A broadly neutralizing humanized ACE2-targeting antibody against SARS-CoV-2 variants

Abstract

The successive emergences and accelerating spread of novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lineages and evolved resistance to some ongoing clinical therapeutics increase the risks associated with the coronavirus disease 2019 (COVID-19) pandemic. An urgent intervention for broadly effective therapies to limit the morbidity and mortality of COVID-19 and future transmission events from SARS-related coronaviruses (SARSr-CoVs) is needed. Here, we isolate and humanize an angiotensin-converting enzyme-2 (ACE2)-blocking monoclonal antibody (MAb), named h11B11, which exhibits potent inhibitory activity against SARS-CoV and circulating global SARS-CoV-2 lineages. When administered therapeutically or prophylactically in the hACE2 mouse model, h11B11 alleviates and prevents SARS-CoV-2 replication and virus-induced pathological syndromes. No significant changes in blood pressure and hematology chemistry toxicology were observed after injections of multiple high dosages of h11B11 in cynomolgus monkeys. Analysis of the structures of the h11B11/ACE2 and receptor-binding domain (RBD)/ACE2 complexes shows hindrance and epitope competition of the MAb and RBD for the receptor. Together, these results suggest h11B11 as a potential therapeutic countermeasure against SARS-CoV, SARS-CoV-2, and escape variants.

Fig. 1. Characterization of hACE2-blocking MAb h11B11.

a h11B11 blocks SARS-CoV-RBD and SARS-CoV-2-RBD binding to hACE2. The quantified data were shown in the right panel. Mean values of three biological replicates ± SEM (standard error of the mean) are shown (unpaired t test, two tailed). Data are representative of three independent experiments. b, c The binding kinetics of h11B11 to wild-type and mutated hACE2 was assessed using a single-cycle model. The kinetic parameters were labeled accordingly. Values represent mean ± SEM of three independent assessments. n.d. means the values are not detectable due to poor binding ability. d Measurement of enzymatic kinetic constants of hACE2 in the presence of h11B11 and control IgG protein. Actual data points and Michaelis–Menten plots for hACE2 hydrolysis of Mca-AFK-Dnp in the absence (purple) or presence (orange, green, and blue) of h11B11 protein. Isotype IgG (yellow) and inhibitor (black) were used as control. The initial velocity conditions were limited to 12 min. The value in the x-axis of d represents the concentrations of substrate (Mca-AFK-Dnp).

Fig. 2. h11B11 effectively inhibits original coronaviruses and novel lineages of SARS-CoV-2 in vitro.

a, b Inhibiting potencies of MAb to original SARS-CoV, SARS-CoV-2, or epidemic SARS-CoV-2 variant pseudovirus were evaluated in a luciferase reporter assay. IC50 was calculated by fitting the relative fluorescence intensity of host cells from serially diluted antibodies to a sigmoidal dose–response curve. c h11B11 was tested against 34 pseudotyped SARS-CoV-2 variants emanated from GISAID. d IC50 to live SARS-CoV-2 was fit the CPE from serially diluted antibody to a sigmoidal dose–response curve after 3 days of incubation. The values in the x-axis of a, b, and d represent concentrations of the antibody. IC50 in (a–d) represents mean ± SEM (standard error of the mean) of triplicated independent assessments.

Fig. 3. The effective protection of h11B11 in ACE2 humanized mice challenged with SARS-CoV-2.

a Groups of hACE2 mice that infected SARS-CoV-2 were divided into pre-exposure, postexposure, and control groups with eight animals in each group. Before the challenge, the animals of prophylactic (one day before infection) and treatment settings (one day after challenge) were intraperitoneally infused with h11B11. Mice in the control group were given PBS as a control. SARS-CoV-2 titers in the lungs were measured by qRT-PCR with two replicates and values represent mean ± SEM (standard error of the mean) (n = 5) (unpaired t test, two tailed). b Histopathological analysis of lung samples at 5 d.p.i. (n = 3). Data are one representative result of two independent experiments. Scale bar: 100 and 200 μm.

Fig. 4. The safety of h11B11 in vitro and in cynomolgus monkeys.

a HEK293-hACE2 cells were incubated with 10 μg/ml h11B11 antibody for the indicated times, followed by western blot analysis of indicated proteins. b HEK293-hACE2 cells were incubated with 10 μg/ml h11B11 antibody for the indicated times, followed by membrane–cytoplasm fractionation analysis of indicated proteins. The antibody used for western blot in a, b was anti-ACE2 pAb. Data are one representative result of two independent experiments. c HEK293-hACE2 cells were incubated with serially diluted h11B11 antibody or anti-IgG control antibody. After incubation at 37 °C for 4 h (c) or 24 h (d), the hACE2–antibody complex was detected by flow cytometry. Mean values of three biological replicates mean ± SEM (standard error of the mean) are shown. Data are representative of three independent experiments. e, f The blood pressure of cynomolgus monkeys in low and high groups after the second dosing. 1001, 1002, 1101, and 1102 represent animal numbers. SP is the systolic pressure and DP is the diastolic pressure. Mean is the average of SP and DP. The value was tested once. g, h The hematology and serum chemistry (toxicology) assays of four animals for 3 weeks. Cynomolgus monkeys were injected 60 or 180 mg/kg h11B11 once a week for three times. WBC white blood cell, RBC red blood cell, MONO monocyte, LYMPH lymphocyte, AST aspartate transaminase, ALT alanine transaminase, TBIL total bilirubin.

Fig. 5. Structural basis of the competitive binding of h11B11 and RBDs with hACE2.

a The overall structure of h11B11-Fab and hACE2. The Fab is shown as the surface with HCDR1, HCDR2, and HCDR3 from the heavy chain (cyan) colored in red, orange, and purple, while the LCDR1, LCDR2, and LCDR3 from light chain (pink) are colored in blue, green, and gray, respectively. The hACE2 is shown as a cartoon. The N-terminal helix (NTH) is marked by a dotted line. b Superimposition of h11B11/hACE2 complex and HCoV-NL63-RBD/hACE2 (PDB:3KBH), SARS-CoV-RBD/hACE2 (PDB:2AJF), and SARS-CoV-RBD/hACE2 (PDB:6LZG) reveal the competition between h11B11 and RBDs. The ACE2, HCoV-NL63-RBD, SARS-CoV-RBD, and SARS-CoV-RBD are colored differently as indicated. c–e Competitive binding surfaces of h11B11 and RBDs on hACE2. The overlapping epitopes bound by both h11B11 and RBDs are colored in purple. The surfaces in contact with MAb are colored in yellow, while others in contact with RBDs are colored differently as indicated.