science.sciencemag.org/cgi/content/full/science.abd0827/DC1

Supplementary Materials for

Studies in humanized mice and convalescent humans yield a SARS-

CoV-2 antibody cocktail

Johanna Hansen*, Alina Baum*, Kristen E. Pascal, Vincenzo Russo, Stephanie Giordano,

Elzbieta Wloga, Benjamin O. Fulton, Ying Yan, Katrina Koon, Krunal Patel, Kyung Min

Chung, Aynur Hermann, Erica Ullman, Jonathan Cruz, Ashique Rafique, Tammy Huang,

Jeanette Fairhurst, Christen Libertiny, Marine Malbec, Wen-yi Lee, Richard Welsh, Glen

Farr, Seth Pennington, Dipali Deshpande, Jemmie Cheng, Anke Watty, Pascal Bouffard,

Robert Babb, Natasha Levenkova, Calvin Chen, Bojie Zhang, Annabel Romero

Hernandez, Kei Saotome, Yi Zhou, Matthew Franklin, Sumathi Sivapalasingam, David

Chien Lye, Stuart Weston, James Logue, Robert Haupt, Matthew Frieman, Gang Chen,

William Olson, Andrew J. Murphy, Neil Stahl, George D. Yancopoulos, Christos A.

Kyratsous†

*These authors contributed equally to this work.

†Corresponding author. Email: christos.kyratsous@regeneron.com

Published 15 June 2020 on Science First Release

DOI: 10.1126/science.abd0827

This PDF file includes:

Materials and Methods

Figs. S1 to S7

Tables S1 to S5

Caption for Data S1

References

Other Supplementary Material for this manuscript includes the following:

(available at science.sciencemag.org/cgi/content/full/science.abd0827/DC1)

MDAR Reproducibility Checklist (.pdf)

Data S1 (.txt)

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Materials and Methods

Convalescent human donors: Whole blood was collected from 3 convalescent human donors (ages 18-60 years); 3-4 weeks post laboratory-confirmed PCR positive test for SARS-CoV-2 and symptomatic COVID19 disease. Samples were received from the National Centre for Infectious Diseases (NCID), Singapore and collections were in accordance to the National Healthcare Group, Domain Specific Review Board E/2020/00210.

Immunization. Thirty-two VelocImmune® (VI) mice were immunized using DNA encoding full length (FL) SARS-CoV-2 spike protein and a recombinant protein of spike receptor binding domain (RBD) with an inline fusion of mouse Fc tag on the C-terminus (RBD-mFc). Pre-immune sera were collected from the mice prior to the initiation of immunization. The mice were primed with intradermal (id) injection of the DNA plasmid and boosted on days 9, 12 and day 14 post with a subcutaneous injection on the footpad (fp) of the RBD protein mixed with CpG oligodeoxynucleotides and aluminum phosphate adjuvants. All mice were bled on day 21 and anti-serum titers were determined using an immunoassay as described below. On day 22, three days prior to antibody isolation mice received a final boost via a intravenous (iv), fp and intraperitoneal (ip) injection of RBD-mFc without adjuvant and FL DNA by iv routes.

Anti-serum Titer Determination. Antibody titers in serum against the immunogen were determined using a direct coating ELISA. Briefly, 96-well microtiter plates (Thermo Scientific) were coated a recombinant SARS-CoV-2 spike RBD protein with hFc tag on the C-terminus (RBD-hFc) in phosphate-buffered saline (PBS, Irvine Scientific) overnight at 2 µg/ml. Plates were washed with phosphate-buffered saline containing 0.05% Tween 20 (PBS-T, Sigma-Aldrich) and blocked with 1% bovine serum albumin (BSA, Sigma-Aldrich) in PBS for 1 hour at room temperature (RT). The plates were washed with PBS-T. Pre-immune and immune anti-sera in a 1:3 fold serial dilutions in 1% BSA-PBS were added to the plates and incubated for1 hour at RT. The plates were then washed and a secondary reagent goat anti-mouse IgG-Fc polyclonal antibodies with Horseradish Peroxidase- (HRP) conjugated (Jackson ImmunoResearch) was added to the wells and incubated for one hour at RT. Plates were washed and developed using TMB/H2O2 as substrate according to manufacturer’s recommended protocol and the plates were read on a spectrophotometer (Victor, Perkin Elmer) and absorbance at 450 nm were recorded. The dilution-factor dependent mouse IgG binding (OD450 nm) was graphed using Graphpad PRISM software and the titers were extrapolated. Here, the titer is defined as the dilution factor of a serum sample that produces a binding signal equivalent to two times of the background signal of a well with the absence of serum samples.

VelocImmune® mouse and human single cell antibody isolation. Anti-SARS-CoV-2 antibodies were generated by either immunization of VelocImmune® mice or by isolation from PBMCs of human donors previously infected with SARS-CoV-2. For convalescent donor samples, whole blood was received from patients 3-4 weeks post laboratory-confirmed PCR positive test for SARS-CoV-2 and symptomatic COVID-19 infection. Red blood cells were lysed in ACK lysis buffer (Life Technologies), and PBMCs were isolated by a Ficoll gradient. From the PBMCs, single B cells that bind the nCoV spike protein were isolated by fluorescent-

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activated cell sorting (FACS). Isolated B cells (from humans or VelocImmune® mice) were single well plated and mixed with antibody light and heavy variable region-specific PCR primers (8). cDNAs for each single B cell were synthesized via a reverse transcriptase (RT) reaction. Each resulting RT product was then split and transferred into two corresponding wells for subsequent antibody heavy and light chain PCRs. One set of the resulting RT products was first amplified by PCR using a 5’ degenerate primer specific for antibody heavy variable region leader sequence or a 5’ degenerate primer specific for antibody light chain variable region leader sequence and a 3’ primer specific for antibody constant region, to form an amplicon. The amplicons were then amplified again by PCR using a 5’ degenerate primer specific for antibody heavy variable region framework 1 or a 5’ degenerate primer specific for antibody light chain variable region framework 1 and a 3’ primer specific for antibody constant region, to generate amplicons for cloning. Antibody variable regions specific for the SARS-CoV-2 spike isolated from either VelocImmune® mice or human donors were cloned into expression vectors containing human heavy constant region and light constant region for production in CHO cell lines. The expression vectors expressing full-length heavy and light chain pairs were transfected into CHO cells, expressed in supernatants and screened in functional assays. Lead antibodies were selected based on potent neutralization of SARS-CoV-2 spike (S) pseudotyped VSV.

Next Generation Sequencing of antibody repertoires. The variable heavy and light chains are separately amplified by PCR from miniprep DNA with the forward primers in the signal sequence and reverse primers in the constant regions of the antibody. The 1st PCR products are cleaned up using AMPure beads and run on the LabChip GX Touch HT Nucleic Acid Analyzer (Perkin Elmer) to both quantify and verify presence of the products, these are than normalized with water to 10 ng/ul. The normalized 1st PCR products are than further amplified by PCR to add unique barcodes and the P5 and P7 regions to each sample that will allow the library to cluster on the illumina flowcell. The unique barcodes will let us demultiplex the solutions after the sequencing run is completed. The 2nd PCR products are cleaned up using AMPure beads and quantified individually using LabChip GX Touch HT Nucleic Acid Analyzer (Perkin Elmer), they are than normalized to a final concentration of 10 nM or 20 nM and pooled together in one tube. The pooled libraries are further quantified using the high sensitivity dsDNA HS assay kit on the Qubit fluorometer (Thermo Scientific) before loading onto a Miseq sequencer (Illumina) for sequencing using Miseq Reagent Kits v3 (2x300 cycles). For bioinformatic analysis, Raw Illumina sequences were de-multiplexed and filtered based on quality, length and perfect match to corresponding first PCR primer.s Overlapping paired-end reads were merged and analyzed using IgBLAST (NCBI, v2.2.25+) to align rearranged light chain sequences to human germline V and J gene database. Productive and non-productive joining events were noted along with the presence of stop codons. CDR3 sequences and likely non-template nucleotides were extracted using International Immunogenetics Information System (IMGT) boundaries.

