A human antibody of potent efficacy against SARS-CoV-2 in rhesus macaques showed strong blocking activity to B.1.351

Abstract

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), which causes coronavirus disease-2019 (COVID-19), interacts with the host cell receptor angiotensin-converting enzyme 2 (hACE2) via its spike 1 protein during infection. After the virus sequence was published, we identified two potent antibodies against the SARS-CoV-2 receptor binding domain (RBD) from antibody libraries using a phage-to-yeast (PtY) display platform in only 10 days. Our lead antibody JMB2002, now in a Phase 1 clinical trial (ChiCTR2100042150), showed broad-spectrum in vitro blocking activity against hACE2 binding to the RBD of multiple SARS-CoV-2 variants, including B.1.351 that was reportedly much more resistant to neutralization by convalescent plasma, vaccine sera and some clinical-stage neutralizing antibodies. Furthermore, JMB2002 has demonstrated complete prophylactic and potent therapeutic efficacy in a rhesus macaque disease model. Prophylactic and therapeutic countermeasure intervention of SARS-CoV-2 using JMB2002 would likely slow down the transmission of currently emerged SARS-CoV-2 variants and result in more efficient control of the COVID-19 pandemic.

Keywords: B.1.1.7; B.1.351; D614G; JMB2002; SARS-CoV-2; broad-spectrum; neutralizing antibody; phage-to-yeast; rhesus macaque disease model.

Figure 1.

Identification of neutralizing antibodies with a PtY display platform. We first used our preconstructed naïve phage displayed human scFv library to screen binders with biotinylated SARS-CoV-2 RBD protein in the solution phase. After enrichment of phage binders, the scFv DNA from enriched binders was cloned into the yeast display plasmid, resulting in display of scFv on the yeast cell surface. We then performed FACS to isolate potential blocking antibodies that could prevent binding of the SARS-CoV-2 RBD to hACE2. The 0.013% gate contained blocking antibodies with high affinity toward RBD. That is, higher Y axis signal represented higher affinity to labeled RBD, whereas lower X signal represented higher potency in blocking the binding of differently labeled hACE2 to RBD. The potential blocking antibodies were sent for sequencing and transient expression. The purified antibodies were evaluated for affinity, blocking activity, biophysical properties, and virus-neutralizing activity

Figure 2.

Characterization of potential blocking antibodies. (a) Blocking assay was performed by immobilizing 1 µg/ml hACE2 on a plate. Serially diluted antibodies and biotinylated SARS-CoV-2 RBD protein were added for competitive binding to hACE2. IC₅₀ values were calculated with Prism V8.0 software using a four-parameter logistic curve fitting approach. (b) Epitope binning was carried out by BLI. Biotinylated SARS-CoV-2 RBD was immobilized onto the SA sensor, and a high concentration of the primary antibody was used to saturate its own binding site. Subsequently, a second antibody was applied to compete for the binding site on the SARS-CoV-2 RBD protein. Data were analyzed with Octet Data Analysis HT 11.0 software. (c) Neutralization activities of Ab2001.08 and Ab2001.10 were assessed by live virus assay. Live SARS-CoV-2 and serially diluted (3-fold) antibodies were added to VERO E6 cells. The PRNT₅₀ values were determined by plotting the plaque number (neutralization percentage) against the log antibody concentration in Prism V8.0 software

Figure 3.

Effects of Fc modification on the ADE activity of JMB2002. (a) Binding of Ab2001.08 and JMB2002 to FcγRs was determined by BLI. His-tagged FcγR was loaded onto the HIS1K sensor, and serially diluted antibodies bound to the receptor on the biosensor. K_D values were determined with Octet Data Analysis HT 11.0 software using a 1:1 global fit model. (b-d) ADE activity was measured using a pseudotyped SARS-CoV-2 system containing a luciferase reporter. Pseudotyped viruses were preincubated with serially diluted antibodies for 1 h. The mixture was added to FcγR-expressing cells and incubated at 37°C for 20–28 h. Infection of cells with pseudotyped SARS-CoV-2 was assessed by measuring cell-associated luciferase activity. Trastuzumab was used as the irrelevant IgG control

Figure 4.

Characterization of JMB2002. Binding affinity of JMB2002 for the SARS-CoV-2 RBD (a)/S1 (b) prototype and its variants was determined by BLI. JMB2002 was loaded onto the AHC sensor, and serially diluted antigens were bound to JMB2002 on the biosensor. K_D values were determined with Octet Data Analysis HT 11.0 software using a 1:1 global fit model. Blocking activity was assessed using ELISA with hACE2-coated plates. A mixture of biotinylated SARS-CoV-2 RBD (c)/S1 (d) proteins and JMB2002 was added for competitive binding to hACE2. IC₅₀ values were calculated by Prism V8.0 software using a four-parameter logistic curve fitting approach. Values are displayed as the mean ± standard deviations from three independent experiments. (e) The pseudovirus neutralization activity of JMB2002 was evaluated using a pseudotyped SARS-CoV-2 system, which contained a luciferase reporter. Pseudotyped viruses were preincubated with serially diluted antibodies for 1 h. The mixture was added to hACE2-expressing cells and incubated at 37°C for 20–28 h. Infection of cells with pseudotyped SARS-CoV-2 was assessed by measuring cell-associated luciferase activity. IC₅₀ values were calculated by plotting the inhibition rate against the log antibody concentration in Prism V8.0 software

Figure 5.

Prophylactic and therapeutic efficacies of JMB2002 against SARS-CoV-2 infection in rhesus macaques. (a) Schematic representation of the design of the in vivo animal experiment. Five monkeys were divided into three groups: the control group (one animal, C1), prophylactic group (two animals, PA1 and PA2), and therapeutic group (two animals, AC1 and AC2). In the prophylactic group, a single dose of 20 mg/kg JMB2002 was intravenously injected into the animals before SARS-CoV-2 infection. The next day, all monkeys were infected with virus (1 × 10⁵ TCID₅₀) via intratracheal inoculation. In the therapeutic group, 50 mg/kg JMB2002 was injected at 1 and 3 dpi, whereas in the control group, a single dose of 20 mg/kg irrelevant IgG control was administered at 1 dpi. (b) The viral load in oropharyngeal swabs was monitored for 7 days by qRT-PCR. The dotted line indicates the copy number detection limit. (c) Histopathological and immunohistochemical characterization of lung tissues. All animals were euthanized and necropsied at 7 dpi. The tissue samples were collected, fixed in 10% formalin solution, embedded in paraffin, sectioned, and stained with hematoxylin and eosin or Masson’s trichrome before observation by light microscopy. Scale bar = 100 μm