Rapid identification of a human antibody with high prophylactic and therapeutic efficacy in three animal models of SARS-CoV-2 infection

Significance Effective therapies are urgently needed for COVID-19. We rapidly (within a week) identified a fully human monoclonal germline-like antibody (ab1) from phage-displayed libraries that potently inhibited mouse ACE2-adapted SARS-CoV-2 replication in wild-type BALB/c mice and native virus in transgenic mice expressing human ACE2 as well as in hamsters when administered before virus challenge. It was also effective when administered after virus infection of hamsters, although at lower efficacy than when used prophylactically. Ab1 was highly specific and did not bind to human cell membrane-associated proteins. It also exhibited good developability properties including complete lack of aggregation. Ab1 has potential for prophylaxis and therapy of COVID-19 alone or in combination with other agents.


Materials and Methods
Generation, Expression and Characterization of SARS-CoV-2 RBD-Fc, S1-Fc, ACE2-Fc and CR3022 Fab. The SARS-CoV-2 surface glycoprotein and the anti-SARS-CoV antibody IgG1 CR3022 (1) were synthesized by IDT (Coralville, Iowa). MERS-CoVspecific IgG1 m336 antibody was expressed in human mammalian cell as described previously (2). The ACE2 gene was ordered from OriGene (Rockville, MD). The RBD domain (residues 330-532) and S1 domain (residues 14-675) and ACE2 (residues 18-740) genes were cloned into plasmid which carries a CMV promotor with an intron, human IgG1 Fc region and Woodchuck posttranscriptional regulatory element (WPRE) to generate the RBD-Fc, S1-Fc and ACE2-Fc expression plasmids. The RBD-avi-his protein with an avi tag followed by a 6×His tag at C-terminal was subcloned similarly. These proteins were expressed with Expi293 expression system (Thermo Fisher Scientific) and purified with protein A resin (GenScript) and by Ni-NTA resin (Thermo Fisher Scientific). The Fab CR3022 antibody gene with a His tag was cloned into pCAT2 plasmid (developed in house) for expression in HB2151 bacteria and purified with Ni-NTA resin. Protein purity was estimated as >95% by SDS-PAGE and protein concentration was measured spectrophotometrically (NanoVue, GE Healthcare).

Selection, Expression, and Purification of the RBD-specific Fabs and VHs and
Conversion to IgG1s or Fc Fusion Proteins. The naïve human antibody phage display libraries were made based on the antibody cDNA from total of 490 healthy donors peripheral blood monocytes (PBMCs) and splenocytes. The Fab and scFv libraries were constructed by randomly pairing antibody VH and VL gene, and the VH libraries -by grafting CDRs into stable VH scaffolds. These libraries contain very large transformants (size for each ~10 11 ) and are highly diverse. For panning, the libraries were preabsorbed on streptavidin-M280-Dynabeads in PBS for 1 h at room temperature (RT) and incubated with 50 nM biotinylated SARS-CoV-2 RBD for 2 h at room temperature with gentle agitation. Phage particles binding to biotinylated antigen were separated from the phage library using streptavidin-M280-Dynabeads and a magnetic separator (Dynal). After washing for 20 times with 1 ml of PBS containing 0.1% Tween-20 and another 20 times with 1 ml of PBS, bound phage particles were eluted from the beads using 100 mM triethanolamine followed by neutralization with 1 M, pH 7.5 Tris-HCl. For the 2 nd round of panning, 10 nM (2 nM for the 3 rd round) of biotinylated antigen was used as antigen.
After the 3 rd round of panning against 2 nM biotinylated antigen, 96 individual clones were screened for binding to RBD-Fc fusion protein by phage ELISA. Panels of Fabs and VHs were selected and sequenced. For conversion to Fc-fusion, the VH gene was subcloned into pSecTag B vector (already containing human Fc fragment). For conversion to IgG1, Fab VH and VL gene was inserted into pDR12 vector which contains the IgG1 CH1-CH3 and CL domains. Both VH-Fc and IgG1 were expressed as previously described (3). Protein purity was estimated as >95% by SDS-PAGE and protein concentration was measured spectrophotometrically (NanoVue, GE Healthcare).

