A Super-Potent Tetramerized ACE2 Protein Displays Enhanced Neutralization of SARS-CoV-2 Virus Infection

Abstract

Approaches are needed for therapy of the severe acute respiratory syndrome from SARS-CoV-2 coronavirus (COVID-19). Interfering with the interaction of viral antigens with the angiotensin converting enzyme 2 (ACE-2) receptor is a promising strategy by blocking the infection of the coronaviruses into human cells. We have implemented a novel protein engineering technology to produce a super-potent tetravalent form of ACE2, coupled to the human immunoglobulin g1 Fc region, using a self-assembling, tetramerization domain from p53 protein. This high molecular weight Quad protein (ACE2-Fc-TD) retains binding to the SARS-CoV-2 receptor binding spike protein and can form a complex with the spike protein plus anti-viral antibodies. The ACE2-Fc-TD acts as a powerful decoy protein that out-performs soluble monomeric and dimeric ACE2 proteins and blocks both SARS-CoV-2 pseudovirus and SARS-CoV-2 virus infection with greatly enhanced efficacy. The ACE2 tetrameric protein complex promise to be important for development as decoy therapeutic proteins against COVID-19. In contrast to monoclonal antibodies, ACE2 decoy is unlikely to be affected by mutations in SARS-CoV-2 that are beginning to appear in variant forms. In addition, ACE2 multimeric proteins will be available as therapeutic proteins should new coronaviruses appear in the future because these are likely to interact with ACE2 receptor.

A tetrameric ACE2 protein broadly neutralizes SARS-CoV-2 spike variants of concern with elevated potency

“…2 A–D). The ACE2-Fc-TD quad protein was more potent in virus neutralization than ACE2-Fc for the Wuhan virus as previously shown ( Miller et al, 2021 ) but also for neutralizing the VOC B.1.1.7 and B.1.351 as well as the D614G pseudotyped virus. Comparing the IC 50 for virus neutralization demonstrates a significant improved potency of the tetrameric ACE2 compared to the dimeric form ( Fig.…”

“…ACE-Fc-TD protein preparation was carried out as previously described ( Miller et al, 2021 ) and also commercially sourced active ACE2-Fc was used as a control (ACRO biosystems - AC2-H5257).…”

“…Sole use of inhalational therapeutics however will not address the multiorgan tropism of SARS-CoV-2 and the use of truncated ACE2 peptides may allow future mutational escape. We have recently described an engineered ACE2 fused to Fc and the p53 tetramerization domain ( Miller et al, 2021 ). This tetravalent ACE2-Fc-TD protein, termed a quad, displayed effective in vitro neutralization of pseudotyped virus displaying spike protein of the original Wuhan SARS-CoV-2 strain.…”

“…Accordingly, we have characterized the binding and pseudotyped virus neutralization capacity of a novel tetrameric form of ACE2-Fc-TD ( Miller et al, 2021 ). The binding properties of this protein was analyzed with recombinant spike proteins corresponding to those from the original SARS-CoV-2 Wuhan isolate and from B.1.1.7 (UK identified VOC) and B.1.351 (South African VOC).…”

Characterisation of a novel ACE2-based therapeutic with enhanced rather than reduced activity against SARS-CoV2 variants

“…Plain ACE2-Fc fusions were developed against SARS-CoV-1 in 2003 and was also proposed for SARS-CoV-2 38,39 . Recently, a tetravalent ACE2-Fc was described which showed improved neutralisation efficiency compared to standard ACE2-Fc formats, without ACE2 domain engineering 40 . These ACE2-Fc fusions retained catalytic activity of ACE2 and maintained full Fc effector function.…”