Background
Many neutralizing monoclonal antibodies capable of providing sterilizing immunity against SARS-CoV-2 have been developed and temporarily approved for emergency clinical use as prophylactics. However, the rapid evolution of SARS-CoV-2 progressively renders these treatments obsolete. Several groups have been exploring the use of ACE2-based Fc fusion proteins to obtain a pan-variant neutralizing molecule. In this work we designed ACE2-Fc candidates effective (IC50<100pM) against all variant tested.
Methods
A catalytically inactivated ACE2 peptidase domain was fused to the N-terminal end of a human IgG1 Fc, either directly or via a flexible linker. In silico predicted point mutations were introduced into the ACE2 sequence to increase both spike receptor binding domain (RBD) affinity and protein stability. Using pseudovirus and full virus-based neutralization assays, all candidates were functionally validated and compared to the monoclonal antibody Sipavibart (AZD3152), against a large panel of pre-Omicron and Omicron-derived variants, including JN.1 and KP.2.
Results
We identified 3 points of improvement: (1) the addition of the flexible linker which systematically improved both the production (up to 10-fold) and neutralizing activity (5-fold); (2) a combination of four ACE2 mutations which, benefited the neutralizing activity (10-fold); (3) the addition of a second ACE2 at the Fc C-terminal end, to generate a tetrameric protein, greatly improved the potency (117-fold). The combination of the linker and binding mutations synergized to increase the neutralizing activity (27-fold) but did not improve the potency of the tetrameric molecule suggesting that its activity was already optimal. Finally, while Sipavibart showed loss of activity against JN.1 and KP.2 variants (IC50 of 660 pM and >3456 pM, respectively), our ACE2 molecules demonstrated increased potency against these latest variants (IC50 down to 12 pM and 4 pM, respectively).
Conclusions
Our results demonstrate the relevance of ACE2 recombinant proteins to neutralize all SARS-CoV-2 variants. While the development and validation of traditional monoclonal antibodies is long and costly. ACE2-Fc molecules could provide an off-the-shelf solution ready to protect the immunocompromised and other risk groups against the rapidly evolving SARS-CoV-2 variants and any coronaviruses using ACE2 as receptor.
Table or Figure