Anti-SARS-CoV-2-S antibody binding specificity assay. A Luminex binding assay was performed to determine the binding of anti-SARS-COV-2-S antibodies to a panel of antigens. For this assay, antigens were amine-coupled or captured by streptavidin to Luminex microspheres as follows: approximately 10 million MagPlex microspheres (Luminex Corp., MagPlex Microspheres, Cat. No. MC10000 and MC12000), were resuspended by vortexing in 500 µL 0.1M NaPO4, pH 6.2 (activation buffer) and then centrifuged to remove the supernatant.

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Microspheres were protected from light, as they are light sensitive. The microspheres were resuspended in 160 µL of activation buffer and the carboxylate groups (-COOH) were activated by addition of 20 µL of 50 mg/mL of N-hydroxysuccinimide (NHS, Thermo Scientific, Cat. No. 24525) followed by addition of 20 µL of 50 mg/mL 1-ethyl-3-[3 dimethylaminopropyl] carbodiimide (EDC, ThermoScientific, Cat. No. 22980) at 25 °C. After 10 minutes, the pH of the reaction was reduced to 5.0 with the addition of 600 µL 50 mM MES, pH 5 (coupling buffer), and the microspheres were vortexed and centrifuged to remove supernatant. The activated microspheres were immediately mixed with 500 µL of 25 µg/mL of the protein antigen or Streptavidin in coupling buffer and incubated for two hours at 25 °C. The coupling reaction was quenched by addition of 50 µL of 1M Tris-HCl, pH 8.0 and the microspheres were vortexed, centrifuged, and washed three times with 800 µL of PBS 0.005% (Tween20 0.05%), to remove uncoupled proteins and other reaction components. Microspheres were resuspended in 1 mL of PBS 2% BSA 0.05% Na Azide at 10 million microspheres/mL. For Streptavidin capture of antigens, 500 µL of 12.5 µg/mL of biotinylated protein in PBS was added to Streptavidin-coupled microspheres and incubated for one hour at 25 °C. Microspheres were vortexed, centrifuged, and washed three times with 800 µL of PBS, and then blocked using 500 µL 30mM Biotin (Millipore-Sigma, Cat. No. B4501) in 0.15M Tris pH 8.0. Microspheres were incubated for 30 minutes then vortexed, centrifuged, and washed three times with 800 µL of PBS. Microspheres were resuspended in 1 mL of PBS 2% BSA 0.05% Na Azide at 10 million microspheres/mL. Microspheres for the different proteins and biotinylated proteins were mixed at 2700 beads/ml, and 75 µL of microspheres were plated per well on a 96 well ProcartaPlex flat bottom plate (ThermoFisher, Cat. No: EPX-44444-000) and mixed with 25 µL of individual anti-SARS-CoV-2 supernatant containing antibody. Samples and microspheres were incubated for two hours at 25oC and then washed twice with 200 µL of DPBS with 0.05% Tween 20. To detect bound antibody levels to individual microspheres, 100 µL of 2.5 µg/mL R-Phycoerythrin conjugated goat F(ab')2 anti-human kappa (Southern Biotech, Cat# 2063-09) in blocking buffer (for antibodies with murine Fc regions) or 100 µL of 1.25 µg/mL R-Phycoerythrin AffiniPure F(ab')₂ Fragment Goat Anti-Mouse IgG, F(ab')₂ Fragment Specific (Jackson Immunoresearch, Cat. No: 115-116-072) in blocking buffer (for antibodies with human Fc regions), was added and incubated for 30 minutes at 25 °C. After 30 minutes, the samples were washed twice with 200 µl of washing buffer and resuspended in 150 µL of wash buffer. The plates were read in a Luminex FlexMap 3Dâ (Luminex Corp.) and Luminex xPonentâ software version 4.3 (Luminex Corp.). Generation of recombinant VSV. Non-replicative pseudoparticles were generated using a VSV genome encoding the mNeonGreen fluorescent reporter gene (23) instead of the native viral glycoprotein (VSV-G). Infectious particles complemented with VSV-G (VSVΔG:mNeon/VSV-G) were recovered and produced using standard techniques with minor modifications (24-26). Briefly, HEK293T cells (ATCC CRL-3216) were transfected with the genomic clone driven by a T7 promoter and helper plasmids expressing the VSV-N, VSV-P, VSV-G, VSV-L, and T7 RNA polymerase with Lipofectamine LTX reagent (Life Technologies). After 48 hours, the transfected cells were co-cultured with BHK-21 cells (ATCC CCL-10) transfected with VSV-G using the SE cell Line 4D-Nucleofector X Kit L (Lonza). Cells were monitored for mNeonGreen expression or cytopathic effect (CPE) indicative of virus replication. Virus was then plaque purified, expanded, and titered in BHK-21 cells transiently expressing VSV-G. Fully replicative VSV-SARS-CoV-2-S virus was generated by replacing the VSV glycoprotein with the native

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SARS-CoV-2 sequences encoding residues 1-1255 of the spike protein (MN908947.3). VSV-SARS-CoV-2-S virus was recovered as described above but the HEK293T cells were instead co-cultured with BHK-21 cells transfected with both VSV-G and hACE2. VSV-SARS-CoV-2-S virus was plaque purified and titered in Vero cells (ATCC CCL-81) and expanded in Vero E6 cells (ATCC CRL-1586). After collection, stocks of both viruses were centrifuged at 3000xg for 5 minutes to clarify, sucrose cushioned to concentrate 10-fold, aliquoted, and frozen at -80C. Pseudotyping of VSV. Non-replicative pVSV-SARS-CoV-2-S (mNeon) pseudoparticles were generated using modified methods from those previously described(27-29). Human codon-optimized CoV-SARS-2 spike (MN908947.3) was synthesized (Genscript) and resulting product was cloned into an expression plasmid. A total of 1.2 x 107 HEK293T cells (ATCC CRL-3216) were seeded overnight in 15-cm dishes in DMEM high glucose media (Life Technologies) containing 10% heat-inactivated fetal bovine serum (Life Technologies), and Penicillin/-Streptomycin-L-Glutamine (Life Technologies). The following day, the cells were transfected with 15ug spike expression plasmid with Lipofectamine LTX (Life Technologies) following the manufacturer’s protocol. At 24 hours post transfection, the cells were washed with phosphate buffered saline (PBS) and infected at a MOI of 1 with the VSVΔG:mNeon/VSV-G virus diluted in 10mL Opti-MEM (Life Technologies). The cells were incubated 1 hour at 37C with 5% CO2. Cells were washed three times with PBS to remove residual input virus and overlaid with DMEM high glucose media (Life Technologies) with 0.7% Low IgG BSA (Sigma), sodium pyruvate (Life Technologies), and Gentamicin (Life Technologies). After 24 hours at 37C with 5% CO2, the supernatant containing pseudoparticles was collected, centrifuged at 3000xg for 5 minutes to clarify, aliquoted, and frozen at -80C. Cleavage site mutants were cloned into the spike expression plasmid using site-directed mutagenesis and pseudoparticles were produced as described above. Neutralization assays with VSV based pseudoparticles and virus. Unless otherwise noted, all reagents obtained from Life Technologies. Vero cells (ATCC: CCL-81) were seeded in 96-well black, clear bottom tissue culture treated plated (Corning: 3904) at 20,000 cells/well in DMEM high glucose media containing 10% heat-inactivated fetal bovine serum, and 1X Penicillin/Streptomycin/L-Glutamine 24 hours prior to assay. Cells were allowed to reach approximately 85% confluence before use in assay. Antibodies were diluted in DMEM high glucose media containing 0.7% Low IgG BSA (Sigma), 1X Sodium Pyruvate, and 0.5% Gentamicin (this will be referred to as “Infection Media”) to 2X assay concentration and diluted 3-fold down in Infection media, for an 11-point dilution curve in the assay beginning at 10 ug/mL (66.67 nM). pVSV-SARS-CoV-2-S (mNeon) was diluted 1:1 in Infection media for a fluorescent focus (ffu) count in the assay of ~1000 ffu. Antibody dilutions were mixed 1:1 with pseudoparticles for 30 minutes at room temperature prior to addition onto Vero cells. Cells were incubated at 37C, 5% CO2 for 24 hours. Supernatant was removed from cells and replaced with 100 uL PBS, and fluorescent foci were quantitated using the SpectraMax i3 plate reader with MiniMax imaging cytometer. Exported values were analyzed using GraphPad Prism (v8.2.0). For replicative VSV-SARS-CoV-2-S virus neutralization assays, antibodies were diluted as described above but in VSV media (DMEM high glucose media containing 3% heat-inactivated fetal bovine serum and Penicillin/-Streptomycin-L-Glutamine). An equal volume of media containing 2000 pfu of VSV-SARS-CoV-2-S virus was mixed with the antibody dilutions and

