*4.3. Monoclonal Phage ELISA*

Individual phage clones from either the third or fourth round were tested for binding to the SARS-2 RBD-coated plate. Several 96-Well Half-Area Microplates (Corning, Cat. 3690, New York, NY, USA) were coated overnight at 4 ◦C, with 30 μL per well of 1 μg/mL SARS-2 RBD, and each well was blocked with 5% skimmed milk in PBS for 1 h at room temperature. The amplified phages of individual clones from the third or fourth rounds of panning were added and incubated for 1 h at 37 ◦C. After washing four times with PBST, the horseradish peroxidase-conjugated anti-M13 antibody (1:5000, Sino Biological, Cat. 11973-MM05, Beijing, China) was incubated for 1 h at 37 ◦C. After washing four times with PBST, a TMB substrate solution (Sigma-Aldrich, Cat. T0440, St. Louis, MO, USA) was added for 8 min, and the reaction was stopped with 1 N sulfuric acid (Merck, Cat. 100731, Darmstadt, Germany). The absorbance was measured at 450 nm using a SpectraMax 190 Microplate Reader (Molecular Devices, Sunnydale, CA, USA).

### *4.4. Production of Fab Proteins*

An in-house bacterial expression vector (pKFAB) was used to construct the Fab expression vectors. The Fab fragments and pKFAB vector were amplified by a polymerase chain reaction (PCR) for each primer set. The PCR products were treated with DpnI (New England Biolabs, Cat. R0176L, Ipswich, MA, USA) for 1 h at 37 ◦C, separated on a 1.2% agarose gel, and the single band was purified using a Wizard SV Gel and PCR Clean-Up System (New England Biolabs, Cat. A9282, Ipswich, MA, USA). The fragments were assembled following the Gibson assembly protocol (New England Biolabs, Cat. E2611, Ipswich, MA, USA). The assembled products were used to transform *E. coli* DH5α competent cells (Enzynomics, Cat. CP010, Daejeon, Korea). The individual colonies of the transformed cells were isolated and the sequences of the isolated clones were verified.

Top10F' Competent Cells (Invitrogen, Cat. C303003, Carlsbad, CA, USA) were transformed with the Fab expression vectors and the transformants were grown in 200 mL of TB (Terrific Broth) (Thermofisher Scientific, Cat. 22711022, Waltham, MA, USA) media supplemented with 100 μg/mL ampicillin at 37 ◦C until the OD600 reached 0.5. The logphase cultures were then induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) (DAWINBIO, Cat. I0355-005, Hanam, Gyeonggi, Seoul) and incubated overnight at 30 ◦C. The cells were collected and resuspended in 16 mL of 1× TES (50 mM Tris-HCl, 1 mM EDTA, 20% Sucrose, pH 8.0). After incubation for 30 min on ice, 24 mL of 0.2× TES was added and incubated for 1 h on ice. The periplasmic fractions were collected after centrifugation at 12,000 rpm for 30 min and filtered through a 0.22 μm filter (Milipore, Cat. SCGP00525, Carrigtwohill, Co., Cork, Ireland). The periplasmic extracts were loaded on a column packed with 0.5 mL of ProL (rProtein L) Agarose resin (Amicogen, Cat. 3010125, Jinju, Gyeongnam-do, Korea). The column was washed with 10 column volumes (CVs) of PBS and eluted with 30 CVs of Buffer W (100 mM Glycine, pH 2.5). The eluted proteins were neutralized with 1M Tris-HCl (pH 9.0) (Biosesang, Cat. TR2016-050-90, Seongnam, Gyeonggi, Korea). The eluted protein was concentrated and buffer-exchanged with PBS

using Amicon Ultra-15 Centrifuge Filter Units (Milipore, Cat. UFC903024, Carrigtwohill, Co., Cork, Ireland).

