Highly synergistic combinations of nanobodies that target SARS-CoV-2 and are resistant to escape

  1. Fred D Mast
  2. Peter C Fridy
  3. Natalia E Ketaren
  4. Junjie Wang
  5. Erica Y Jacobs
  6. Jean Paul Olivier
  7. Tanmoy Sanyal
  8. Kelly R Molloy
  9. Fabian Schmidt
  10. Magdalena Rutkowska
  11. Yiska Weisblum
  12. Lucille M Rich
  13. Elizabeth R Vanderwall
  14. Nicholas Dambrauskas
  15. Vladimir Vigdorovich
  16. Sarah Keegan
  17. Jacob B Jiler
  18. Milana E Stein
  19. Paul Dominic B Olinares
  20. Louis Herlands
  21. Theodora Hatziioannou
  22. D Noah Sather
  23. Jason S Debley
  24. David Fenyö
  25. Andrej Sali
  26. Paul D Bieniasz
  27. John D Aitchison  Is a corresponding author
  28. Brian T Chait  Is a corresponding author
  29. Michael P Rout  Is a corresponding author
  1. Center for Global Infectious Disease Research, Seattle Children's Research Institute, United States
  2. Laboratory of Cellular and Structural Biology, The Rockefeller University, United States
  3. Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, United States
  4. Department of Chemistry, St. John’s University, United States
  5. Department of Bioengineering and Therapeutic Sciences, Department of Pharmaceutical Chemistry, California Institute for Quantitative Biosciences, University of California, San Francisco, United States
  6. Laboratory of Retrovirology, The Rockefeller University, United States
  7. Center for Immunity and Immunotherapies, Seattle Children’s Research Institute, United States
  8. Institute for Systems Genetics and Department of Biochemistry and Molecular Pharmacology, NYU Grossman School of Medicine, United States
  9. AbOde Therapeutics Inc, United States
  10. Department of Pediatrics, University of Washington, United States
  11. Division of Pulmonary and Sleep Medicine, Seattle Children’s Hospital, United States
  12. Howard Hughes Medical Institute, The Rockefeller University, United States
  13. Department of Biochemistry, University of Washington, United States
8 figures, 10 tables and 1 additional file

Figures

Approach.

(A) Schematic of our strategy for generating, identifying, and characterizing large, diverse repertoires of nanobodies that bind the spike protein of SARS-CoV-2. The highest quality nanobodies were assayed for their ability to neutralize SARS-CoV-2 pseudovirus, SARS-CoV-2 virus, and viral entry into primary human airway epithelial cells. We also measured the activities of homodimers/homotrimers and mixtures. (B) A network visualization of 374 high-confidence CDR3 sequences identified from the mass spectrometry workflow. Nodes (CDR3 sequences) were connected by edges defined by a Damerau–Levenshtein distance of no more than 3, forming 183 isolated components. A thicker edge indicates a smaller distance value, that is, a closer relation. (C) Dendrogram showing sequence relationships between the 116 selected nanobodies, demonstrating that the repertoire generally retains significant diversity in both anti-S1 (green) and anti-S2 (blue) nanobodies, albeit with a few closely related members. Scale, 0.2 substitutions per residue.

Figure 2 with 2 supplements
Biophysical characterization of anti-SARS-CoV-2 spike nanobodies.

(A) Each nanobody plotted against their affinity (KD) for their antigen separated into three groups based on their binding region on SARS-CoV-2 spike protein. The data points highlighted in blue correspond to nanobodies that neutralize. The majority of nanobodies have high affinity for their antigen with KDs below 1 nm. 10 nanobodies are not included in this plot as they were unable to be analyzed successfully using surface plasmon resonance (SPR). (B) SPR sensorgrams for each of the three targets on SARS-CoV-2 spike protein of our nanobody repertoire, showing three representatives for each binding region. (C) The association rate of each nanobody (kon) versus the corresponding dissociation rate (koff). The majority of our nanobodies have fast association rates (~10+5–10+7 M–1 s–1), with many surpassing the kon of high-performing monoclonal antibodies (~10+4–10+5 M–1 s–1). (D) Each nanobody plotted against their Tm as measured by differential scanning fluorimetry (DSF), revealing all but two nanobodies fall within a Tm range between 50 and 80°C, where the bulk of our nanobodies have a Tm ≥ 60°C. No data could be collected for two nanobodies, and 10 nanobodies exhibited two dominant peaks in the thermal shift assay and were not included in this plot (a full summary of this data can be seen in Tables 13). The KD (E) and Tm (F) of six nanobodies were assessed pre- and post-freeze-drying, revealing no significant change in affinity or Tm after freeze-drying. (G) SPR sensorgrams comparing the kinetic and affinity analysis of seven nanobodies against wild-type spike S1 (Wuhan strain), spike 20I/S1 501Y.V1 (alpha variant), and 20H/spike S1 501Y.V2 (beta variant).

