulations starting from the 20 highest scoring models. <b>(B)</b> RMSD after 40 ns simulation plotted versus the RMSD deviation of the model from the crystal structure; simulations started from the RosettaDock models at two different temperatures; at the higher temperature (300 and 340K).</p

Root-mean-square deviation from the position of Cα atoms in the respective models of G3:HER2_IV complex as a function of simulation time.

<p>While some models rapidly move away from the initial pose, others only deviate from the initial structure when temperature is increased. The only model that even after 20 ns simulation at a temperature of 390 K remains close to the initial structure is model r37, that is, the one closest to the correct structure.</p