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incubated for 30 minutes at room temperature. The mixture was then added onto Vero cells and incubated at 37C, 5% CO2 for 24 hours. The cells were fixed (PBS with 2% paraformaldehyde) for 20 minutes, permeabilized (PBS with 5% fetal bovine serum and 0.1% Triton-X100) for 15 minutes and blocked (PBS with 3% bovine serum albumin) for 1 hour. Infected cells were immunostained with a rabbit anti-VSV serum (Imanis Life Sciences) and an Alexa Fluor® 488 secondary antibody in PBS + 3% bovine serum albumin. Fluorescent foci were quantitated using the SpectraMax i3 plate reader with MiniMax imaging cytometer. PRNT50 Assays with SARS-CoV-2 virus. Monoclonal antibodies and antibody combinations were serially diluted in DMEM (Quality Biological), supplemented with 10% (v/v) heat inactivated fetal bovine serum (Sigma), 1% (v/v) penicillin/streptomycin (Gemini Bio-products) and 1% (v/v) L-glutamine (2 mM final concentration, Gibco) (VeroE6 media) to a final volume of 250 μL. Next, 250 μL of VeroE6 media containing SARS-CoV-2 (WA-1) (1000 PFU/mL) was added to each serum dilution and to 250 μL media as an untreated control. The virus-antibody mixtures were incubated for 60 min at 37°C. Following incubation, virus titers of the mixtures were determined by plaque assay. Finally, we calculated the 50% plaque reduction neutralization titer (PRNT50) values, the serum dilutions at which plaque formation was reduced by 50% relative to that of the untreated control, using a 4-parameter logistic curve fit to the percent neutralization data (GraphPad Software, La Jolla, CA).

SARS-CoV-2 spike protein receptor binding domain (RBD) ELISA. The SARS-CoV-2 protein used in the experiments was comprised of the receptor binding domain portion of the SARS-CoV-2 spike protein (aa Arg319-Phe541) expressed with 6X histidine and two myc epitope tags at the c-terminus (SARS-CoV-2 RBD-mmH SEQ ID: MN908947.3). SARS-CoV-2 RBD-mmH was coated at 1mg/ml in PBS on a 96-well microtiter plate overnight at 4°C. Nonspecific binding sites were subsequently blocked using a 0.5% (w/v) solution of BSA in PBS. Antibody supernatants or media alone were diluted 1:40 or 1:50 in PBS+0.5% BSA and transferred to the washed microtiter plates. After one hour of incubation at RT, the wells were washed, and plate-bound antibody was detected with either goat-anti-human IgG antibody conjugated with horseradish peroxidase (HRP) (Jackson Immunoresearch), or anti-mouse IgG antibody conjugated with horseradish peroxidase (HRP) (Jackson Immunoresearch). The plates were then developed using TMB substrate solution (BD Biosciences) according to manufacturer’s recommendation and absorbance at 450nm was measured on a Victor X5 plate reader.

SARS-CoV-2 blocking ELISA to hACE2. The SARS-CoV-2 protein used in the experiments was comprised of the receptor binding domain (RBD) portion of the SARS-CoV-2 spike protein (aa Arg319-Phe541) expressed with the Fc portion of the human IgG1 at the c-terminus (SARS-CoV-2 RBD-hFc SEQ ID: MN908947.3). The human ACE2 protein used in the experiments was purchased from R&D systems and is comprised of aa Gln18-Ser740 with a c-terminal 10X-Histidine tag (hACE2-His; accession#Q9BYF1). A monoclonal anti-Penta-His antibody (Qiagen) was coated at 1mg/ml in PBS on a 96-well microtiter plate overnight at 4°C. The hACE2-His receptor was added at 0.2 mg/ml in PBS and bound for 2 hours at RT. Nonspecific binding sites were subsequently blocked using a 0.5% (w/v) solution of BSA in PBS. In other microtiter plates, a constant amount of 10 or 15pM (as indicated in the experimental table) of

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SARS-CoV-2 RBD-hFc protein was bound with antibodies diluted 1:10 or 1:20 in PBS +0.5% BSA. These antibody-protein complexes, after a one-hour incubation, were transferred to the microtiter plate coated hACE2-His. After 1.5 hours of incubation at RT, the wells were washed, and plate-bound SARS-CoV-2 was detected with goat-anti-human IgG antibody conjugated with horseradish peroxidase (HRP) (Jackson). The plates were then developed using TMB substrate solution (BD Biosciences, #555214) according to manufacturer’s recommendation and absorbance at 450nm was measured on a Victor X5 plate reader. Data analysis was performed by calculating the % blocking of the antibody vs the constant alone. In the calculation, binding signal of the sample of the constant SARS-CoV-2 RBD-hFc without the presence of the antibody for each plate was referenced as 100% binding or 0% blocking; and the baseline signal of the sample of media only without the presence of SARS-CoV-2 RBD-hFc was referenced as 0% binding or 100% blocking.

BIAcore surface plasmon resonance analysis. Binding kinetics and affinities for anti-spike mAbs were assessed using surface plasmon resonance technology on a Biacore T200 instrument (GE Healthcare, Marlborough, MA) using a Series S CM5 sensor chip in filtered and degassed HBS-EP running buffer (10 mM HEPES, 150 mM NaCl, 3mM EDTA, 0.05% (v/v) polysorbate 20, pH 7.4). A capture sensor surfaces were prepared by covalently immobilizing with a mouse anti-human Fc mAb (REGN2567) on to the chip surface using the standard amine coupling chemistry, reported previously (30). Following surface activation, the remaining active carboxyl groups on the CM5 chip surface were later blocked by injecting 1M ethanolamine, pH8.0 for 7 minutes. A typical resonance unit (RU) signal of about ~10,000 RU was achieved after the immobilization procedure. The binding of anti-spike mAbs to their respective target was performed on a CM5 sensor surface immobilized with either an anti-human Fc mAb at 37ºC. At the end of each cycle, the anti-human Fc surface was regenerated using a 12 second injection of 20mM phosphoric acid. The binding to SARS-CoV2 spike protein RBD ectodomain expressed with a C-terminal myc-myc-hexahistidine tag (RBD.mmh), spike protein RBD ectodomain expressed with a C-terminal mouse IgG2a Fc tag (RBD.mFc), or ecto foldon Trimer expressed with a C-terminal myc-myc-hexahistidine (SARS-CoV2 Spike ECD foldon) was determined for each SARS-CoV2 mAb. Following the capture of the SARS-CoV2 mAb on the anti-human Fc mAb immobilized surface, different concentrations of RBD.mmh (3.33nM - 90nM, three-fold serial dilution), RBD.mFc (1.11nM – 30nM, three-fold serial dilution) or SARS-CoV2 Spike ECD foldon (0.78nM – 25nM, three-fold serial dilution) were injected for 3 minutes at a flow rate of 50 µL/min with a 6-10 minutes of dissociation phase in the running buffer. All of the specific SPR binding sensorgrams were double-reference subtracted as reported previously (31) and the kinetic parameters were obtained by globally fitting the double-reference subtracted data to a 1:1 binding model with mass transport limitation using Biacore T200 Evaluation software v 3.1 (GE Healthcare) or Biacore Insight Evaluation software (GE Healthcare). The dissociation rate constant (kd) was determined by fitting the change in the binding response during the dissociation phase and the association rate constant (ka) was determined by globally fitting analyte binding at different concentrations. The equilibrium dissociation constant (KD) was calculated from the ratio of the kd and ka. The dissociative half-life (t½) in minutes was calculated as ln2/(kd*60).

