An ACE2-IgG4 Fc Fusion Protein Demonstrates Strong Binding to All Tested SARS-CoV-2 Variants and Reduced Lung Inflammation in Animal Models of SARS-CoV-2 and Influenza

Background: The continued emergence of SARS-CoV-2 variants has caused concern that a constantly evolving virus will escape vaccines and antibody therapies. New approaches are needed. Methods: We created and manufactured an ACE2 extracellular domain (ECD) fragment Fc fusion drug candidate, G921, and engineered the compound for enhanced delivery of drug to peripheral tissues by minimizing the size of the ACE2 ECD and by incorporating an Fc domain to enhance transcytosis. G921 was assessed for binding, neutralization, in vivo anti-inflammatory effect, and pharmacokinetic profile. Results: G921 was expressed as an IgG4 Fc fusion protein presenting two ACE2 domains to ACE2 ligands while avoiding risk of infection via antibody-dependent enhancement. G921 strongly binds to the SARS-CoV-2 Wuhan-Hu-1 spike protein and demonstrates further diminished off rate to the spike protein from each of the currently identified variants of concern. G921 demonstrates ACE2 enzymatic activity comparable to positive control and binding to the neonatal Fc receptor (FcRn) without binding to low affinity Fc-gamma receptors (FcγRs). G921 is effective in a concentration-dependent manner in a focus reduction neutralization assay with EC50=16.3±4.2 µg/mL without cytotoxicity in Vero E6 cells when tested at 200 µg/mL in an MTS cell proliferation assay. G921 demonstrates statistically significant reduction of lung inflammation in relevant models of both SARS-CoV-2 and influenza. The pharmacokinetic profile demonstrated dose-dependent exposure with a multi-day half-life in monkeys and rats. Conclusion: G921 data are consistent with both antiviral and anti-inflammatory modes of action. G921 is a novel approach for the prevention and treatment of COVID-19 and possible other diseases characterized by deficiency of ACE2.

There is moderate bronchointerstitial pneumonia similar in histology to infected + PBS control animals but less extensive, with interstitial and peribronchiolar involvement. Areas of inflammation are smaller, and emphysema is present but is less frequent, and less severe as in this image. Areas of normal lung tissue can often be seen between areas of inflammation which differs from PBS control. (D) Quantitation of histology data (mean +/-SEM). G921 treated group demonstrates reduced lung inflammation area relative to infected animal treated with PBS (P=0.0169 student t test).

G921 Cloning and Protein Expression
Codon optimized DNA sequence encoding G921 containing amino acid 19-615 of the ACE2 extracellular domain with a leader peptide from murine light chain and directly fused to the IgG4 Fc at the hinge domain was cloned into expression vector pXLG6. A stable CHO cell pool expressing G921 was established from which a stable cell line clone was isolated and employed for G921 expression (ExcellGene SA, Monthey, Switzerland). G921 was purified from stable cell line supernatant using a combination of protein A affinity and ion exchange chromatography. Non-reduced and reduced SDS-PAGE analysis of G921 was performed using NuPAGE 3-8% Tris-Acetate and 4-12% Bis-Tris SDS gels respectively. Analytical size exclusion chromatography was done on an Agilent Bio SEC-3, 300Å column in 50 mM MES, 0.2M Arginine pH 6.0. Dynamic light scattering studies using a Stunner instrument (Unchained Labs, Pleasanton, CA) confirmed that G921 has a molecular weight consistent with a homodimeric molecule.

Cynomolgus Monkey pk study
Non-compartmental analysis of G921 concentrations in serum was performed by using the Phoenix WinNonlin 6.4 software. Maximum G921 serum concentration was reached 24 hours post subcutaneous dose or immediately post end of IV infusion. G921 serum concentrations declined at an estimated t1/2 of 44.1 and 41.0 hours in the male and female, respectively when administered by subcutaneous injection, and at an estimated t1/2 of 44.7 to 79.7 hours in males and 47.6 to 73.4 hours in females when administered via IV infusion. Over the dose range, exposure to G921 increased dosedependently and in a more-than dose proportional manner in both sexes and both routes of administration. There were no evident sex-related differences in any of the measured pharmacokinetic parameters. When comparing the subcutaneous versus intravenous infusion routes of administration at 100 mg/kg, systemic exposure from IV infusion route animals were 3.3 to 18.8fold higher than from subcutaneous route animals

ACE2 Enzymatic Activity
In vitro ACE2 enzymatic activity was assessed using the Biovision ACE2 Activity Assay Kit (cat #K897-100). ACE2 cleaves a synthetic MCA fluorophore-based peptide substrate to release a free fluorophore which is quantified using a fluorescence microplate reader (Molecular Devices SpectraMax iD3). The MCA peptide substrate contains a highly fluorescent 7-methoxycoumarin group that is efficiently quenched by resonance energy transfer to the 2,4-dinitrophenyl group as part of the intact peptide and released after cleavage.

FcγR and Neonatal Receptor (FcRn) Binding
Binding assessment was performed in 1X kinetics binding buffer ( followed by baseline at pH 6.0, association with protein at pH 6.0 and dissociation at pH 6.0.

Efficacy Testing in Influenza virus model
Mice were lightly anesthetized by isoflurane. For intranasal inoculation of influenza virus, A/PR/8/34 H1N1, 50 µL of the dose 6.1 Lg EID50 was instilled into the nares in one bolus (25 µL/nare) on Day 0. Mice were then moved to their home cage to recover until fully awake. Sham mice were given saline intranasally.
Test article or vehicle at the same volume was administrated on Day 3 via IV route (40mg/kg). A daily maintenance dose was delivered via SC route on Days 4-8 (20mg/kg). Lungs harvested on day 7 for groups to be analyzed by histology. On Day 7, lungs from the vehicle and G921 treated groups were fixed in 10% formalin, embedded, and stained for H&E. The inflamed fraction of diseased lung in affected animals was scored as described.