Highly potent anti-SARS-CoV-2 multivalent DARPin therapeutic candidates

Abstract

Globally accessible preventive and therapeutic molecules against SARS-CoV-2 are urgently needed. DARPin molecules are an emerging class of novel therapeutics based on naturally occurring repeat proteins (∼15 kDa in size) and can be rapidly produced in bacteria in large quantities. Here, we report the identification of 380 DARPin molecules specifically targeting the SARS-CoV-2 spike protein selected from a naïve library of 1012 DARPin molecules. Using extensive biophysical and biochemical characterization, (pseudo)virus neutralization assays and cryo-EM analysis, 11 mono-DARPin molecules targeting either the receptor binding domain (RBD), the S1 N-terminal-domain (NTD) or the S2 domain of the SARS-CoV-2 spike protein were chosen. Based on these 11 mono-DARPin molecules, 31 anti-SARS-CoV-2 multi-DARPin molecules were constructed which can broadly be grouped into 2 types; multi-paratopic RBD-neutralizing DARPin molecules and multi-mode DARPin molecules targeting simultaneously RBD, NTD and the S2 domain. Each of these multi-DARPin molecules acts by binding with 3 DARPin modules to the SARS-CoV-2 spike protein, leading to potent inhibition of SARS-CoV-2 infection down to 1 ng/ml (12 pM) and potentially providing protection against viral escape mutations. Additionally, 2 DARPin modules binding serum albumin, conferring an expected half-life of about 3 weeks in humans, were included in the multi-DARPin molecules. The protective efficacy of one multi-DARPin molecule was studied in a Golden Syrian hamster SARS-CoV-2 infection model, resulting in a significant reduction in viral load and pathogenesis. In conclusion, the multi-DARPin molecules reported here display very high antiviral potency, high-production yield, and a long systemic half-life, and thereby have the potential for single-dose use for prevention and treatment of COVID-19.

Ensovibep, a novel trispecific DARPin candidate that protects against SARS-CoV-2 variants

“…Among all tested single mutations, only F486V caused a strong decrease in potency for ensovibep, when compared to the wildtype. This finding is in line with our previous structural analysis 15 . Most importantly, F486 is also a critical residue for the virus itself, allowing binding to the ACE2 receptor and thus cell infection.…”

“…In addition, we also evaluated less frequent single point-mutations (i.e. G446V, G476S, T478I, P479S, V483A, F486V, Q493K, F490S) within the epitope region of the three RBD binders of ensovibep 15 or mutations known to impact other therapeutics 10,19,20 . Among all tested single mutations, only F486V caused a strong decrease in potency for ensovibep, when compared to the wildtype.…”

“…We have applied our DARPin® platform that allows fast generation and cost-effective production of multi-specific therapeutics to generate anti-SARS-CoV-2 multi-specific DARPin® antivirals, with very high neutralizing potencies, which were described previously 15 . In brief, DARPin® molecules are structurally completely different from antibodies and consist of a single chain of linked DARPin® domains; in this case the molecules have two N-terminal human serum albumin (HSA) binding domains for systemic half-life extension and three spike protein binding domains at the C-terminus.…”

Structural investigation of ACE2 dependent disassembly of the trimeric SARS-CoV-2 Spike glycoprotein

“…This hypothesis is supported by the recent manuscript deposited in bioRxiv.org, which describes a similar effect triggered by engineered DARPin molecules (Walser et al, 2020). Therefore, we suppose that the soluble forms of hACE2 may not only block the infection and replication of SARS-CoV-2, but also destroy the trimeric Spike adaptors that are responsible for viral host membrane fusion.…”

Coronavirus entry: how we arrived at SARS-CoV-2

“…Development of novel therapeutic strategies can often follow an understanding of virus entry pathways. As with many viruses, specific inhibition of the receptor interaction, along with less specific inhibition of membrane fusion events are logical points in virus entry to target and there are examples of each which have been studied for COVID-19 [ 58 , 59 , 60 , 61 ]. While these remain promising approaches, it is the inhibition of S protein cleavage-activation that is closest to therapeutic use in humans.…”