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Competition Binning Analyses by Bio-Layer Interferometry (BLI). Epitope binning of the anti-COVID19 mAbs was conducted in a pre-mix sandwich format (32) involving competing mAbs against one another in a pairwise combinatorial manner for binding to SARS CoV-2 RBD-MMH protein using a ForteBio Octet HTX biolayer interferometry instrument (Molecular Devices ForteBio LLC, Fremont, CA) with running buffer of 10 mM HEPES, 150 mM NaCl, 0.05% (v/v) Tween-20, pH 7.4, 1 mg/mL BSA. Assays were performed at 30 ºC with continuous agitation at 1000 rpm. After obtaining an initial baseline in running buffer 20 μg/mL of anti-COVID19 mAbs was captured onto anti-human Fc (AHC) biosensor tips for 300 s. To block remaining free unsaturated binding sites on AHC biosensor tips, all sensors were exposed for 240 s to blocking solution well containing 100 μg/mL irrelevant IgG1. Following this process, biosensors were immersed into wells containing pre-mix solution of 100nM SARS CoV-2 RBD-MMH protein and 600 nM of anti-COVID19 mAb binding site of a second mAbs for 300 s. Binding response at each step was recorded and specific signal was normalized by subtracting self-blocking mAb competing control from dataset. Data analysis was performed with Octet Data Analysis HT 10.0 software using the Epitope Binning.

HDX-MS analysis. Experiments were performed on an integrated platform consisting of a LEAP HDX PAL system for deuterium labeling and quenching, a Waters Acquity Binary Solvent Manager for sample digestion and loading, a Waters Acquity Binary Solvent Manager for analytical gradient, and a Thermo Q Exactive HF mass spectrometer for peptide identification and mass measurement. Protein samples were prepared as 4 µM to 15 µM RBD alone or RBD premixed with each mAb in a 1:1 ratio in PBS buffer (10 mM sodium phosphate, 137 mM NaCl, 3 mM KCl, pH 7.4). Non-deuterated samples, prepared as 10 μL of each sample incubated with 90 μL PBS buffer, were used to identify peptide sequences and determine peptide masses without deuterium exchange. For HDX reactions, 10 μL of each sample above was incubated with 90 μL D2O labeling solution (10 mM sodium phosphate, 137 mM NaCl, 3 mM KCl, pD 7.0, equivalent to pH 7.4 at 25°C) for various times. For REGN10989, 10987, 19834 and 10933, the incubation time periods were 5 min and 10 min. For rest of the mAbs, a single incubate time of 10 min was used to increase experimental throughput. All experiments were performed in duplicate. After incubation, samples were quenched by adding 100 μL of quench buffer (0.5 M TCEP, 4 M urea and 0.5% formic acid, pH 2.1) and incubated for 90 s at 20 °C. The quenched samples were injected into the LC on the LEAP HDX PAL system for online pepsin/protease XIII digestion using a flow rate of 0.1 mL/min. The digested peptides were trapped in a Waters Acquity BEH C18 VanGuard Pre-column (2.1 mm x 5 mm) and further separated by a Waters Acquity BEH C18 column (2.1 mm x 50 mm) at -5 °C using a gradient from 1% to 95% mobile phase B at 0.2 mL/min flow rate (Mobile phase A was 0.5% formic acid, 4.5% acetonitrile in water; mobile phase B was 0.5% formic acid in acetonitrile). The eluted peptides were analyzed by Q Exactive HF mass spectrometry in LC-MS/MS mode for peptide mapping and LC-MS mode for HDX data acquisition. The LC-MS/MS data of non-deuterated samples were searched against a database containing sequences of the RBD protein, pepsin and protease XIII using the Byonic search engine (Protein Metrics) with parameters for non-specific enzymatic digestion. The identified peptide list was

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then imported into the HDExaminer software (version 3.1) together with the LC-MS data from all deuterated samples to calculate the deuterium uptake level of individual peptides. Mass spectra of each peptide were examined manually to ensure that deuterium uptake levels were calculated using the correct isotopic patterns of corresponding peptides. HDX-MS data were obtained for RBD peptides representing 84-87% of its amino acid sequence. Peptide deuterium uptake percentages calculated from RBD-mAb and RBD-only samples were compared. Any peptide that exhibited a reduction in deuterium uptake of 5% or greater upon mAb binding was defined as protected. Cryo-EM sample preparation and data collection. Fab fragments of REGN10933 and REGN10987 antibodies were isolated using FabALACTICA kit (Genovis). 600 µg of the REGN10933 fab and 600 µg of REGN10987 fab were mixed with 300 µg of SARS-CoV-2 RBD and incubated on ice for ~1 hour then injected into a Superdex 200 increase gel filtration column equilibrated to 50 mM Tris pH 7.5, 150 mM NaCl. Peak fractions containing the REGN10933 fab - REGN10987 fab - RBD complex were collected and concentrated using a 10 kDa MWCO centrifugal filter. For cryo-EM grid preparation, the protein sample was diluted to 1.5 mg/mL and 0.15% PMAL-C8 amphipol was added. 3.5 µL of protein was deposited onto a freshly plasma cleaned UltrAufoil grid (1.2/1.3, 300 mesh). Excess solution was blotted away using filter paper and plunge frozen into liquid ethane using a Vitrobot Mark IV. The Cryo-EM grid was transferred to a Titan Krios (Thermo Fisher) equipped with a K3 detector (Gatan). Movies were collected using EPU (Thermo Fisher) at 105,000x magnification, corresponding to a pixel size of 0.85 Å. A dose rate of 15 electrons per pixel per second was used and each movie was 2 seconds, corresponding to a total dose of ~40 electrons per Å2. Cryo-EM data processing. All cryo-EM data processing was carried out using cryoSPARC v2.14.2 (33). 2,821 movies were aligned using patch motion correction and patch CTF estimation. 2,197 aligned micrographs were selected for further processing on the basis of estimated defocus values and CTF fit resolutions. An initial set of particles picked using blob picker were subjected to 2D classification to generate templates for template picking. 989,553 particles picked by template picking were subjected to multiple rounds of 2D classification to remove unbound fabs and particles containing an incomplete complex. Ab initio reconstruction with three classes generated a single class containing 61,707 particles that corresponded to the REGN10933 fab - REGN10987 fab - RBD complex. Heterogenous refinement of the particles in this class followed by non-uniform refinement resulted in a 3.9 Å resolution (FSC=0.143) map containing 48,140 particles that was used for model building. Into this map, we manually placed models of the RBD (taken from PDB code 6M17) and the two Fabs (taken from prior Regeneron antibody structures, except for the lambda light chain of REGN10987 which came from PDB code 5U15). These models were then manually rebuilt using Coot and real-space refined against the map using Phenix. ADCC NK cell purification and purity assessment. Unstimulated human NK cells were isolated from leukocyte-enriched whole blood by density gradient centrifugation using NK RosetteSep Human NK Cell Enrichment Cocktail (Stemcell Technologies) and SepMate tubes (Stemcell Technologies) according to the manufacturer’s instructions. To evaluate NK cell enrichment, cells were stained with LIVE/DEAD Fixable Aqua Dead Cell Stain (Thermo Fisher) according to the manufacturer’s instructions, followed by incubation for 30 min on ice with a