#### *4.5. Determination of Apparent Affinity by ELISA*

Several 96-Well Half-Area Microplates (Corning, Cat. 3690, New York, NY, USA) were coated overnight at 4 ◦C, with 30 μL per well of 2 μg/mL SARS-2 RBD. After rinsing them twice with tap water, the wells were blocked with 5% skimmed milk in PBS for 1 h at room temperature. Serially diluted anti-SARS-2 RBD Fabs or IgGs were added and incubated for 1 h at 37 ◦C. After washing the plates four times with PBST, the HRP-conjugated human kappa light-chain antibody (1:5000, Bethyl laboratories, Cat. A80-115P, Montgomery, TX, USA) or HRP-conjugated human IgG Fc (1:5000, Abcam, Cat. ab97225, Cambridge, USA) were added to the plates and incubated at 37 ◦C for 1 h. After washing the plates four times with PBST, a TMB substrate solution was incubated for 8 min, and the reaction was stopped with 1 N sulfuric acid. The absorbance was measured at 450 nm using a SpectraMax 190 Microplate Reader. A plot was created using a nonlinear regression with Graphpad Prism 7 (GraphPad Software, San Diego, CA, USA), and half-maximal effective concentration (*EC50*) values were determined accordingly.

#### *4.6. ELISA-Based Neutralizing Assay*

A 96-Well Half-Area Microplate (Corning, Cat. 3690, New York, NY, USA) was coated overnight at 4 ◦C, with 30 μL per well of 2 μg/mL SARS-2 RBD. After rinsing them twice with tap water, the wells were blocked with 5% skimmed milk in PBS for 1 h at room temperature. Both anti-SARS-2 RBD Fabs or IgGs and biotinylated human ACE2 (Acrobiosystems, Cat. AC2-H82E6, Newark, NJ, USA) were added and incubated for 1 h at 37 ◦C. After washing the plates four times with PBST, High Sensitivity Streptavidin-HRP (1:5000, Thermofisher, Cat. 21130, Waltham, MA, USA) was added to the plates and incubated for 1 h at 37 ◦C. After washing the plates four times with PBST, a TMB substrate solution was incubated for 8 min, and the reaction was stopped with 1 N sulfuric acid. The absorbance was measured at 450 nm using a SpectraMax 190 Microplate Reader. Graphpad Prism 7 (GraphPad Software, San Diego, CA, USA) was used to plot data using a two-way ANOVA algorithm.

#### *4.7. Flow Cytometry-Based Neutralizing Assay*

Calu-3 cells were obtained from the American Type Culture Collection (Manassas, VA, USA). Calu-3 cells were seeded in a 96-well plate (Corning, Cat. 3894, New York, NY, USA) at a density 1 × 106 cells per well. Afterward, 50 <sup>μ</sup>g/mL, 100 <sup>μ</sup>g/mL Fabs, or 50 <sup>μ</sup>g/mL IgGs were mixed with 5 μg/mL SARS-2 RBD-mFc (mouse IgG2a Fc-tagged SARS-2 RBD) (Acrobiosystems, Cat. SPD-C5259, Newark, NJ, USA), and the mixture was then incubated with cells for 1 h at 4 ◦C. After washing, the cell was labeled with the PE anti-mouse IgG2a antibody (Biolegend, Cat. 407108, San Diego, CA, USA) and incubated for 1 h at 4 ◦C. The cells were analyzed by FACS canto II (BD Biosciences, San Jose, CA, USA). Data were analyzed by FlowJo (downloadable at https://www.flowjo.com/solutions/flowjo/ downloads (accessed on 1 January 2021)).

#### *4.8. Conversion to IgG and Production of IgG Proteins*

The light- and heavy-chain vectors (pcDNA3.4) were used as the backbone vectors. The VL and VH genes were individually amplified by polymerase chain reaction (PCR) from each Fab. The PCR products (VL and VH) were purified with an Expin PCR SV Mini Kit (Geneall, Cat. 103-102, Seoul, Korea) and digested with the following restriction enzymes (New England Biolabs, Ipswich, MA, USA): for VH, EcoRI (Cat. R3101S) and NheI (Cat. R3131S); for VL, XhoI (Cat. R0146S) and BsiWI (Cat. R3553S). The digestion products were separated on a 1.2% agarose gel, and the single band was purified with an Expin Gel SV Kit (Geneall, Cat. 102-102, Seoul, Korea). The fragments were ligated with the same restriction enzyme-digested vector using T4 DNA ligase (Promega, Cat. M1801, Madison, WI, USA). The ligation mixtures were used to transform *E. coli* DH5α Competent Cells (Enzynomics, Cat. CP010, Daejeon, Korea). Individual colonies of the transformed cells were isolated and the sequences of selected clones were confirmed by sequencing.

