Identification of an anti–SARS–CoV-2 receptor-binding domain–directed human monoclonal antibody from a naïve semisynthetic library

There is a desperate need for safe and effective vaccines, therapies, and diagnostics for SARS– coronavirus 2 (CoV-2), the development of which will be aided by the discovery of potent and selective antibodies against relevant viral epitopes. Human phage display technology has revolutionized the process of identifying and optimizing antibodies, providing facile entry points for further applications. Herein, we use this technology to search for antibodies targeting the receptor-binding domain (RBD) of CoV-2. Specifically, we screened a naïve human semisynthetic phage library against RBD, leading to the identification of a high-affinity single-chain fragment variable region (scFv). The scFv was further engineered into two other antibody formats (scFv-Fc and IgG1). All three antibody formats showed high binding specificity to CoV-2 RBD and the spike antigens in different assay systems. Flow cytometry analysis demonstrated specific binding of the IgG1 format to cells expressing membrane-bound CoV-2 spike protein. Docking studies revealed that the scFv recognizes an epitope that partially overlaps with angiotensin-converting enzyme 2 (ACE2)–interacting sites on the CoV-2 RBD. Given its high specificity and affinity, we anticipate that these anti-CoV-2 antibodies will be useful as valuable reagents for accessing the antigenicity of vaccine candidates, as well as developing antibody-based therapeutics and diagnostics for CoV-2.


Figure S5
. Figure S5: ELISA based binding of soluble S protein with II62 antibody with increasing concentration of antibody. BSA used as negative control.  Incubation of SARS-CoV-2 virus with 100ug/ml of C) scFv, D) scFv-Fc and E) IgG formats of II62 mAb respectively, followed by 1 h adsorption on Vero E6 cells. CPE was observed after 72 hours. No significant reduction in the CPE (neutralizing activity) was observed for any of the antibody format.

Figure S8
Figure S8: Plaque based neutralization assay: Assay plates fixed with formaldehyde and stained by crystal violet. A) Plaques formation in infected Vero E6 cells with mAb at 100µg/ml concentration incubated with 100 PFU of SARS-CoV-2 Wuhan-1 strain B) Control cells infected with 100 PFU of virus. No significant difference was seen in presence or absence of II62-IgG1 indicating that the II62-IgG1 is not neutralizing.

Figure S9
Figure S9: RBD-ACE2 competition assay: ACE2 protein coated on the ELISA plate and followed by incubation of a mix of RBD-His and increasing concentration of II62 antibody formats (scFv-Fc and IgG1). We have shown that II62 mAb binds with RBD-His. So, in pre-incubation of mAb and RBD, the mAb will occupy the epitope on the RBD in a concentration dependent manner. If the epitope on the RBD is same or overlapping with the ACE2 binding site, the RBD-His will not be available for binding to coated ACE2 protein and will be removed during washing. However, we do see a decrease in binding of RBD-His with coated ACE2 with increasing concentration of the Abs used. The inhibitory effect was more prominent in case of scFv-Fc as compared to IgG1. It suggests that the two binding sites, Ab epitopes and ACE2, on RBD are partially overlapping as competition was seen more prominent when higher concentrations of II62 mAb was used. Figure S10 Figure S10: Differential binding of II62-scFv-Fc with mammalian and Baculovirus expressed RBD: The RBD protein expressed using baculovirus expression system in insect cell lines (RBD-His from Sinobiological) binds better to the II62-scFv-Fc compared to the same RBD when expressed in mammalian Expi293 cells.

Library amplification and panning
The panning procedure was performed as described in library manual. Briefly, 96-well ELISA binding plates were coated overnight with purified protein of receptor binding domain (RBD) of SARS-CoV-2 at 2μg/ml in coating buffer, which was blocked directly with MPBS (PBS containing 5% skimmed milk powder) at room temperature for 1.5 h. After removal of the blocking solution, the plate was washed three times with PBS. Then 100μl of the primary library phage (10 12 ) diluted in 2% MPBS was added to ten 10 wells of antigen coated 96 well ELISA plates. After 1 hr of binding at RT, non-specific binding phages were washed away with PBS containing 0.1% Tween for 10 times (20 times in the second panning and 30 times in the third panning). Then 250 μl of the eluted phages were used to infect 10 ml of E. coli TG1 cultured in 2XTY media (OD 600 = 0.4 to 0.5) for 30 minutes at 37°C without shaking. Then, about 5-8 × 10 11 cfu (colony-forming units) per milliliter of helper phage M13K07 was added and incubated for 30 minutes at 37°C. After that, bacteria were collected by centrifuging for 10 min at 4000 r/min, re-suspended in 100 mL of fresh 2XYT with 100 μg/ml ampicillin and 50 μg/ml kanamycin, and incubated overnight at 30°C. Next morning, bacterial cells were removed by centrifugation at 8000 rpm for 20 min at 4°C, and subsequently phages were precipitated from the supernatant by adding polyethylene glycol (PEG)-NaCl solution (20 % Polyethylene glycol 6000, 2.5 M NaCl). The precipitated phages were titrated and were used for next round of biopanning. A total of three rounds of biopanning process were performed.