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blood cell phenotyping cocktail of fluorophore-conjugated antibodies (anti-CD56, anti-CD3, anti-CD19, and anti-CD14). Cells were then fixed in Cytofix fixation buffer (BD Biosciences) for 30 min, filtered through an AcroPrep 40uM Advance Filter Plate (Pall Corporation) and acquired on a Cytoflex flow cytometer (Beckman Coulter). Data were analyzed with FlowJo software (BD). Live, single-cell, lymphocyte events were then either plotted as CD3 versus CD56 or as CD14 versus CD19. Cell populations after NK cell enrichment were determined as follows: CD56+ CD3- (NK cells), CD56- CD3+ (T cells), CD56+ CD3+ (NK T cells), CD19+ (B cells), and CD14+ (monocytes). ADCC Target Cell Generation. For generation of target cells the Jurkat cell line (ATCC, TIB-152), stably transduced with human CD20 (Uniprot accession #: P11836, amino acids M1 to P297), was subsequently engineered to express full length SARS-CoV-2 spike protein (Uniprot accession #: P0DTC2, amino acids M1 to T1273). After puromycin selection, a population of cells expressing SARS-CoV-2 spike protein at a high, uniform, level were isolated via fluorescent activated cell sorting (FACS) using SARS-CoV-2 (2019-nCoV) anti-spike antibody (Sino Biological, cat#40150-R007), followed by incubation with Alexa Fluor® 647 Anti-Rabbit IgG secondary antibody (Jackson ImmunoResearch, Cat# 111-606-046). Cells were maintained in RPMI + 10% FBS + penicillin/streptomycin/glutamine (P/S/G) + 250 mg/mL hygromycin, supplemented with 1 µg/mL puromycin for cells expressing SARS-CoV-2 spike protein. ADCC assay. Jurkat or Jurkat/SARS-CoV-2-spike cells were resuspended in assay media (RPMI supplemented with 1% BSA, 100 U/ml penicillin, 100 μg/ml streptomycin, and 292 μg/ml L-glutamine) and added in triplicate to opaque, white 96-well flat-bottom plates (ThermoFisher) at a concentration of 5x103 cells/well. Anti-spike antibodies or IgG1 isotype control were serially diluted following a 12-point 1:4 titration in assay media with the 12th point containing no antibody, such that their concentration in the assay ranged from 190.7 fM to 200 nM and added to the assay plate. Human NK cells were diluted in assay media and added to the assay plate at a final concentration of 2.5 x 104 cells/well. Control samples containing all components except the antibody was incorporated into each experiment to determine the background signal of the assay (ie, nonspecific lysis of target cells in the presence of NK cells). To assess spontaneous lysis, untreated Jurkat cells alone (target cells) and NK cells alone (effector cells) were incubated in separate wells. Plates were incubated at 37°C, 5% CO2 for 3.5 hours. After incubation, the plates were equilibrated to room temperature for 10 minutes, followed by the addition of CytoTox Glo reagent (Promega) to the wells for 15 minutes while shaking. The luminescence signal was measured as a readout of cytotoxicity using an ENVISION plate reader. The cytotoxic response was calculated as follows: Cytotoxicity (%) = (Experimental Signal – SBS (target cells) – SBS (effector cells))/(Max signal (target cells w/digitonin) – SBS (target

cells)) x100 where SBS represents the spontaneous background signal. From this value, background was subtracted: (Value) – (Average of No antibody). For EC50 determinations, % Cytotoxicity was analyzed using a 4-parameter logistic equation over a 12-point response curve with GraphPad Prism. The experiment was run in triplicate with 3 donors. Differentiation of Primary Monocyte-Derived Macrophages. Frozen CD14+ cells obtained from Lonza were thawed, resuspended in assay media (RPMI 1640 supplemented with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 292 μg/ml L-glutamine, NaPyr, HEPES, NEAA, and 10 µM BME) supplemented with 100 ng/ml M-CSF, and plated at 5.5 x 104

11

cells/well into clear-bottom, collagen-coated 96-well plates for differentiation into phagocytes over 7 days, with fresh M-CSF (100 ng/ml) added on day 4. ADCP Assay. Target cells and monocyte-derived phagocytes were incubated in PBS supplemented with either CellTrace CFSE dye or CellTrace Violet dye, respectively, for 15 minutes at 37°C, 5% CO2. After a series of washes in assay media (RPMI 1640 + 10% FBS + 1% P/S/G + NEAA + NaPyr + HEPES + 10 µM BME), CFSE-labeled target cells resuspended in assay media and 6 x 105 cells/well were added in duplicate to 96-well U-bottom plates, followed by the addition of anti-spike mAbs or an IgG1 isotype control (final concentrations ranging from 0.1pM to 20nM). Target cells without antibody were included as a control for background phagocytosis. Effector cells without target cells or antibody were included as a background control. After 15 minutes of incubation on ice the mixture of target cells, with or without titrated antibody, was then transferred to plates containing the violet-labelled macrophages and plates were incubated at 37°C, 5% CO2 for 30 minutes. Wells were then washed with PBS 3 times, followed by addition of 4.21% formaldehyde in PBS supplemented with 2.5uM DRAQ5. After a 20-minute incubation wells were washed with PBS and imaged in both the 488 nm (CFSE-labelled target cells) and 375 nm (violet-labelled phagocytes) excitation channels using an Opera Phenix High-Content Screening System. Image analysis was performed in Harmony software. Image segmentation in the 375 nm excitation channel was used to identify the phagocyte population. Image segmentation in the 488 nm excitation channel was used to identify the target cells. Phagocytosis was quantified by identifying the phagocyte population which contained target cells within them. Percent phagocytosis was calculated by comparing number of macrophages undergoing phagocytosis to total phagocyte cell number. From this value, background was subtracted: (Value) – (Average of No antibody). For EC50 determinations, % ADCP was analyzed with GraphPad Prism using a 4-parameter logistic equation over a 10-point dose response curve, the last point containing no antibody. Experiment was performed in duplicate across two donors. A representative donor is shown.

12

Fig. S1. VelocImmune® mice elicit robust anti-SARS-CoV-2 antibody titers post immunization. VelocImmune® mice (n=32) were immunized with SARS-CoV-2 as immunogen, primed with DNA coding full-length of SARS-Cov-2 spike protein followed by three boost injections of recombinant RBD protein. Anti-SARS-CoV-2 titers of the bleeds collected one week after the 2nd boost injection were assayed by a direct coating ELISA with SARS-CoV-2 protein (nCoV RBD.hFc) coated plates on day 21. Fig. S2. Antibody repertoire and CDR length for human and mouse-derived neutralizing antibodies. V gene frequencies in the Heavy (A) and Light (B, C) chain repertoires of isolated neutralizing antibodies to SARS-CoV-2 for VelocImmune® mice (Black bars; N=185) and convalescent human donors (Gray Bars; N=68). The gene segments were arranged left to right on the x axis according to their position on the human chromosome from distal to proximal relative to their constant regions. The CDR sequence/length for Heavy (D) and Light (B, C) chain were extracted using International Immunogenetics Information System (IMGT) boundaries. Neutralization is defined as >70% with 1:4 dilution of antibody (~2µg/ml) in VSV pseudoparticle neutralization assay.

Fig. S3. Effect of cleavage site mutations on neutralization and infectivity. (A) Top, the WT amino acid sequence of the S1/S2 polybasic cleavage site is underlined. Substitutes incorporated into SARS-CoV-2 S FurMut and FurKO mutants are marked in red. (B) Four-point neutralization curves (Log M: -7.48, -8.48, -9.48, and -10.48) for the lead antibodies in Vero cells. (B) Infectivity of the WT and cleavage mutants in Vero and Calu-3 cells at 24 and 48 hours, respectively. Infectivity of the mutants was normalized to the infectivity of the WT virus in each cell type. Fig. S4. Neutralization potency of antigen-binding fragment (Fab) and full-length antibodies. VSV-spike pseudoparticle neutralization by either full length antibodies or corresponding Fab in Vero cells. Fig. S5. Anti-SARS-CoV-2-spike antibodies induce ADCC. NK cells were purified from three healthy donor leukopaks. NK cells from donor 1 (A, D), donor 2 (B, E) and donor 3 (C, F) were mixed with either parental Jurkat cells (A, B, C) or Jurkat cells expressing SARS-CoV-2 spike Protein (D, E, F). A titration of Anti-SARS-CoV-2-spike Protein antibodies or an IgG1 isotype control were added to cells, incubated for 3.5h, and cell lysis assessed using CytoTox Glo reagent. Percent cytotoxicity was defined as 100 x [experimental signal – (NK alone signal + target alone signal)] / (Target maximum signal – target alone signal). From this value the average percent cytotoxicity in absence of antibody was subtracted. Each condition was tested in triplicate. Fig. S6. Anti-SARS-CoV-2-spike antibodies induce ADCP. Macrophages were differentiated from primary human monocytes with M-CSF, labeled with CellTrace violet and mixed with CFSE-labeled parental Jurkat cells (A) or Jurkat cells expressing SARS-CoV-2 spike Protein (B). A titration of anti-SARS-CoV-2-spike Protein antibodies or an IgG1 isotype control were added to cells, incubated for 30min, washed and subsequently imaged using a phenix confocal microscopy platform. Phagocytosis was defined as 100 x (macrophage containing engulfed target cell) / (Total number of macrophages). From this value, background was subtracted: (Value) –

