Unbiased approach to identify and assess efficacy of human SARS-CoV-2 neutralizing antibodies

Abstract

Coronavirus disease 2019 (COVID-19) continues to significantly impact the global population, thus countermeasure platforms that enable rapid development of therapeutics against variants of SARS-CoV-2 are essential. We report use of a phage display human antibody library approach to rapidly identify neutralizing antibodies (nAbs) against SARS-CoV-2. We demonstrate the binding and neutralization capability of two nAbs, STI-2020 and STI-5041, against the SARS-CoV-2 WA-1 strain as well as the Alpha and Beta variants. STI-2020 and STI-5041 were protective when administered intravenously or intranasally in the golden (Syrian) hamster model of COVID-19 challenged with the WA-1 strain or Beta variant. The ability to administer nAbs intravenously and intranasally may have important therapeutic implications and Phase 1 healthy subjects clinical trials are ongoing.

Figure 1

Rapid Discovery of Neutralizing Antibodies. (A) The G-MAB phage display library was panned for SARS-CoV-2 Spike S1 subunit-binding scFv fragments. Following confirmation of binding activity and blocking of S1:ACE2 interactions by candidate scFvs, the most potent of these candidates were converted to IgG1 antibodies bearing the LALA Fc modification. Candidate nAbs were characterized for binding of Spike S1 subunit and neutralization of related clinical SARS-CoV-2 isolates. Affinity maturation of potent nAbs was carried out in parallel to biophysical profiling, cell line development, and evaluation of protective efficacy for the parental nAbs, S1D2 and S7E3 to yield STI-2020 and STI-5041, respectively. Artwork credit: William SooHoo. (B) Affinity measurements of STI-2020 and STI-5041 for Spike S1 domain from the following isolates and VOCs: USA/WA-1/2020(WA-1) isolate, D614G 2020001 isolate, B.1.1.7 VOC (Alpha), B.1.351 VOC (Beta). The antibody affinities were measured using SPR on a BIAcore T200 instrument using a 1:1 binding model. (C) Spike protein derived from the WA-1 and 2020001 (D614G) SARS-CoV-2 isolates were independently expressed on the surface of HEK 293 cells. Serially-diluted STI-2020 or STI-5041 were assayed for Spike protein binding by flow cytometry. To quantify antibody binding, mean fluorescent intensity was measured for each dilution tested and the EC₅₀ value was calculated for each nAb. Representative replicate experiments are shown. (D) STI-2020 and STI-5041 were evaluated in neutralizing test for potency against SARS-CoV-2 USA/WA-1/2020, 2020001 (D614G), B.1.1.7 VOC (Alpha), B.1.351 VOC (Beta) and B.1.617.2 (Delta).

