key: cord-0812302-uommxryg
authors: Rothenberger, Sylvia; Walser, Marcel; Malvezzi, Francesca; Mayor, Jennifer; Ryter, Sarah; Moreno, Hector; Liechti, Nicole; Hälg, Silvan; Bosshart, Andreas; Iss, Chloe; Calabro, Valérie; Cornelius, Andreas; Hospodarsch, Tanja; Neculcea, Alexandra; Looser, Thamar; Herrup, Rachel; Lusvarghi, Sabrina; Neerukonda, Sabari Nath; Vassell, Russell; Wang, Wei; Mangold, Susanne; Reichen, Christian; Radom, Filip; Dawson, Keith M.; Lewis, Seth; Steiner, Daniel; Weiss, Carol D.; Amstutz, Patrick; Engler, Olivier; Stumpp, Michael T.
title: Multispecific DARPin® therapeutics demonstrate very high potency against SARS-CoV-2 variants in vitro
date: 2021-05-26
journal: bioRxiv
DOI: 10.1101/2021.02.03.429164
sha: 779e411a220ef2a0e3a7a47b2b31bdd4a8649278
doc_id: 812302
cord_uid: uommxryg

The SARS-CoV-2 virus responsible for the COVID-19 pandemic has so far infected more than 160 million people globally, and continues to undergo genomic evolution. Emerging SARS-CoV-2 variants show increased infectivity and may lead to resistance against immune responses of previously immunized individuals or existing therapeutics, especially antibody-based therapies. Several monoclonal antibody therapeutics authorized for emergency use or in development have been shown to lose potency against some SARS-CoV-2 variants. Cocktails of two different monoclonal antibodies constitute a promising approach to protect against novel emerging variants as long as both antibodies are potent, but come with increased development complexity and therefore cost. As an alternative, we developed two multispecific DARPin® therapeutics, each combining three independent DARPin® domains binding the SARS-CoV-2 spike protein in one molecule, to potently neutralize the virus and overcome virus escape. Here, we show in a panel of in vitro studies that the multispecific DARPin® therapeutic design incorporated in our clinical candidate ensovibep (MP0420), achieved high neutralization potencies against the circulating SARS-CoV-2 variants B.1.1.7 (UK variant), B.1.351 (South African variant), P.1 (Brazilian variant), B.1.429 (South Californian variant), B.1.526 (New York variant), R.1 (Japanese variant), A.23.1 (Ugandan variant), and B.1.617 (Indian variant), and there is strong evidence that ensovibep also potently neutralizes the Indian variant B.1.618 based on the analysis of the key point mutations in the spike protein of this variant. Additionally, viral passaging experiments show potent protection by ensovibep and MP0423 against development of escape mutations. Finally, we demonstrate that the cooperative binding of the individual modules in a multispecific DARPin® antiviral is key for potent virus inhibition and protection from escape variants. These results, combined with the relatively small size and high production yields of DARPin® molecules, suggest that ensovibep is a highly valuable alternative to monoclonal antibody cocktails for global supply and demonstrate the strength of the DARPin® platform for achieving potent and lasting virus inhibition for SARS-CoV-2 and possibly other viruses.

As the COVID-19 pandemic progresses, new circulating variants of SARS-CoV-2 have been described 57 that show resistance against the immune response of previously immunized individuals 1,2 or available 58 therapeutics 3,4 . In many regions around the globe, governments are implementing lock-down measures 59 to contain rapidly spreading SARS-CoV-2 spike variants associated with increased transmissibility 5 . independently. Amongst those, mutations K417E/N/T, L452R, E484K and N501Y are of major concern, 64 as these residues are associated with an enforced receptor binding 7 , and thus increased transmissibility 65 and/or immune escape of the virus 3, [8] [9] [10] [11] . 66