Figure 2—figure supplement 1
Binding of nanobody candidates to immobilized antigen.

All nanobody candidates identified by (a) S1, (b) S1-RBD, or (c) S2 purification were expressed in bacterial periplasm, which was bound to the respective immobilized antigen protein. After washes, loaded input (L) and elution (E) samples were analyzed by Coomassie stained SDS-PAGE. Positive binders (blue) displayed a nanobody band in the elution, while negative candidates (gray) had none.

Figure 2—figure supplement 2
Quantified antigen binding of nanobody candidates.

All nanobody candidates were expressed in bacterial periplasm, which was bound to immobilized S1, S1-RBD, or S2 antigen protein. Bound nanobody was quantified by Coomassie staining after SDS-PAGE. Binding intensity against each antigen was normalized to the maximum observed binding among all nanobodies. Candidates with >20% maximum activity (blue) were selected for follow-up, while others (gray) were generally discarded.

Figure 3 with 1 supplement
Epitope characterization of nanobodies against SARS-CoV-2 spike.

(A) Major epitope bins are revealed by a clustered heat map of Pearson’s correlation coefficients computed from the response values of nanobodies binding to the spike RBD in pairwise cross-competition assays on a biolayer interferometer. Correlated values (red) indicate that the two nanobodies respond similarly when measured against a panel of 11 RBD nanobodies that bind to distinct regions of the RBD. A strong correlation score indicates binding to a similar/overlapping region on the RBD. Anticorrelated values (blue) indicate that a nanobody pair responds divergently when measured against nanobodies in the representative panel and indicate binding to distinct or non-overlapping regions on the RBD. (B) As in (A), but for 16 S1 non-RBD-binding nanobodies. (C) As in (A), but for 19 S2-binding nanobodies. (D) A network visualization of anti-S1-RBD nanobodies. Each node is a nanobody and each edge is a response value measured by biolayer interferometry from pairwise cross-competition assays. Orange nodes represent 11 nanobodies used as a representative panel for clustering analysis in (A). Blue nodes represent the other nanobodies in the dataset. The average shortest distance between any nanobody pair in the dataset is 1.64. An average clustering coefficient of 0.831 suggests that the measurements are well distributed across the dataset. The small world coefficient of 1.031 indicates that the network is more connected than to be expected from random, but the average path length is what you would expect from a random network, together indicating that the relationship between nanobody pairs not actually measured can be inferred from the similar/neighboring nanobodies. (E, F) As in (D) but for S1 non-RBD and S2 nanobodies, respectively. These are complete networks with every nanobody measured against the others in the dataset. (G) Mass photometry (MP) analysis of spike S1 monomer incubated with different anti-spike S1 nanobodies. Two examples of an increase in mass as spike S1 monomers (black line) are incubated with 1–3 nanobodies. The accumulation in mass upon addition of each different nanobody on spike S1 monomer is due to each nanobody binding to non-overlapping space on spike S1, an observation consistent with Octet binning data. As a control, using MP, each individual nanobody was shown to bind spike S1 monomers on its own (data not shown).

Figure 3—source data 1

Normalized response values from epitope binning of nanobodies.

https://cdn.elifesciences.org/articles/73027/elife-73027-fig3-data1-v2.xlsx
Figure 3—figure supplement 1
Mass photometry (MP) of non-RBD S1 nanobodies.