13

(Average of No antibody), in order to derive % ADCP. Each condition was tested in duplicate with two donors. A representative donor is shown. Fig. S7. Epitope bin analysis from a matrix of pre-mix binding assays for different anti-SARS-CoV-2 mAbs. Epitope binning was performed against nine anti-SARS-CoV-2 mAb as described in the materials and method. There are three phases (I, II, and III) for each graph. In phase I anti-SARS-CoV-2 mAb (20ug/ml) was loaded to the anti-human Fc probe. In phase II human IgG1 blocking mAb solution (100ug/ml). In phase III solution of 100nM SARS CoV-2 RBD-MMH pre-mix complex of each 600 nM anti-SARS-CoV-2 mAb binding site flowed over the

14

Table. S1. VSV Pseudoparticle neutralization and SARS-COV-2 blocking ELISA to hACE2. Neutralization IC50 (M) shown for mab treatment of non-replicating pVSV-SARS-CoV-2-S-mNeon to Vero cells (Fig. 2A), or live SARS-COV-2 virus (Fig. 2D). In addition, an ELISA-based blocking assay shows the ability of anti-SARS-CoV-2 antibodies to block the binding of the SARS-CoV-2 spike protein RBD to hACE2 (0.2µg/ml). Table. S2. SPR-Biacore kinetics of anti-SARS-CoV-2 spike monoclonal antibodies. Equilibrium dissociation constants (KD) for different SARS-CoV-2-S antibodies were determined using a real-time surface plasmon resonance-based Biacore T200 instrument using a Series S CM5 sensor chip. Binding kinetics parameters for the nine monoclonal antibodies binding to nCoV RBD.mmh, nCoV RBD.mFc at 37°C are shown. For the top four mabs binding kinetics parameters for binding to SARS-CoV-2 spike ECD foldon 37°C is also shown (NT = not tested). Table S3. Anti-SARS-CoV-2-spike antibodies induce ADCC. ADCC was assessed as described in Figure 1. Max (% Cytotoxicity) was calculated for each NK donor and target cell line and is the highest mean % Cytotoxicity value within the tested dose range. Not calculated (NC) was reported when an EC50 could not be calculated because data did not fit a 4-parameter logistic equation. Not determined (ND) was reported when no dose dependent response was observed. Table S4. Anti-SARS-CoV-2-spike antibodies induce ADCP. ADCP was assessed as described in Figure 2. Max % ADCP is the highest mean % ADCP value within the tested dose range. Not determined (ND) was reported when no dose dependent response was observed. Table S5. CryoEM data statistics. Data collection and refinement statistics are reported for the REGN10987 + REGN10933 + SARS-CoV-2 RBD complex structure shown in Fig. 4. Data S1. VH and VL sequences of described antibodies

Pre-immune Post immunization100

1000

10000

100000

1000000

Ant

ibod

y tit

ers

Fig. S1.

Fig. S1. VelocImmune® mice elicit robust anti-SARS-CoV-2 antibody titers post immunization.VelocImmune® mice (n=32) were immunized with SARS-CoV-2 as immunogen, primed with DNA coding full-length of SARS-Cov-2 spike protein followed by three boost injections of recombinant RBD protein. Anti-SARS-CoV-2 titers of the bleeds collected one week after the 2nd boost injection were assayed by a direct coating ELISA with SARS-CoV-2 protein (nCoV RBD.hFc) coated plates on day 21.

IGKV

4-1

IGKV

1-5

IGKV

1-6

IGKV

1-9

IGKV

3-11

IGKV

1-12

IGKV

3-15

IGKV

1-16

IGKV

1-17

IGKV

3-20

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1-33

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1-39

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IGKV

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IGKV

1-6

IGKV

1-9

IGKV

3-11

IGKV

1-12

IGKV

3-15

IGKV

1-16

IGKV

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1-39

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hBST

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2-8

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3-21

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1-51

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6-57

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1-44

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1-2

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1

IGHV

2-5

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3-7

IGHV

1-8

IGHV

3-9

IGHV

3-11

IGHV

3-15

IGHV

1-18

IGHV

3-20

IGHV

3-23

IGHV

2-26

IGHV

3-30

IGHV

3-30

-3

IGHV

4-31

IGHV

3-33

IGHV

4-34

IGHV

4-39

IGHV

1-46

IGHV

3-48

IGHV

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IGHV

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IGHV

3-64

IGHV

3-66

IGHV

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human

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IGHV

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IGHV

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IGHV

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KCDR3 (AA) LCDR3 (AA)

VelocImmune® Derived

Human Derived

A

B C

D E F

Fig. S2.

Fig. S2. Antibody repertoire and CDR length for human and mouse-derived neutralizing antibodies. V gene frequencies in the Heavy (A) and Light (B, C) chain repertoires of isolated neutralizing antibodies to SARS-CoV-2 for VelocImmune® mice (Black bars; N=185) and convalescent human donors (Gray Bars; N=68). The gene segments were arranged left to right on the x axis according to their position on the human chromosome from distal to proximal relative to their constant regions. The CDR sequence/length for Heavy (D) and Light (B, C) chain were extracted using International Immunogenetics Information System (IMGT) boundaries. Neutralization is defined as >70% with 1:4 dilution of antibody (~2µg/ml) in VSV pseudoparticle neutralization assay.

7 8 9 10 110

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KCDR3 (AA)

% S

eque

nces

%mBST%hBSTmBST

hBST

7 8 9 10 110

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KCDR3 (AA)

Num

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human

Table. S1. VSV Pseudoparticle neutralization and SARS-COV2 blocking ELISA to hACE2. Neutralization IC50 (M) shown for mab treatment of non-replicating pVSV-SARS-CoV-2-S-mNeon to Vero cells (Fig. 2A), or live SARS-COV-2 virus (Fig. 2D). In addition, an ELISA-based blocking assay shows the ability of anti-SARS-CoV2 antibodies to block the binding of the SARS-COV2 Spike protein RBD to hACE2 (0.2µg/ml).

Table S1.

