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[Preprint]. 2021 Sep 10:2021.09.10.459744.
doi: 10.1101/2021.09.10.459744.

Clinical grade ACE2 as a universal agent to block SARS-CoV-2 variants

Gerald Wirnsberger  1 , Vanessa Monteil  2 , Brett Eaton  3 , Elena Postnikova  3 , Michael Murphy  3 , Benedict Braunsfeld  4 , Ian Crozier  3 , Franz Kricek  5 , Janine Niederhöfer  1 , Alice Schwarzböck  1 , Helene Breid  1 , Anna Sanchez Jimenez  1 , Agnes Bugajska-Schretter  1 , Alexander Dohnal  1 , Christine Ruf  5 , Romana Gugenberger  1 , Astrid Hagelkruys  6 , Nuria Montserrat  7   8 , Michael R Holbrook  3 , Chris Oostenbrink  4 , Robert H Shoemaker  9 , Ali Mirazimi  2 , Josef M Penninger  6   10
Affiliations

Clinical grade ACE2 as a universal agent to block SARS-CoV-2 variants

Gerald Wirnsberger et al. bioRxiv. .

Update in

  • Clinical grade ACE2 as a universal agent to block SARS-CoV-2 variants.
    Monteil V, Eaton B, Postnikova E, Murphy M, Braunsfeld B, Crozier I, Kricek F, Niederhöfer J, Schwarzböck A, Breid H, Devignot S, Klingström J, Thålin C, Kellner MJ, Christ W, Havervall S, Mereiter S, Knapp S, Sanchez Jimenez A, Bugajska-Schretter A, Dohnal A, Ruf C, Gugenberger R, Hagelkruys A, Montserrat N, Kozieradzki I, Hasan Ali O, Stadlmann J, Holbrook MR, Schmaljohn C, Oostenbrink C, Shoemaker RH, Mirazimi A, Wirnsberger G, Penninger JM. Monteil V, et al. EMBO Mol Med. 2022 Aug 8;14(8):e15230. doi: 10.15252/emmm.202115230. Epub 2022 Jul 4. EMBO Mol Med. 2022. PMID: 35781796 Free PMC article.

Abstract

The recent emergence of multiple SARS-CoV-2 variants has caused considerable concern due to reduced vaccine efficacy and escape from neutralizing antibody therapeutics. It is therefore paramount to develop therapeutic strategies that inhibit all known and future SARS-CoV-2 variants. Here we report that all SARS-CoV-2 variants analyzed, including variants of concern (VOC) Alpha, Beta, Gamma, and Delta, exhibit enhanced binding affinity to clinical grade and phase 2 tested recombinant human soluble ACE2 (APN01). Importantly, soluble ACE2 neutralized infection of VeroE6 cells and human lung epithelial cells by multiple VOC strains with markedly enhanced potency when compared to reference SARS-CoV-2 isolates. Effective inhibition of infections with SARS-CoV-2 variants was validated and confirmed in two independent laboratories. These data show that SARS-CoV-2 variants that have emerged around the world, including current VOC and several variants of interest, can be inhibited by soluble ACE2, providing proof of principle of a pan-SARS-CoV-2 therapeutic.

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Conflict of interest statement

Competing interests: J.M.P. declares a conflict of interest as a founder and shareholder of Apeiron Biologics. G.W., R.G., A.S., J.N, A.S.J, and H.B. are employees of Apeiron Biologics. Apeiron holds a patent on the use of ACE2 for the treatment of various diseases and is currently testing APN01 (soluble recombinant human ACE2) for treatment in COVID-19. The other authors do not declare any conflict of interest.

Figures

Figure 1.
Figure 1.. Increased affinity of APN01 interactions with SARS-CoV-2-RBD variants.
(a) Schematic depicts structure of the SARS-CoV-2 spike protein S1 domain. Indicated is the amino terminal domain (NTD), the receptor binding domain (RBD) in blue and within the RBD the receptor binding motif (RBM) in purple. Numbers above depict domain boundaries. Mutations within the RBD/RBM are indicated below with observed amino acid exchanges. Shown in red are mutations observed in Variants of Concern (VOC). (b) PyMOL rendered visualization of the SARS-CoV-2 RBD. Rendering depicts the SARS-CoV-2 RBD with mutation sites shown in green. (c) ELISA analysis showing the binding strength of SARS-CoV-2 RBD carrying the indicated mutations to APN01. Axis labels indicate the SARS-CoV-2 RBD variant substitutions tested. (d) Surface Plasmon Resonance analysis to derive kinetic constants (ka, kd) and affinity values (KD) of SARS-CoV-2 RBD/APN01 interaction. The table lists both the tested variants and the introduced amino acid substitution as well as the designation of the respective Variants of Concern mutations tested in this study. Reference strain RBD sequence corresponds to the Wuhan SARS-CoV-2 isolate (e) Representative SPR sensorgram images for the SARS-CoV-2 RBD/APN01 interaction.
Figure 2.
Figure 2.. Increased binding affinity of APN01 to full-length pre-fusion trimeric Spike proteins from SARS-CoV-2 variants of concern.
(a) PyMOL rendering of the trimeric full-length SARS-CoV-2 Spike protein. One RBD is shown in red. Indicated in green are positions mutated in the various strains of SARS-CoV-2 used in experiments in this study. Depicted in yellow are the glycan-modifications of the spike protein. (b) Table lists the SARS-CoV-2 strains used in this study (c) Representative sensorgram images for the SPR analysis conducted with full-length trimeric spike proteins in pre-fusion state with APN01. Reference strain corresponds to original Wuhan viral isolate spike sequence. Indicated are VOC Alpha, Beta, Gamma, and Delta, as well as the variant Kappa. (d) Tables listing ka, kd, as well as KD values for the interaction of APN01 and full-length trimeric spike proteins. Values are derived from calculations based upon the Langmuir (upper table) or Bivalent Analyte sensorgram fitting (lower table).
Figure 3.
Figure 3.. Increased neutralisation potency of APN01 towards SARS-CoV-2 variants.
(a) Table depicts source of the tested viral isolates, as well as multiplicity of infection (MOI) and the infection time used in these experiments for both VeroE6 and Calu-3 cells. (b) Panels depict both neutralization of the indicated SARS-CoV-2 isolates (blue line) as well as cytotoxicity of APN01 (red line) in VeroE6 cells. Analysis was done in quadruplicate with mean and standard deviations shown. Y-axis depicts the percentage of neutralization and cytotoxicity, respectively. (c) Table depicts IC50 and IC90 values for APN01 mediated neutralization of viral infection in VeroE6 and Calu-3 cells. (d) Same as (a) but conducted with epithelial lung cancer cell line Calu-3.
Figure 4.
Figure 4.. Increased potency of APN01 against SARS-CoV-2 VOCs.
(a,b) Diagrams depict the level of infection with the indicated SARS-CoV-2 isolates at MOI 0.01 (a) and MOI 1 (b) of VeroE6 cells in the presence of increasing concentrations of APN01 as compared to infections in the absence of APN01. Shown are means of triplicate analyses with standard deviations. Statistical significance is indicated by asterisks (p-value < 0.05: *; p-value < 0.001: *** as calculated with one-way ANOVA) (c) List and source of strains used at the Karolinska Institutet and IC50 and IC90 values obtained for the indicated MOIs. See Materials and Methods section for a detailed list of viral mutations for the strains used.
See this image and copyright information in PMC

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