MP analysis of spike S1 monomer incubated with different non-RBD-binding anti-spike S1 nanobodies. There is either an accumulation in mass upon addition of each different nanobody on spike S1 monomer that is due to each nanobody binding to non-overlapping space on spike S1 or not, suggesting overlapping epitopes. These results, coupled with surface plasmon resonance (SPR) binning data, assisted the binning analysis for non-RBD anti-S1 nanobodies.

Figure 4 with 1 supplement
Diverse and potent nanobody-based neutralization of SARS-CoV-2.

Nanobodies targeting the S1-RBD, S1 non-RBD, and S2 portions of spike effectively neutralize lentivirus pseudotyped with various SARS-CoV spikes and their variants from infecting ACE2 expressing HEK293T cells. (A) Of the 116 nanobodies, monomers that neutralize SARS-CoV-2 pseudovirus with IC50 values 20 nM and lower are displayed. (B) Representative nanobodies targeting the non-RBD portions of S1 and (C) the S2 domain of SARS-CoV-2 neutralize SARS-CoV-2 pseudovirus. (D–F) Oligomerization of RBD, S1 non-RBD, and S2 nanobodies significantly increases neutralization potency. (G) Summary scatter plot of all nanobody IC50s across the major domains of SARS-CoV-2 spike and where tested, across SARS-CoV-2 variant 20H/501Y.V2 and SARS-CoV-1. Representative published nanobodies were also tested in our neutralization assays and show similar potency towards SARS-CoV-2 pseudovirus. From Xiang et al., 2020: (1) Nb-21 (IC50 2.4 nM); (2) Nb-34 (IC50 5.6 nM); and (3) Nb-93 (IC50 123 nM). From Wrapp, 2020a: (4) VHH-72 (IC50 2.5 μM). (H–K) Representative SARS-CoV-2 RBD targeting nanobodies cross-neutralize the 20I/501Y.V1/alpha variant with H69-, V70-, Y144- amino acid deletions and N501Y, A570D, D614G, P681H, T716I, S982A, and D1118H amino acid substitutions in spike (H); the 20H/501Y.V2/beta variant with L18F, D80A, K417N, E484K, and N501Y amino acid substitutions in spike; (I) 20J/501Y.V3/gamma variant with L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y, T1027I, and V1176F amino acid substitutions in spike (J); and SARS-CoV-1 spike pseudotyped lentivirus (K). In all cases, n ≥ 2 biological replicates of each nanobody monomer/oligomer with a representative biological replicate with n = 4 technical replicates per dilution are displayed. See also Table 1, Table 2, Table 4, and Table 5.

Figure 4—figure supplement 1
Correlation between pseudovirus assays.

IC50s of the 25 nanobodies tested in both lentiviral and VSV-based pseudovirus assays with wild-type SARS-CoV-2 spike were plotted to compare the values obtained from each system, showing a strong correlation.

Authentic SARS-CoV-2 neutralization by anti-spike nanobodies.

(A) Nanobodies neutralize the authentic SARS-CoV-2 virus with similar kinetics as the SARS-CoV-2 pseudovirus. Neutralization curves are plotted from the results of a focus-forming reduction neutralization assay with the indicated nanobodies. Serial dilutions of each nanobody were incubated with SARS-CoV-2 (MOI 0.5) for 60 min and then overlaid on a monolayer of Vero E6 cells and incubated for 24 hr. LaM2, an anti-mCherry nanobody (Fridy et al., 2014), was used as a non-neutralizing control. After 24 hr, cells were collected and stained with anti-spike antibodies and the ratio of infected to uninfected cells was quantified by flow cytometry. (B) A schematic of an air-liquid interface (ALI) culture of primary human airway epithelial cells (AECs) as a model for SARS-CoV-2 infection. Cells were incubated with nanobodies and then challenged with SARS-CoV-2 (MOI 0.5). After daily treatment with nanobodies for three more days, the cultures are harvested to isolate RNA and quantify the extent of infection. (C) Potent neutralization of authentic SARS-CoV-2 in AECs. The AECs were infected with the indicated concentrations of anti-SARS-CoV-2 spike nanobodies. The infected cultures were maintained for 5 days with a daily 1 hr incubation of nanobodies before being harvested for RNA isolation and determination of the SARS-CoV-2 copy number by qPCR. SARS-CoV-2 copy number was normalized to total RNA measured by spectrophotometry. Mock-treated samples exposed to infectious and UV-inactivated SARS-CoV-2 virions served as positive and negative controls. Recombinant soluble angiotensin converting enzyme 2 (rACE2) was used as a positive treatment control. The indicated nanobodies were used at 1, 10, and 100× their IC50 values determined in pseudovirus neutralization assays (Table 1 and Table 4).