Freestyle 293 cells were cultured in Freestyle 293 Expression Medium (Thermofisher Scientific, Cat. 12338018, Waltham, MA, USA) in a humidified 8% CO2 incubator at 37 ◦C and 125 rpm. On the day of transfection, Freestyle 293 cell density was approximately 2.0 × 106 cells/mL. Cells were transfected with plasmid DNA and these were mixed by DNA/PEI (polyethylenimine, Sigma-Aldrich, Cat. 913375, St. Louis, MO, USA) in a 1:2 ratio in the medium. Culture supernatants were collected after five days by centrifugation and filtration (0.22 μm, Polyethersulfone, Milipore, Cat. SLGPR33RB, Burlington, MA, USA).

Antibodies were purified from the culture supernatants using HiTrap MabSelect SuRe (GE Healthcare, Cat. 11-0034-94, Chicago, IL, USA) columns. Briefly, equilibration was carried out using Buffer A (1xPBS). The sample was loaded onto the equilibrated column. Following the sample loading, the column was washed with Buffer A until a stable baseline was established. Following the wash step, the protein was eluted with Buffer B (IgG elution buffer or 100 mM citrate buffer, pH 3.0). Following the elution, the IgG was brought to neutral pH with 1 M Tris base, pH 9.0, and dialyzed into a final buffer composition of PBS (pH 7.4) (Thermofisher Scientific, Cat. 10010023, Waltham, MA, USA). Each antibody was separated on 4–12% Bis-Tris gels (Thermofisher Scientific, Cat. NP0321, Waltham, MA, USA) with reducing or non-reducing conditions and stained with Sun-Gel Staining Solution (LPS Solution, Cat. SGS01, Daejeon, Korea).

### *4.9. Size-Exclusion Chromatography*

The separation of the IgGs using size-exclusion chromatography (SEC) was performed using a Waters Alliance 2695 (Waters, Milford, MA, USA) connected to a Biosuite highresolution SEC column (7.5 mm × 300 mm, 10 μm particle size, Waters, Milford, MA, USA). The separation was conducted using an isocratic elution with PBS, pH 7.4, at a flow rate of 1 mL/min. The effluent detection was conducted using a UV/Vis detector 2489 at 280 nm.

#### *4.10. Determination of Melting Temperature by a Protein Thermal Shift (PTS) Assay*

To each well of a MicroAmp Fast Optical 96-Well Reaction Plate (Applied Biosystems, Cat. 4346906, Foster City, CA, USA), 12.5 μL of anti-SARS-2 RBD Fabs or anti-SARS-2 RBD IgGs, 5 μL of Protein Thermal Shift Buffer and 2.5 μL of Protein Thermal Shift Dye (10×, Applied Biosystems, Cat. 4461146, Foster City, CA, USA) were mixed. As a negative control, PBS was mixed with the Protein Thermal Shift Dye. The plate was sealed with a MicroAmp Optical Adhesive Film (Applied Biosystems, Cat. 4306311, Foster City, CA, USA) and centrifuged at 1000 rpm for 1 min. The measurement was conducted using a real-time PCR instrument (ViiA 7 Real-Time PCR System, Thermofisher Scientific, Waltham, MA, USA). The instrument was set up according to the manufacturer's instructions. All the experiments were performed at least in triplicate.