ELISA Blocking

AntibodyPseudoparticleNeutralization

IC50 (M)

Live VirusNeutralization

IC50 (M)IC50 (M) Blocking nCoV RBD.hFc

% Blocking nCoV RBD.hFc

REGN10989 7.23E-12 7.38E-12 4.33E-11 98

REGN10987 4.06E-11 4.21E-11 2.31E-10 95

REGN10933 4.28E-11 3.74E-11 6.93E-11 99

REGN10934 5.44E-11 2.83E-11 6.36E-11 96

REGN10977 5.15E-11 NT 7.22E-11 65

REGN10964 5.70E-11 NT 6.40E-11 95

REGN10954 9.22E-11 NT 7.36E-11 95

REGN10984 9.73E-11 NT 7.91E-11 90

REGN10986 9.91E-11 NT 8.58E-11 98

Table S2. SPR-Biacore kinetics of anti-SARS-CoV2 spike monoclonal antibodies.Equilibrium dissociation constants (KD) for different SARS-CoV-2-S antibodies were determined using a real-time surface plasmon resonance-based Biacore T200 instrument using a Series S CM5 sensor chip. Binding kinetics parameters for the nine monoclonal antibodies binding to nCoVRBD.mmh, nCoV RBD.mFc at 37°C are shown. For the top four mabs binding kinetics parameters for binding to SARS-CoV2 Spike ECD foldon 37°C is also shown (NT = not tested).

aAntibody was captured on an anti-human Fc mAb-coupled sensor surface and different concentrations of specific antigen were injected.NT= Not Tested

Binding kinetic parameters for antigen binding to their specific antibody at 37oC

Antibody Test ligand

Surface density of antibody captured

(RU)

Antigen bound (RU) ka (M-1s-1) kd (s-1) KD (M) t½ (min)

REGN10989a

nCoV RBD.mmh 91 + 0.7 20 3.81 x 106 1.39 x 10-2 3.65 x 10-9 0.8

nCoV RBD.mFc 86 + 0.6 41 9.48 x 106 8.75 x 10-5 9.23 x 10-12 132

SARS-CoV2 Spike ECD foldon 88.0 + 0.9 57.5 2.38 x 106 9.82 x 10-5 4.12 x 10-11 117.6

REGN10987a

nCoV RBD.mmh 87 + 0.8 10 8.07 x 105 3.65 x 10-2 4.52 x 10-8 0.3

nCoV RBD.mFc 83 + 0.8 29 9.18 x 106 2.74 x 10-4 2.98 x 10-11 42.2

SARS-CoV2 Spike ECD foldon 82.5 + 0.5 46.1 1.34 x 106 5.74 x 10-5 4.28 x 10-11 201.2

REGN10933a

nCoV RBD.mmh 100 + 1.0 24 3.00 x 106 1.01 x 10-2 3.37 x 10-9 1.1

nCoV RBD.mFc 94 + 1.0 46 6.90 x 106 9.65 x 10-5 1.40 x 10-11 119.7

SARS-CoV2 Spike ECD foldon 83.7 + 0.8 52.8 1.90 x 106 7.90 x 10-5 4.17 x 10-11 146.2

REGN10934a

nCoV RBD.mmh 98 + 0.9 17 8.13 x 106 3.95 x 10-2 4.86 x 10-9 0.3

nCoV RBD.mFc 91 + 1.2 43 1.46 x 107 8.33 x 10-5 5.70 x 10-12 138.7

SARS-CoV2 Spike ECD foldon 79.7 + 0.7 40.1 2.85 x 106 1.06 x 10-4 3.71 x 10-11 109.0

REGN10977a

nCoV RBD.mmh 341±1.4 122 4.35 x 105 1.31 x 10-3 3.01 x 10-9 8.8

nCoV RBD.mFc 146 ± 0.7 93 2.50 x 106 1.07 x 10-4 4.28 x 10-11 107.9

SARS-CoV2 Spike ECD foldon NT NT NT NT NT NT

REGN10964a

nCoV RBD.mmh 333 ± 4.6 121 3.68 x 106 2.08 x 10-3 5.64 x 10-10 5.6

nCoV RBD.mFc 123 ± 0.3 84 1.05 x 107 1.26 x 10-4 1.20 x 10-11 91.7

SARS-CoV2 Spike ECD foldon NT NT NT NT NT NT

REGN10954a

nCoV RBD.mmh 372 ± 2.0 131 5.68 x 105 1.35 x 10-3 2.38 x 10-9 8.6

nCoV RBD.mFc 142 ± 0.8 105 3.02 x 106 1.12 x 10-4 3.69 x 10-11 103.1

SARS-CoV2 Spike ECD foldon NT NT NT NT NT NT

REGN10984a

nCoV RBD.mmh 423 ± 0.7 144 3.28 x 105 1.82 x 10-3 5.55 x 10-9 6.3

nCoV RBD.mFc 158 ± 0.7 110 2.07 x 106 8.36 x 10-5 4.04 x 10-11 138.2

SARS-CoV2 Spike ECD foldon NT NT NT NT NT NT

REGN10986a

nCoV RBD.mmh 349 ±1.5 129 8.24 x 105 5.83 x 10-4 7.07 x 10-10 19.8

nCoV RBD.mFc 125 ± 0.7 83 4.59 x 106 5.79 x 10-5 1.26 x 10-11 199.5

SARS-CoV2 Spike ECD foldon NT NT NT NT NT NT

Table S2.

Fig. S3. Effect of cleavage site mutations on neutralization and infectivity. (A) Top, the WT amino acid sequence of the S1/S2 polybasic cleavage site is underlined. Substitutes incorporated into SARS-CoV-2 S FurMut and FurKOmutants are marked in red. (B) Four-point neutralization curves (Log M: -7.48, -8.48, -9.48, and -10.48) for the lead antibodies in Vero cells. (C) Infectivity of the WT and cleavage mutants in Vero and Calu-3 cells at 24 and 48 hours, respectively. Infectivity of the mutants was normalized to the infectivity of the WT virus in each cell type.

A

B C

Fig. S3.

Vero Calu-3

-13 -12 -11 -10 -9 -8 -7 -60

20

40

60

80

100

Antibody Concentration Log(10) [M]

% N

eutra

lizat

ion

mAb 1mAb 2

mAb 6

mAb 3mAb 4

mAb 7

mAb 9mAb 8

mAb 5

hACE2.hFc

IgG1 ControlA

Strain Cleavage Site Sequence Cleavage Site Type

SARS-CoV-2 S WT Q677 TNSPRRAR | SV687 Polybasic

SARS-CoV-2 S FurMut Q677 TILR | SV687 Monobasic

SARS-CoV-2 S FurKO Q677 TNSPGSASSV687 KO

REGN10989-WTREGN10989-FurMutREGN10989-FurKOREGN10987-WTREGN10987-FurMutREGN10987-FurKOREGN10933-WTREGN10933-FurMutREGN10933-FurKOREGN10934-WTREGN10934-FurMutREGN10934-FurKOIgG1 Control-WTIgG1 Control-FurMutIgG1 Control-FurKO

Fig. S4. Neutralization potency of antigen-binding fragment (Fab) and full-length antibodies. VSV-spike pseudoparticle neutralization by either full length antibodies or corresponding Fab in Vero cells.

Fig. S4.

-13 -12 -11 -10 -9 -8 -7-40

-20

0

20

40

60

80

100

Total mAb Concentration Log10 [M]

% N

eutra

lizat

ion REGN10933

REGN10987REGN10989IgG1 Control

REGN10987 (Fab)REGN10989 (Fab)

REGN10933 (Fab)

REGN10934

REGN10934 (Fab)

Fig. S5. Anti-SARS-CoV2-Spike antibodies induce ADCC. NK cells were purified from three healthy donor leukopaks. NK cells from donor 1 (A, D), donor 2 (B, E) and donor 3 (C, F) were mixed with either parental Jurkatcells (A, B, C) or Jurkat cells expressing SARS-CoV-2 Spike Protein (D, E, F). A titration of Anti-SARS-CoV2-Spike Protein antibodies or an IgG1 isotype control were added to cells, incubated for 3.5h, and cell lysis assessed using CytoTox Glo reagent. Percent cytotoxicity was defined as 100 x [experimental signal – (NK alone signal + target alone signal)] / ( Target maximum signal – target alone signal). From this value the average percent cytotoxicity in absence of antibody was subtracted. Each condition was tested in triplicate.

A B C

D E F

Fig. S5.