Other mutations confer resistance against monoclonal antibody therapeutics, including N439K, where 67 independent lineages were originally detected in Scotland, subsequently in Romania and then spread 68 in many countries such as the USA 3 . Further amino acid changes, which may reduce therapeutic options 69 are Y453F, which evolved in Danish mink farms 12 , and less common mutations, such as G446V, G476S, 70 T478I, P479S, V483A, F486V, F490S and Q493K, originally described by Baum et al. 13 . Additional 71 mutations which occur globally at high frequencies such as A222V, S477N, D614G and Q675H 14-16 are 72 considered less of a concern to date. 73

To circumvent the loss of potency of single monoclonal neutralizing antibodies, antibody cocktails were 74 generated, providing a higher resistance against escape mutations 13,17,18 . However, this strategy is 75 associated with increased manufacturing costs and limited global supply capacities 19 . 76

We have applied our DARPin® platform that allows fast generation and cost-effective production of 77 multispecific therapeutics to generate anti-SARS-CoV-2 multispecific DARPin® antivirals, with very high 78 neutralizing potencies, which were described previously 20 . In brief, DARPin® molecules are structurally 79 completely different from antibodies and consist of a single chain of linked DARPin® domains; in this 80 selective pressure through passage 2 to 4, the monovalent DARPin® binder RBD-2, and the individual 167 monoclonal antibodies S309 and REGN10933 lost the capacity to protect cells, which manifested in 168 complete CPE up to the highest concentration tested. In contrast, ensovibep and MP0423, as single 169 molecules or in combination, and the cocktail of two monoclonal antibodies remained effective and 170 protected cells from CPE throughout the 4 passages. 171

While the application of ensovibep and MP0423 as single agents required higher concentrations, 172 respectively 2 µg/mL and 10 µg/mL, after 4 passages, the combination of the two DARPin® antivirals 173 Novel emerging variants of the virus are an ever-present threat, as they may escape the antibodies 186 generated during immunization or developed as therapeutics. 187

Here, we showed that our multispecific DARPin® clinical candidate ensovibep gives high levels of 188 protection against the currently most frequently reported SARS-CoV-2 mutations as well as recently 189 identified variants. Neutralization tests using VSV-based as well as lentivirus-based pseudoviruses 190 carrying SARS-CoV-2 spike variants as well as infectious SARS-CoV-2 showed inhibitory potencies for 191 ensovibep that were comparable to that for the corresponding wild type. While MP0423 also remained 192 active against some tested variants, a partial potency decrease was observed for most of the tested 193 variants of concern. Of note, this lowered potency is still in an active potency range described by many 194 monoclonal antibodies. 195

In the present study we investigated a panel of single mutations, shown to be of concern because they 196 may be associated with increased transmissibility, or have an impact on disease severity, or affect 197 neutralization by some polyclonal or monoclonal antibodies 13,25,26 . Among all mutations tested, only 198 F486V caused a strong decrease in potency for ensovibep, when compared to the wild-type. In 199 accordance, deep sequencing of the viral passaging supernatant for the monovalent RBD binder (RBD-200 2, incorporated in ensovibep) also revealed F486L as a potential mutation for the virus to escape the 201 treatment (Suppl. Fig. 4 ). These findings are in line with our previous structural analysis 20 showing that 202 F486 is one of the key binding residues for the interaction with ensovibep and the RBD. Most 203 importantly, F486 is a critical residue for the virus itself, allowing an efficient binding to the ACE2 204 receptor and thus cell infection. Therefore, mutations of the phenylalanine at position 486 will decrease 205 the affinity between the RBD and human ACE2 receptor and lower the infectivity of the virus 10,27-29 . To 206 date, mutations at position F486 are not present at high frequency in circulating SARS-CoV-2 strain. 207

Interestingly, the single point mutation F486V does not affect the neutralizing capacity of MP0423, 208 despite the fact that it contains a RBD binder for which F486 is a critical residue. These findings, 209 summarized in Suppl. Fig.2 , highlight the strength of the tri-epitopic DARPin design. In this approach, a 210 loss of binding of one DARPin domain might be compensated by the binding via the two remaining 211