Figure 5—source data 1

Neutralization data from authentic SARS-CoV-2 experiments.

https://cdn.elifesciences.org/articles/73027/elife-73027-fig5-data1-v2.xlsx
Figure 6 with 3 supplements
Epitope coverage of the 21 structural models of anti-spike SARS-CoV-2 nanobodies and neutralization potential of each epitope.

(A) The structure of SARS-CoV-2 full spike (PDB ID: 6VYB) solved via cryo-EM with one RBD in the up position overlaid with the crystal structure of RBD bound to ACE2 (PDB ID: 6M0J). Key elements of spike are colored as follows: RBD (white), S1-NTD (gray), and S2 (light blue). All 21 modeled nanobody footprints are colored gold on full spike with the ACE2 footprint (RBM) colored green. Full coverage of the 18 anti-RBD modeled nanobody footprints on RBD is seen in four different orientations. All 21 nanobodies are categorized into 10 groups based on their footprint on spike where groups 1–7 are anti-RBD nanobodies; group 8 contains an anti-S1-NTD nanobody and groups 9 and 10 contain anti-S2 nanobodies. (B) Heatmap of neutralizing epitopes on the structure of SARS-CoV-2 full spike (PDB ID: 6VYB). Epitopes are colored from pale yellow (epitopes with weak neutralization against SARS-CoV-2) to dark red (strong neutralization against SARS-CoV-2).

Figure 6—source data 1

PDB files of structural models of anti-spike SARS-CoV-2 nanobodies.

https://cdn.elifesciences.org/articles/73027/elife-73027-fig6-data1-v2.zip
Figure 6—figure supplement 1
Mapping of spike substitutions in rVSV/SARS-CoV-2/GFP escape mutants obtained in the presence of the corresponding nanobody.

(A) Mapped on to the structure of SARS-CoV-2 spike trimer in complex with one ACE2 molecule (PDB ID: 7KNB, used for all SARS-CoV-2 spike trimer representations) is the position of neutralization-resistant amino acid substitutions (in red), also known as ‘escape mutants’ that were generated in response to cultivation of rVSV/SARS-CoV-2/GFP in the presence of each nanobody, and were subsequently shown to confer resistance to the same nanobody. (B) Structure of the SARS-CoV-2 RBD (PDB ID: 6M0J) showing the positions of amino acid residues (in green) that form the ACE2 binding site, for reference. (C) Structure of the SARS-CoV-2 RBD (PDB ID: 6M0J) showing the positions (in red) of the location of substitutions that confer resistance for each nanobody tested in two orientations 90° apart. For structure pairs of S1-RBD-16 and S1-RBD-23 escape mutants, the rotation is 90° from the structure to the left in the pair. (D) The location of four key non-RBD escape mutants S172G, Y170H, T315I, and S982R resulting from assays performed with the anti-S1 non-RBDs S1-49, S1-3, S1-30, and anti-S2 (S2-10) nanobodies, respectively. (E) Infectious rVSV/SARS-CoV-2/GFP yield (IU/ml) following two passages in the presence of the indicated individual nanobodies or nanobody combinations, at 100 IC50 of the individual nanobodies, or 50 IC50 of each of the nanobodies in the combinations. Each data point represents an independent titer measurement. Red open circles represent virus escapes while blue circles represent nanobody combinations for which no escapes (titer = 0) were detected. The location of two escape mutants K378Q (F) and Y508H (G) mapped onto SARS-CoV-2 spike trimer for the two corresponding nanobodies S1-RBD-9 and S1-RBD-15, respectively, revealing an exposed putative nanobody binding site on RBD when in the ‘up’ position that is hidden when RBD is in the ‘down’ position. (H) A close-up of escape mutant S172G on each monomer of SARS-CoV-2 spike trimer revealing a larger crevice between the NTD of spike S1 and RBD when the RBD is in the ‘down’ position compared to the ‘up’ position. (I) Three orientations of SARS-CoV-2 spike trimer revealing the position in all three orientations of escape mutant S982R revealing the putative binding site for nanobody S2-10 is accessible regardless of whether the RBD is the ‘up’ or ‘down’ position.