#### *4.11. Production of SARS-CoV-2 Spike pseudovirus*

Plasmids encoding the SARS-CoV-2 spike protein (D614) were purchased from Sino Biological (pCMV3-SARS-CoV-2 Spike, Cat. VG40589-UT, Beijing, China). The SARS-CoV-2 spike protein (D614G) was made by site-directed mutagenesis. The mutation was confirmed by full-length spike gene sequencing. The SARS-CoV-2 pseudoviruses were produced by co-transfection HEK-293T cells with pMDLg/pRRE (Addgene plasmid, 12251), pRSV-Rev (Addgene plasmid, 12253), pCDH-CMV-Nluc-copGFP-Puro (Addgene plasmid, 73037), and plasmids encoding either SARS-CoV-2 spike (D614) or SARS-CoV-2 spike (D614G) by using polyetherimide. Sixty hours post-infection, SARS-CoV-2 spike pseudoviruses containing culture supernatants were harvested, filtered (0.45 μm pore size, Millipore, Cat. S2HVU01RE, Burlington, MA, USA), and stored at −80 ◦C in 1 mL aliquots until use.

#### *4.12. Pseudovirus Neutralization Assay*

Derivatives of HEK-293T cells expressing ACE2 were generated by transducing HEK-293T cells with ACE2 (Addgene plasmid, 145839). Cells were used as single cell clones derived by limiting dilution from the bulk populations. The HEK-293T cells expressing ACE2 were seeded at a density of 1.5 × 104 cells/well in 96-well luminometer-compatible tissue culture plates (Corning, Cat. 3610, New York, NY, USA) 24 h before infection. For the neutralization assay, 30 uL of pseudoviruses (~1 × 106 RLU) was incubated with serial dilutions of the test antibody (12 dilutions in a threefold stepwise manner) for 1 h at 37 ◦C, together with the virus control, and then added to the 96-well 293T-ACE2 cells. After 24 h of incubation, the inoculum was replaced with fresh medium. Luciferase activity was measured 72 h after infection. Briefly, cells were washed twice, carefully, with PBS and lysed with 40 μL/well of a Passive Lysis buffer (Promega, Cat. E1941, Madison, WI, USA). Luciferase activity in lysates was measured using the Nano-Glo Luciferase Assay System (Promega, Cat. N1130, Madison, WI, USA). Specifically, 40 μL of the substrate in a Nano-Glo buffer was mixed with 40 μL of cell lysate and incubated for 3 min at RT. NanoLuc luciferase activity was measured using a Filter max F5 (Molecular Devices, San Jose, CA, USA) with an integration time of 1000 ms. The IC50 values were calculated with nonlinear regression using GraphPad Prism 7 (GraphPad Software, Inc., San Diego, CA, USA).

#### *4.13. Authentic Virus Neutralization Assay*

The SARS-CoV-2 virus (NCCP43326) for this study was provided by the National Culture Collection for Pathogens (Osong Health Technology Administration Complex, Cheongju, Chungbuk-do, Korea). Vero cells were seeded in a 96-well plate (Greiner Bio-One, Cat. 655180, Kremsmünster, Austria) at a density of 1 × <sup>10</sup><sup>4</sup> cells per well. Serially, twofold-diluted mAbs and 100TCID50 (median tissue culture infectious dose) SARS-CoV-2 virus were incubated at RT for 0.5 h. mAb–virus mixtures were added to the Vero cells and incubated at 37 ◦C for 72 h. After 72 h of incubation, the supernatant was replaced with 100 μL of CellTiter-Glo® 2.0 Reagent (Promega, Cat. G9241, Madison, WI, USA) and incubated at RT for 10 min. Luciferase activity in lysates was measured using the CellTiter-Glo® 2.0 Assay (Promega, Cat. G9241, Madison, WI, USA). The luminescent signal was measured using a GloMax® Discover Microplate Reader (Promega, Cat. GM3000, Madison, WI, USA) and the IC50 values were calculated by nonlinear regression using GraphPad Prism 7 (GraphPad Software, Inc., San Diego, CA, USA). This experiment was conducted at Chungbuk National University in a BSL3 facility (KCDC (Korea Center for Disease Control)-14-3-07).