ELISA.
For phage ELISA, the SARS-CoV-2 RBD-Fc (residues 330-532) protein was coated on a 96-well plate (Costar, half-area, #3690) at 100 ng/well in PBS overnight at 4 o C. Phage from each round of panning (polyclonal phage ELISA) or clones randomly picked from the infected TG1 cells (monoclonal phage ELISA) were incubated with immobilized antigen. Bound phage were detected with horseradish peroxidase (HRP) conjugated anti-M13-HRP polyclonal Ab (Pharmacia, Piscataway, NJ). For the soluble Fab/VH binding assay, 200 ng RBD-Fc was coated and HRP-conjugated mouse anti-FLAG tag Ab (Sigma-Aldrich) was used to detect Fab/VH binding. For the IgG1 or VH-Fc binding assay, 200 ng RBD-his was coated and HRP-conjugated goat anti-human IgG Fc (Sigma-Aldrich) was used for detection. For the competition ELISA with hACE2, 2 nM of human ACE2-mouse Fc (Sino Biological) was incubated with plate-coated RBD-Fc in the presence of serially diluted IgG1 or VH-Fc. After washing, bound ACE2-mouse Fc was detected by HRP-conjugated anti mouse IgG (Fc specific) (Sigma-Aldrich). For the competition ELISA between ab1 and other antibodies, ~20 nM Fab ab1 was incubated with RBD-Fc in the presence of different concentrations of antibodies in IgG1 or VH-Fc formats. After washing, detection was made by using HRP conjugated anti-FLAG tag antibody. For the competition ELISA with CR3022, 10 nM Fab CR3022 was incubated with serially diluted IgG1 or VH-Fc antibodies, and the mixtures were added to RBD-Fc coated wells. After washing, bound Fab CR3022 was detected by HRP-conjugated anti-FLAG tag antibody. All the colors were developed by 3,3′,5,5′-tetramethylbenzidine (TMB, Sigma) and stopped by 1 M H2SO4 followed by recording absorbance at 450 nm.
Experiments were performed in duplicate and the error bars denote ± SD, n =2. The association was monitored for 300 s. Meanwhile, the signals of 100 nM hACE2 or CR3022 binding to the RBD-Fc coated sensor in the absence of IgG1 ab1 was independently recorded. Competition was determined by the percentage of signals in the presence of ab1 to signals in the absence of ab1 (< 0.7 is considered to be competitive) (4).

SARS-CoV and SARS-CoV-2 Reporter Gene Neutralization Assay. Full-length viruses
expressing luciferase were designed and recovered via reverse genetics and described previously (10,11). Viruses were tittered in Vero E6 USAMRID cells to obtain a relative were sacrificed, and lung viral titer was determined by the plaque assay.

Evaluation of IgG1 ab1 Protective Efficacy in a hACE2 Mouse Model of Infection.
Human ACE2 transgenic 6-9 week old C3B6 mice were treated intraperitoneally with 0.3 mg (15 mg/kg) of antibody (5 mice) or negative controls (6 mice) 15 hours prior to intranasal infection with 10 5 PFU of SARS-CoV-2. No weight loss was observed over the course of the two-day infection. Lung tissue was homogenized in PBS and virus replication assessed by plaque assay on VeroE6 cells. The assay limit of detection was 100 PFU.  The RBD sequences were obtained from https://www.ncbi.nlm.nih.gov/genbank/sars-cov-2-seqs/. Liabilities were evaluated online (opig.stats.ox.ac.uk/webapps/sabdabsabpred/TAP.php) (14).     Competition of ab1 with hACE2 tested by Blitz. 100 nM hACE2-Fc was monitored to bind ab1 saturated sensors (red line), which is compared to its independent binding signal to RBD sensor in the absence of ab1(green line). (D) Competition of ab1 with CR3022 tested by Blitz. 100 nM Fab CR3022 was monitored to bind ab1 saturated sensors (red line). The signal was compared to the same concentration of CR3022 binding to the RBD sensor in the absence of ab1 (yellow line). The percentage of signal for CR3022 + ab1 to that of CR3022 alone is ~77%. Thus, there is no competition between CR3022 and ab1.