Figure 2

Pharmacokinetic and bioavailability of Neutralizing Antibody. (A) Neutralization of SARS-CoV-2 Spike-pseudotyped VSV by STI-2020 and STI-5041. VSVΔG-luciferase was pseudotyped with the indicated spike variant, incubated with STI-2020 or STI-5041 at a range of 0.0005–10 µg/mL for 30 min, then added to 293-ACE2 target cells. Absolute IC₅₀ was calculated from luciferase values and are indicated. Experiments were performed at least three independent times and data presented as the mean ± SD. (B) Epitope binning was performed as described in the Materials and Methods. The sensorgram shows STI-5041 can bind to SARS-CoV-2 S1 and STI-2020 complex (blue line) and indicates that STI-5041 and STI-2020 bind to distinct epitopes. (C–H) Biodistribution: Concentration of STI-2020 (C,D) or 5041 (E,F) in serum and lung lavage or lysates of spleens, lungs, small intestines, and large intestines collected from female CD-1 mice administered STI-2020 IV at doses of 0.5 mg/kg (Dark blue circle), 0.05 mg/kg (Sky blue circle), or 0.005 mg/kg (light blue circle) or IN at doses of 2.5 mg/kg (black circle), 0.5 mg/kg (Maroon circle), 0.05 mg/kg (Red Circle), and 0.005 mg/kg (Rose circle) or STI-5041 administered IV at doses of 2 mg/kg (Dark blue circle), 0.2 mg/kg (Sky blue circle), and 0.02 mg/kg (light blue circle) or IN at doses of 10 mg/kg (Black circle), 2 mg/kg (Maroon circle), 0.2 mg/kg (Red circle), and 0.02 mg/kg (Rose circle) at 24 h post-administration as compared to samples collected from untreated mice. Values represent mean ± SEM (n = 3 animals no treatment group, n = 5 in treatment groups). Significant differences are denoted by * p < 0.05; ** p < 0.01; *** p < 0.001, **** p < 0.0001. Pharmacokinetics: Concentration of STI-2020 (G) or STI-5041 (H) in lungs and isolated serum collected from female CD-1 mice administered STI-2020 or STI-5041 intranasally (IN) at a dose of 5 mg/kg or 20 mg/kg, respectively. Samples from treated mice were collected at the indicated timepoint post-administration; antibody concentrations were quantified by ELISA and compared to samples collected from untreated mice. Overlay of antibody concentrations in lung tissue vs. serum following IN administration of a 5 mg/kg or 20 mg/kg dose. Values represent mean ± SD (n = 3 animals no treatment group, n = 6 per time point in treatment groups).

Figure 3

Efficacy of Intravenous (IV) Delivery of Neutralizing Antibodies in the golden (Syrian) Hamster Model of COVID-19. Female hamsters were inoculated with SARS-CoV-2 USA/WA-1/2020 (A,B) or SARS-CoV-2 Beta variant (C,D) on day 0. One-hour post-infection, animals were administered a single intravenous dose of Isotype control IgG (500 µg) or, For A and B, STI-2020 (100 µg, 300 µg, or 500 µg); For C and D, Isotype control IgG (1,000 µg) or STI-5041 (500 µg, or 1,000 µg). Weight changes were recorded for up to 11 days. (A) Average % weight change ± SEM was plotted for each group. Days in which there was a significant difference in average % weight change compared to Isotype control IgG 500 µg-treated animals are denoted by * (p-value < 0.05). (B) Lung tissues collected from five animals per group and virus titers were determined on day 5. A broken line indicates the detection limit of the assay (< 1.5 TCID₅₀/g). (C) Average % weight change ± SEM was plotted for each group. Days in which there was a significant difference in average % weight change for STI-5041 at 500 µg or 1,000 µg compared to Isotype control IgG 1000 µg-treated animals are denoted by * (p-value < 0.05). (D) Lung tissues collected from five animals per group administered Isotype control IgG (1,000 µg) or STI-5041 (500 µg or 1,000 µg) and virus titers were determined on day 5.

Figure 4

Efficacy of Intranasal (IN) Delivery of Neutralizing Antibodies in the golden (Syrian) Hamster Model of COVID-19. (A,B) Female hamsters were inoculated with SARS-CoV-2 USA/WA-1/2020, and then administrated with 500 µg or 400 µg Isotype control antibody or 500 µg or 400 µg STI-2020 intranasally at 12 h post-infection. (A) Average % weight change ± SEM was plotted for each group. Days in which there was a significant difference in average % weight change for STI-5020 at 500 µg compared to Isotype control IgG 500 µg-treated animals are denoted by * (p-value < 0.05). (B) Upper panels show representative figures of nasal turbinates and nasal septum at 5 d.p.i. Average ± SEM of OE thickness on the nasal septum in the lower graph for STI-5020 at 400 µg compared to Isotype control IgG 400 µg-treated animals are denoted with * (p-value < 0.05). (C) Hamsters were inoculated with SARS-CoV-2 Beta variant, and then administrated with 500 µg Isotype control antibody or 100 µg, 300 µg, or 500 µg STI-5041 intranasally at 12 h post-infection. Average % weight change ± SEM was plotted for each group.