#### *4.14. Measurements of Affinity Using Bio-Layer Interferometry*

Affinity measurement was performed by BLI using an Octet QK384 (ForteBio, Menlo Park, CA, USA) instrument. The anti-SARS-2 RBD human antibody was immobilized at 15 μg/mL in 10× Kinetic Buffer (KB) (ForteBio, Cat. 18-1105, Menlo Park, CA, USA). SARS-2 RBD protein was prepared in six different concentrations (100~0 nM, in twofold serial dilutions) in 10× KB for baseline stabilization. Before the binding measurements, the Anti-Human IgG Fc Capture (AHC; Cat. 18-5060) (ForteBio, Menlo Park, CA, USA) sensor tips were washed with 10× KB for 60 s and incubated in a binding buffer for 300 s (loading step). After a 180 s baseline dip in the same buffer, the binding kinetics were measured by dipping each human IgG (C2 and D12)-coated sensor into a well containing SARS-CoV-2 RBD protein at the above six concentrations. The binding interactions were monitored over a 300 s association step, followed by a 500 s dissociation step, in which the sensors were dipped into new wells containing 10× KB only. Non-specific binding was assessed using sensor tips without human IgGs. Data analysis was performed using Octet Data Analysis Software v6.4 (ForteBio, Menlo Park, CA, USA). Data were fitted to a 1:1 binding model to determine an association rate (Kon, M−1s−1) and a dissociation rate (Koff, s−1), and the equilibrium dissociation constant (KD) was calculated using the kinetic constants as follows: equilibrium dissociation constant (KD, M) = Koff ÷ Kon.

### *4.15. Statistical Analysis*

Statistical analysis was carried out using GraphPad Prism version 7.0 (GraphPad Software, San Diego, CA, USA). All error bars reported are the standard error of the mean (± SEM), unless otherwise indicated. Pairwise comparisons were conducted using an unpaired *t*-test. Differences between groups were considered significant at *p*-values below 0.05 (\* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001).

#### **5. Conclusions**

We selected human anti-SARS-2 RBD mAbs from human synthetic Fab phage display libraries. We characterized the resulting Fabs and IgGs to observe their desirable biophysical properties, such as their high affinity, non-aggregation, and thermal stability. We conducted in vitro assays to assess their neutralizing activities against pseudo-typed and authentic SARS-CoV-2 and identified two clones, C2 and D12, which demonstrated an exceptional ability to block the viral entry into cells. Further refinement of the mAbs should allow for the development of promising human anti-SARS-CoV-2 therapeutic and diagnostic reagents.

#### **6. Patents**

We are in the process of obtaining a patent for the data on the human anti-SARS-2 RBD Fabs and IgGs in Korea (patent application number 10-2020-0161180; application date 26th November 2020).

**Supplementary Materials:** Supplementary materials can be found at https://www.mdpi.com/1422 -0067/22/4/1913/s1.

**Author Contributions:** Conceptualization and experiment design, Y.J.K. and D.Y.K.; investigation, Y.J.K., S.-R.L., M.H.L., H.-Y.C., and K.K.; supervision, D.Y.K.; project administration, D.Y.K. and T.G.L.; writing—original draft preparation, Y.J.K. and D.Y.K.; writing—review and editing, Y.J.K. and D.Y.K.; funding acquisition, T.G.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Osong Medical Innovation Foundation and Chungcheongbukdo (no grant numbers issued).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data sharing is not applicable due to patent-pending and techtransfer issue.

**Acknowledgments:** We thank Hyo Jeong Hong (Kangwon National University, Gangwon-do, Korea) for her work in constructing the human Fab synthetic phage display library. We also thank Min-Seok Song for his support in the neutralization assay on authentic SARS-CoV-2. Lastly, we are grateful to Gu-Sun Park, the Chairman of the Osong Medical Innovation Foundation, for his support throughout the research.

**Conflicts of Interest:** Y.J.K. and D.Y.K. are inventors with the Korean patent application number 10-2020-0161180 (application date 26th November 2020). The authors declare no other conflicts of interest.

#### **Abbreviations**


