The COVID-19 pandemic, caused by SARS-CoV-2, presents a significant global health challenge due to the emergence of new variants and resistance to existing therapeutics. SARS-CoV-2, like other SARS-related coronaviruses (SARSr-CoVs), utilizes the angiotensin-converting enzyme 2 (ACE2) receptor for cell entry. Neutralizing antibodies (NAbs) targeting the receptor-binding domain (RBD) of the spike protein are promising therapeutic candidates, but viral mutations can lead to escape variants. Existing therapies, such as those targeting the RBD, face challenges due to the rapid accumulation of escape mutations in SARS-CoV-2. The need for broadly effective therapies to combat current and future SARSr-CoV threats is therefore paramount. Previous research demonstrated the potential of antibodies targeting ACE2 to block viral infection. However, monoclonal antibodies targeting ACE2 were not previously reported. This study focuses on the identification and characterization of such an antibody, aiming to provide a broad-spectrum antiviral intervention.

Neutralizing antibodies (NAbs) targeting the SARS-CoV-2 spike protein, particularly the receptor-binding domain (RBD), have emerged as promising therapeutics. Several studies have described the isolation and characterization of potent NAbs targeting this region. However, the rapid evolution of SARS-CoV-2, and the development of escape mutations under therapeutic pressure, pose a significant challenge. This phenomenon mirrors the escape mechanisms observed in viruses like HIV-1 and influenza, highlighting the need for broadly neutralizing antibodies that can overcome these mutations. While antibodies targeting the RBD show efficacy, their limitations in addressing emerging variants warrant exploration of alternative strategies, including targeting the ACE2 receptor itself. Studies have shown the potential of ACE2-blocking antibodies to inhibit viral entry, but a detailed characterization of a humanized ACE2-blocking monoclonal antibody with broad neutralizing activity against SARS-CoV and various SARS-CoV-2 lineages has been lacking until this study.

The study employed a multi-faceted approach. First, murine anti-hACE2 antibodies were generated by immunizing BALB/c mice with hACE2 (19-615) soluble antigens. Hybridoma technology was used to identify ACE2-blocking monoclonal antibodies, with clone h11B11 showing significant inhibitory activity. This antibody was then humanized through CDR grafting onto human acceptor germline frameworks. The binding kinetics of the humanized h11B11 and the parental antibody were compared using surface plasmon resonance (SPR). The ability of h11B11 to block SARS-CoV and SARS-CoV-2 RBD binding to hACE2 was evaluated using flow cytometry. SPR analysis determined the affinity of h11B11 to hACE2, as well as its interactions with hACE2 variants. The impact of h11B11 on the carboxypeptidase activity of ACE2 was assessed using an in vitro enzyme activity assay. The broad neutralizing activity of h11B11 was tested against a panel of 39 SARS-CoV and SARS-CoV-2 pseudoviruses, including various epidemic lineages and high-frequency mutants from GISAID. Live virus neutralization assays using SARS-CoV-2 and HCoV-NL63 were also conducted to evaluate antiviral efficacy. In vivo efficacy was assessed in hACE2 transgenic mice using both prophylactic and therapeutic administration regimens. The viral load and lung pathology were examined. Finally, the safety of h11B11 was evaluated in cynomolgus monkeys through repeated intravenous infusions at different doses, monitoring blood pressure and conducting hematology and serum biochemistry analyses. The crystal structure of the h11B11/hACE2 complex was determined to understand the mechanism of action.

The humanized monoclonal antibody h11B11 potently inhibited SARS-CoV and multiple SARS-CoV-2 lineages in vitro. It exhibited strong neutralizing activity against both pseudoviruses and live SARS-CoV-2, with an IC50 of 0.66 ± 0.03 µg/ml for live SARS-CoV-2. A combination of h11B11 and CB6 showed even greater potency (IC50 of 0.06 ± 0.01 µg/ml). h11B11 effectively protected hACE2 transgenic mice from SARS-CoV-2 infection, reducing viral loads and mitigating lung pathology in both prophylactic and therapeutic settings. Importantly, h11B11 did not impact the carboxypeptidase activity of ACE2 in vitro. High-dose injections (up to 180 mg/kg) of h11B11 in cynomolgus monkeys did not result in significant changes in blood pressure or hematology chemistry toxicology. Structural analysis revealed that h11B11 binds to the N-terminal helix (NTH) of hACE2, sterically hindering and competing with the SARS-CoV and SARS-CoV-2 RBDs for binding. The one exception was the S19P variant of hACE2, which showed complete resistance to h11B11 binding. The study also showed that h11B11 does not affect the surface expression of hACE2. Compared to Dalbavancin, which was shown in a separate study to also inhibit SARS-CoV-2 infection, h11B11 demonstrated significantly stronger antiviral activity.

The findings demonstrate that h11B11 is a potent and broadly neutralizing antibody against SARS-CoV and SARS-CoV-2, including variants with mutations known to confer resistance to other therapies. Its ability to effectively block viral entry without interfering with the essential carboxypeptidase activity of ACE2 enhances its safety profile. The preclinical data strongly support further development of h11B11 as a potential therapeutic agent for COVID-19. The observation that h11B11 binds to a relatively conserved region of ACE2, largely unaffected by common mutations in the human population, hints at its potential broad applicability. The lack of significant toxicity at high doses in cynomolgus monkeys further strengthens its potential as a safe and effective therapeutic. The results demonstrate the superior antiviral potency of h11B11 compared to Dalbavancin. The mechanistic insights obtained from structural analysis clarify how h11B11 effectively competes with the viral RBD for ACE2 binding.

This study successfully identified and characterized h11B11, a broadly neutralizing humanized ACE2-blocking monoclonal antibody with potent antiviral activity and a favorable safety profile. Its effectiveness against SARS-CoV-2 variants and its lack of toxicity in preclinical models make it a promising therapeutic candidate for COVID-19. Future research should focus on clinical trials to assess its efficacy and safety in humans and exploring potential combination therapies with other antiviral agents.

The study used a limited number of cynomolgus monkeys in the safety assessment. Although the results were encouraging, more extensive studies adhering to GLP guidelines are needed to fully characterize the safety profile. The observed resistance to h11B11 by the S19P variant of ACE2 highlights the potential for future viral mutations to overcome this therapeutic strategy. Further studies should investigate the potential emergence of h11B11-resistant variants and strategies for mitigating such resistance.