Screening of clones (monoclonal phage ELISA)
After third round of biopanning individual colonies were picked and tested for specificity by phage ELISA. Monoclonal Phage ELISA was carried out by inoculating individual colonies from the third round of selection into 100 μl 2XTY medium containing 100 μg/ml ampicillin and 2% glucose in 96 cell-well plates. The plate was incubated with shaking overnight at 37 °C. Then a small inoculum (about 2 μl) from each well was transferred from this plate to a second 96 cell-well plate as replica plate. After approximately 3 hrs when the OD 600 of culture reached 0.4 to 0.5, 5x10 10 helper phage was added to each well. After 30 mins of incubation the culture was spinned down and pellet was resuspended in 2XTY media containing 100 μg/ml ampicillin and 50 μg/ml kanamycin. The plate was incubated with shaking overnight at 30 °C. 50 μl of the supernatant from each well in the overnight inoculated plate was tested by using phage ELISA. The ELISA plates were coated with 100 μl of RBD protein (1 μg/ml) in 0.1 M NaHCO3 (pH 8.6) and incubated overnight at 4°C or 1 hr at 37°C. As a negative control antigen same concentration of BSA was coated to ELISA plate. The plates were washed once with PBS and blocked with 5% non-fat milk (HiMedia laboratories) for 1.5 h at RT. The plates were then washed three times with PBS. After blocking, 50μl phage-rescued supernatants diluted in 50 μl of 2% MPBS were added to each well and incubated for 1h at RT. Phage supernatant was discarded and the plates were washed four times with PBST (0.1%). After that 100 μl of HRP-anti-M13 (diluted 1:2000) was added (Sigma) and incubated for 1h at RT. The plates were then washed four times with PBST (0.1%) and then, 100 μl of tetramethylbenzidine (TMB) substrate was added to each well and the plates were incubated in the absence of light at RT. The reaction was stopped by adding 2N H 2 SO 4 and absorbance was measured at 450nm.

Expression and purification of RBD and S protein
The RBD and the soluble ectodomain of S protein were expressed in Expi293F cells to produce recombinant protein. The expression plasmids were transiently transfected into cells at a density of 2.5 millions/ml and volume ranging from 100-300 ml using ExpiFectimine 293 transfection reagent kit (Thermo) following manufacturers protocol and scale up. After 5 days, the supernatant was collected and soluble protein was purified by Ni affinity chromatography using Ni ++ ions immobilized on a resin by covalent linkage to nitrilotriacetic acid (NTA) (QIAGEN, Germany). The sample was further purified via gel filtration chromatography with a Superdex 200 column (GE Healthcare) in PBS (pH 7.4) buffer background. Purified proteins were concentrated to about 1 mg/ml, concentration was estimated by absorption at 280 nm using molar extinction coefficient and the amount of protein yield calculated based on the final amount of the purified proteins that is extrapolated to mg/l yield

Purification of scFv, scFv-Fc and IgG
The His-tagged II62-scFv format was further transformed into E.coli HB2151 to express a soluble scFv antibody. Briefly, E.coli HB2151 cells carrying scFv plasmid were grown in 10 ml 2XTY media overnight at 37ºC with shaking at 200 rpm. The next day, 1/100th volume of the overnight culture was inoculated in 1 litre of 2XTY media and grown at 37ºC with shaking at 200 rpm till the OD reached 0.4-0.6 and then the culture was induced with 1 mM IPTG and incubated at 18°C for 16-20 h. The bacteria were subsequently harvested by centrifugation at 8000 rpm for 15 min at 4°C. Cells were harvested and different fractions were prepared. The scFv antibody fragments containing a hexahistidine tag were purified from the periplasmic fraction using Ni ++ ions immobilized on a resin by covalent linkage to nitrilotriacetic acid (NTA) (QIAGEN, Germany).
The scFv-Fc and IgG formats were expressed by transient transfection of Expi293F cells and are described above. Both the antibody formats were purified from culture supernatants using a protein G affinity column. Bound proteins were eluted with 0.1M glycine (pH 2.2), immediately neutralized with 0.1M Tris (pH 9.5) dialyzed with PBS (pH 7.4) and subsequently concentrated using Amicon ultracentrifuge filters (Millipore) with a 30-kDa cut-off. The amount of protein yield calculated based on the final amount of the purified proteins that is extrapolated to mg/l yield