DARPin domains of the molecule. 212

In the same line, some mutations, that are not predicted as key interaction residues for the three 213 distinct RBD binders of ensovibep (e.g. E484K or Q493K), led to a reduction of the potency in one or 214 several of the RBD binders, while the trispecific ensovibep molecule maintained the full neutralization 215 domain is evaluated separately. This cooperativity is an exclusive feature of a trispecific approach, 218 highlighting a key advantage of the multispecific DARPin® design over cocktails of antibodies. 219

The high level of protection against viral escape mutations by ensovibep and MP0423 was further 220 demonstrated in a viral passaging experiment: the single monoclonal antibodies and the monovalent-221

DARPin® RBD-2 binder were rapidly overcome by escape mutants, whereas both multispecific DARPin® 222 antivirals, individually or in combination, maintained potency up to passage 4, to an extent comparable 223 to one of the best monoclonal antibody cocktails with emergency use authorization (EUA) status in the 224

The high level of neutralization of multispecific DARPin® antivirals against mutations and variants 226 evaluated in this study encourages the characterizations of future upcoming mutations and will remain 227 an ongoing topic for COVID-19 diagnostics, therapeutics and vaccines. 228

Pathobiology, Vetsuisse Faculty, University of Bern, CH-3012 Bern, Switzerland). 232

The expression plasmid for the SARS-CoV-2 spike protein was kindly provided by Dr. Giulia Single mutations of the spike protein were generated via two PCR fragments of the spike ORF using 329 high-fidelity Phusion polymerase (New England Biolabs, USA). The first fragment was generated via a 330 generic forward primer (pCAGGS-5) annealing upstream of the spike ORF and the specific reverse 331 primer encoding the mutation. The second fragment was generated using the specific forward primer 332 encoding the mutation and a reverse primer (rbglobpA-R). The two fragments were gel-purified and 333 used as input for an assembly PCR without addition of flanking primers. 334

For multi-mutation spike proteins, a complementary pair of primers (forward and reverse) encoding 335 each mutation was designed. Fragment 1 was generated with forward primer pCAGGS-5 and reverse 336 primer encoding mutation 1. Fragment 2 was generated using forward primer encoding mutation 1 and 337 reverse primer encoding mutation 2. All subsequent fragments were generated analogously. DNA 338 fragments were gel-purified and mixed in equimolar amounts. This mix was used for re-assembly of the 339 full spike ORF using outer primers pCAGGS-5 and rbglobpA-R. 340

For both single as well as multi-mutation spike protein, the full-length spike ORF was isolated from an 341 The neutralizing activity of therapeutic antibodies against SARS-COV-2 variants was measured using 363 lentiviral particles pseudotyped with spike proteins of SARS-COV-2 variants, as previously described 35 . Table 1 . Reported is the mean +/-SEM (standard error of the mean). A) VSV-based pseudovirus assay on variants carrying mul�ple spike muta�ons as specified in Table 1 ; B) VSV-based pseudovirus assay on single muta�ons present in different variants; C) VSV-based pseudovirus assay on single muta�ons present in high frequency, reported as resistance muta�ons for other therapeu�cs or located within the RBD epitope; D) Len�virus-based pseudovirus assay on variants carrying mul�ple spike muta�ons as specifed in Table 1 ; F) Len�virus-based pseudovirus assay on single muta�ons present in different variants, analyzed in a D614G background; F) SARS-CoV-2 neutraliza�on assay on the globally most diffused variants B.1.1.7, B.1.351 and P.1 compared to wild type. 

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The authors thank Dr. Gert Zimmer for the gift of the recombinant VSV (Institute of Virology and 230 Immunology (IVI), CH-3147 Mittelhäusern, Switzerland, Department of Infectious Diseases and

Titer-Glo and real-time Virus neutralization capacity of mono-valent DARPin® candidate and multispecific DARPin® molecules 463 was determined for 100 TCID50 SARS-CoV-2 wild-type French isolate (with the following differences to 464 the Wuhan wild-type: V367F; E990A) by measuring ATP levels of protected cells in a cell viability assay.