-14 -13 -12 -11 -10 -9 -8 -7 -6-10

0

10

20

30

Antibody Concentration Log(10) [M]

% C

ytot

oxic

ity

REGN10934REGN10933REGN10987REGN10989IgG1 Isotype Control

-14 -13 -12 -11 -10 -9 -8 -7 -6-10

0

10

20

30

Antibody Concentration Log(10) [M]

% C

ytot

oxic

ity

REGN10934REGN10933REGN10987REGN10989IgG1 Isotype Control

-14 -13 -12 -11 -10 -9 -8 -7 -6-10

0

10

20

30

Antibody Concentration Log(10) [M]%

Cyt

otox

icity

REGN10934REGN10933REGN10987REGN10989IgG1 Isotype Control

-14 -13 -12 -11 -10 -9 -8 -7 -6-10

0

10

20

30

Antibody Concentration Log(10) [M]

% C

ytot

oxic

ity

REGN10934REGN10933REGN10987REGN10989IgG1 Isotype Control

-14 -13 -12 -11 -10 -9 -8 -7 -6-10

0

10

20

30

Antibody Concentration Log(10) [M]

% C

ytot

oxic

ity

REGN10934REGN10933REGN10987REGN10989IgG1 Isotype Control

-14 -13 -12 -11 -10 -9 -8 -7 -6-10

0

10

20

30

Antibody Concentration Log(10) [M]

% C

ytot

oxic

ity

REGN10934REGN10933REGN10987REGN10989IgG1 Isotype Control

NK Donor 1 NK Donor 2 NK Donor 3

Jurkat / hCD20 Jurkat / hCD20 / SARS-CoV2-Spike Protein Jurkat / hCD20 Jurkat / hCD20 / SARS-

CoV2-Spike Protein Jurkat / hCD20 Jurkat / hCD20 / SARS-CoV2-Spike Protein

EC50 (M) Max (%Cytotoxicity) EC50 (M) Max (%

Cytotoxicity) EC50 (M) Max (% Cytotoxicity) EC50 (M) Max (%

Cytotoxicity) EC50 (M) Max (% Cytotoxicity) EC50 (M) Max (%

Cytotoxicity)

REGN10989 ND 2.6 1.56E-10 15.1 ND 1.9 NC 11.4 ND 1.9 NC 17.5

REGN10987 ND 2.2 1.12E-10 19.8 ND 1.9 3.32E-10 17.1 ND 3.1 1.44E-10 18.2

REGN10933 ND 1.1 1.24E-10 10.6 ND 2.3 NC 8.4 ND 4.9 2.00E-11 8.8

REGN10934 ND 4.6 2.17E-11 7 ND 2 1.60E-10 6.1 ND 0 NC 11.6

IgG1 IsotypeControl ND 4.2 ND 1.9 ND 2.1 ND 1.8 ND 0 NC 7.2

Table S3. Anti-SARS-CoV2-Spike antibodies induce ADCC. ADCC was assessed as described in Figure 1. Max (% Cytotoxicity) was calculated for each NK donor and target cell line and is the highest mean % Cytotoxicity value within the tested dose range. Not calculated (NC) was reported when an EC50 could not be calculated because data did not fit a 4-parameter logistic equation. Not determined (ND) was reported when no dose dependent response was observed.

Table S3.

A B

Fig. S6. Anti-SARS-CoV2-Spike antibodies induce ADCP. Macrophages were differentiated from primary human monocytes with M-CSF, labeled with CellTrace violet and mixed with CFSE-labeled parental Jurkat cells (A) or Jurkat cells expressing SARS-CoV-2 Spike Protein (B). A titration of anti-SARS-CoV2-Spike Protein antibodies or an IgG1 isotype control were added to cells, incubated for 30min, washed and subsequently imaged using a phenix confocal microscopy platform. Phagocytosis was defined as 100 x (macrophage containing engulfed target cell) / (Total number of macrophages). From this value, background was subtracted: (Value) – (Average of No antibody), in order to derive % ADCP. Each condition was tested in duplicate with two donors. A representative donor is shown.

Fig. S6.

-15 -14 -13 -12 -11 -10 -9 -8 -7

0

20

40

60

80

100

Antibody Concentration, Log(10) [M]

% A

DC

P

REGN10933REGN10987REGN10989IgG1 Isotype Control

REGN10934

-15 -14 -13 -12 -11 -10 -9 -8 -7

0

20

40

60

80

100

Antibody Concentration, Log(10) [M]

% A

DC

P

REGN10933REGN10987REGN10989IgG1 Isotype Control

REGN10934

Jurkat/hCD20 Jurkat/hCD20/SARS-CoV-2 Spike Protein

Antibody EC50 [M] Max (% ADCP) EC50 [M] Max (% ADCP)

REGN10989 ND 3.52 4.86E-12 56.0

REGN10987 ND 5.60 6.33E-12 56.7

REGN10933 ND 3.67 5.79E-12 53.4

REGN10934 ND 2.05 2.72E-12 40.1

IgG1 Isotype Control ND 1.97 ND 10.4

Table S4. Anti-SARS-CoV2-Spike antibodies induce ADCP. ADCP was assessed as described in Figure 2. Max % ADCP is the highest mean % ADCP value within the tested dose range. Not determined (ND) was reported when no dose dependent response was observed.

Table S4.

Fig. S7. Epitope bin analysis from a matrix of pre-mix binding assays for different anti-SARS-CoV-2 mAbs. Epitope binning was performed against nine anti-SARS-CoV-2 mAb as described in the materials and method. There are three phases (I, II, and III) for each graph. In phase I anti-SARS-CoV-2 mAb (20ug/ml) was loaded to the anti-human Fc probe. In phase II human IgG1 blocking mAb solution (100ug/ml). In phase III solution of 100nM SARS CoV-2 RBD-MMH pre-mix complex of each 600 nM anti-SARS-CoV-2 mAb binding site flowed over the mAb captured probe.

Bi-directional Competition

Partial competition

No competition

Self-self competition

Pre-mix Competition between anti-SARS-CoV-2 mAbs

Fig. S7.

Phase IMeasure

mAb1 captured

Phase II measure

IgG blocking

mAb (nm)

Phase III, Response of 100 nM SARS CoV-2 RBD-MMH complexed600 nM of mAb2 binding site (nm)

Pre-mix Competition

mAb nm captured mAb

nm mAb bound REGN10977 REGN10989 REGN10933 REGN10964 REGN10984 REGN10986 REGN10954 REGN10934 REGN10987

REGN10977 1.69 ± 0.05

0.27 + .08

0.00 -0.03 -0.01 -0.04 -0.02 0.46 0.33 0.00 0.96

REGN10989 1.95 ± 0.03 0.07 0.00 -0.04 -0.03 -0.04 0.03 -0.01 -0.07 0.30

REGN10933 1.73 ± 0.06 0.10 0.04 0.00 -0.01 -0.01 0.04 0.00 0.25 1.52

REGN10964 1.90 ± 0.03 0.04 0.02 0.04 0.00 -0.01 0.05 0.04 0.38 1.37

REGN10984 1.88 ± 0.04 0.11 0.03 0.15 0.02 0.00 0.08 0.06 0.55 1.13

REGN10986 1.73 ± 0.04 1.09 0.14 0.32 -0.03 -0.06 0.00 -0.04 0.86 1.21

REGN10954 1.83 ± 0.04 0.85 0.03 0.53 -0.01 -0.04 0.02 0.00 1.12 1.16

REGN10934 1.78 ± 0.06 0.07 0.05 0.03 -0.03 0.65 1.23 1.60 0.00 0.31

REGN10987 1.83 ± 0.04 1.29 0.06 0.76 0.85 1.03 1.05 1.07 -0.08 0.00

= Anti-hFc

= RBD.mmh

= mab

Table S5. CryoEM data statistics. Data collection and refinement statistics are reported for the REGN10987 + REGN10933 + SARS-CoV-2 RBD complex structure shown in Fig. 4.

Table S5.

SARS COV-2 RBD: REGN10933:REGN10987 complexPDB XXXXEMDB XXXX

Data collection and processingMagnification 105,000Voltage (kV) 300Electron exposure (e–/Å2) 40Defocus range (μm) 1.6-3.0 Pixel size (Å) 0.85Symmetry imposed C1Initial number of particles 989,553Final selected particles 61,707Map resolution (Å) 3.9FSC threshold 0.143RefinementMap sharpening B factor (Å2) -122Model composition (# of atoms) 7979Model vs. map correlation coefficient 0.64R.m.s. deviationsBond lengths (Å) 0.02Bond angles (°) 1.12ValidationMolProbity score 2.7Rotameric outliers (%) 1.0Ramachandran plotFavored (%) 83.0Allowed (%) 16.3Disallowed (%) 0.7

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