Multifunctional ACE2-nanobody fusion design for pan-specific neutralization and cardiovascular protection in SARS coronavirus infection

Severe acute respiratory syndrome coronaviruses use the ACE2 receptor for viral entry while downregulating its activity, potentially leading to hypertension and major organ injuries. Dual-action technologies based on soluble ACE2 aimed to neutralize the virus while restoring ACE2's normal enzymatic activity. Here, we describe a novel general molecular design, VHH_Spike-ACE2_ECD-VHH_Albumin, in the toolbox of ACE2-centric therapeutic modalities. The optimized nanobody module VHH_Spike afforded strong pan-specific binding against the entire sarbecoviral clade. This correlated with potent <i>in vitro</i> neutralization of pseudotyped virus variants of concern, with IC₅₀ values in the picomolar range. Exogenous enzymatic activity was provided by the ACE2_ECD module, which also contributed binding avidity via intrinsic homodimerization. Persistence of enzymatic activity in circulation was increased <i>in vivo</i> via the nanobody module VHH_Albumin optimized for serum albumin binding. Single-dose therapeutic administration of lead compound 72opt-ACE2-R28 demonstrated virus neutralization in lungs of hamsters at day 5 post-infection with SARS-CoV-2. In hypertensive mice maintained under continuous injection of angiotensin II, a single dose of 72opt-ACE2-R28 normalized systolic blood pressure, maintaining a 30 mmHg reduction after 24 h. Overall, encouraging coronavirus neutralization and hypertension reduction showed magnitudes and timeframes appropriate for treatment of typical acute infections. We discuss molecular bases of interactions with coronavirus spike protein molecules and future scale-up manufacturability toward clinical development of this modular design scaffold with high potential against emerging SARS-CoV-2 variants.