Protein -protein molecular docking study
A detailed molecular modeling was performed to establish the residue level interactions between RBD-mAb proteins. For RBD the crystal structure 6M17 was used. For mAb, based on sequences and its similarity and identity obtained from BLASTp, the 3D model was generated through homology modelling by using MODELLER. The top model was selected to check the robustness and accuracy via using different post-processing tools like PROCHECK and ProSA-Web (Z-score). Although, the crystal structure of RBD is reported, a detailed molecular modelling was carried out for loop movement analysis. A similar protocol was executed for the mAb also. The data has shown that the model is robust enough. Both the structures were optimized and then minimized using the Protein Preparation Wizard module of Maestro (Schrödinger Release 2020-1: Maestro, Schrödinger, LLC, New York, NY, 2020). Molecular dynamics minimization was employed to allow conformational relaxation of the protein structures prior to subjecting them to protein-protein docking calculations. Subsequently, two different algorithms (PyDOCK & Swarmdock) were used to perform the protein-protein dockings to identify the most likely binding interfaces and poses of RBD-mAbs interactions. The docking procedure aimed to generate a set of solutions for candidates with at least one near native structure. Since, rigid docking through PyDock allows some steric clashes, therefore the flexible docking by Swarm dock. was also conducted on relaxed structures of the proteins. The candidate solutions were scored and ranked according to different parameters such as clusters, lowest binding energy, number of conformers, and agreement with known binding sites. Clustering of docked poses was conducted to filter out the most likely complex of RBD-mAb. We quantified the docking results and picked the lowest energy zone (< -45.0 kcal/mol). Only the best-docked pose, which ranged between <-40.0 to <-45.0 kcal/mol, was used for further analysis.

Fluorescent staining
2.5x10 5 293 T cells expressing Spike protein of the SARS-CoV-2 were seeded into 96 well culture plate. For control, 293T cells which do not express Spike protein (untransfected) were used. Cells were pelleted down by centrifugation at 480 x g and 22 o C for 4 minutes. 20 μg of primary antibodies II62 (scFv-Fc and IgG) were made in 5% DMEM (DMEM supplemented with FCS) and 100 μl of it was added to respective wells and mixed 6-8 times by pipetting. Cells were incubated for 1 hour at room temperature. After incubation, 100 μl of 5% DMEM was added to each well and the cells were centrifuged at 480 x g and 22 o C for 4 minutes. Supernatant was aspirated and the cells were washed two more times with 5% DMEM. Secondary antibody (R-Phycoerythrin conjugated AffinityPure F(ab') 2 Fragment Goat Anti-Human IgG from Jackson, Cat No: 109-116-097) was diluted in 5% DMEM to 1:100. In each well 100 μl of diluted secondary antibody was added, mixed 6-8 times by pipetting and incubated in dark at room temperature for 1 hour. After incubation, 100 μl of 5% DMEM was added to each well and the cells were pelleted by centrifugation at 480 x g and 22 o C for 4 minutes. Supernatant was aspirated, cells were given two wash with 5% FACS buffer (1X PBS supplemented with FCS). Finally, cells were resuspended in 200 μl of 4% formaldehyde made in 5% FACS buffer, and then acquired in FACS canto-II machine of BD Bioscience.

Neutralization assay (plaque based neutralization titer assay)
For the plaque based neutralization titer assay, 100 µg/ ml of II62-IgG1 mAb was incubated with 100 PFU of the SARS-CoV-2 Wuhan-1 strain for 1 h at 37°C. The virus and antibodies were then added to a 12-well plate seeded with Vero E6 cells. After 1-h incubation at 37°C, cells were overlaid with 3 ml of 0.8% carboxy methyl cellulose in 2% medium. Plates were incubated for 2 days at 37°C and then fixed with paraformaldehyde and stained with crystal violet for 1 h, and plaques were counted.

CPE based assay:
The virus was obtained from BEI resources (Isolate USA-WA1/2020), passaged once in Vero cells, titrated and 1x10 2 TCID 50 virus (diluted in 50 μl of the serum-free media) was incubated with mAb (100 µg to 0.3 µg) for 90 mins followed by 1-hour adsorption on Vero cells. The cells were subsequently washed and DMEM media supplemented with 2% FBS was added to cells. The presence of cytopathic effect (CPE) was observed in cells using a microscope after incubation for 4-5 days at 37 0 C with 5% CO 2 . Not infected VERO E6 cells were used as positive control. Infected VERO E6 cells were used as negative control.

RBD-ACE2 competition assay:
For ACE2 inhibition assay, Fc conjugated soluble ACE2 was coated on the ELISA plate (100 µl/well of 2 µg/ml) overnight. Next day, the plate was washed and blocked with 2% skim milk for 1 hour. A 100 µl mix of RBD-His and different concentration of II62 (scFv-Fc and IgG1) were pre-incubated for 1 hour. As a negative control unrelated IgG (HIV mAb) was preincubated with RBD protein. The mix was then transferred to ELISA plate after removing milk and washed and incubated for 1 hour at room temperature. The plate was washed and HRP conjugated anti-His antibody was added for 1 hour. and developed with (TMB) substrate.