Figure 6—figure supplement 2
Comparison between RBD-Nb21 interface modeled with Integrative Modeling Platform (IMP) (red) with cryo-EM structure (7N9B) of the co-complex (indigo, taken from Sun et al., 2021).

IMP modeling was done using the 6M0J.E structure of the RBD (residues 333–526), which was subsequently aligned with the corresponding region of the down RBD from 7N9B.A, to compare the modeled and experimental binary complexes. (A) and (B) show the binding modes and the epitopes on the whole spike while (C) and (D) focus on just the 333–526 region of the RBD. Based on the observation that the point mutation E484K/Q significantly inhibits Nb21 binding (Sun et al., 2021), we treat E484 as an effective escape mutant for Nb21 to benchmark our computational model developed in IMP. Both the epitope (defined as all RBD residues within 6 Å of any Nb21 residue) and the binding mode (i.e., relative orientation) of Nb21 agree between the IMP model and the cryo-EM structure to within 2.1 Å backbone RMSD.

Figure 6—figure supplement 3
Nanobody groups resistant or predicted to be resistant against SARS-CoV-2 variants of concern (VOC).

(A) Color-coded and mapped on the structure of SARS-CoV-2 spike trimer with one RBD in the ‘up’ conformation (PDB ID: 7BNN) are the footprints of eight nanobody groups determined, which are mapped relative to the mutations found on four key SARS-CoV-2 VOC. Summarized are the nanobodies that make up each group and where applicable their degree of neutralization. The nanobody footprints are taken from the integrative modeling of these nanobodies on SARS-CoV-2 spike trimer (see Figure 6). (B) Neutralization of authentic SARS-CoV-2 isolate WA-1 and an isolate of the delta variant of concern (B.1.617.2) in a plaque reduction neutralization test assay. Serial dilutions of S1-1 and S1-RBD-35 were incubated with each SARS-CoV-2 variant for 60 min and then overlaid on a monolayer of Vero E6 cells and incubated for 90 min. Following infection, the cell cultures incubated for 48 hr before the number of visualized plaques were counted to determine the half maximal inhibitory concentration of each nanobody.

Figure 7 with 1 supplement
Synergistic neutralization of spike with nanobody cocktails.

(A) An example of additive effects between two anti-SARS-CoV-2 spike nanobodies. S1-23 and S1-27 were prepared in a two-dimensional serial dilution matrix and then incubated with SARS-CoV-2 pseudovirus for 1 hr before adding the mixture to cells. After 56 hr, the expression of luciferase in each well was measured by addition of Steady-Glo reagent and read out on a spectrophotometer. The left panel shows a heatmap of pseudovirus neutralization by a two-dimensional serial dilution of combinations of S1-23 and S1-27. Lines and red numbers demarcate the % inhibition, that is, inhibitory concentration where X% of the virus is neutralized, e.g., IC50. Dark blue regions are concentrations that potently neutralize the pseudovirus, as per the heatmap legend. The right panel shows neutralization curves (with 90% confidence interval bands) and the calculated IC50 of each nanobody alone, or in a 1:1 combination was determined along with a calculated IC50 based on the theoretical additive mixture model of the pair (curve with dotted gray line). The inset shows a difference (synergy) map calculated as the difference between the parameterized 2D neutralization response and that expected in a null model of only additive effects. Here, no difference is observed. (B) S1-1 synergizes with S1-23 in neutralizing SARS-CoV-2 pseudovirus. The left panel shows the heatmap of pseudovirus neutralization observed by a two-dimensional serial dilution of combinations of S1-1 and S1-23. The middle panel shows a heatmap mapping the synergy of neutralization observed for this pair. The lines bounding the darker purple areas demarcate regions in the heatmap where the observed neutralization is greater than additive by the indicated percentages (yellow numbers), as per the heatmap legend. The right panel shows two representations of spike with the accessible S1-1 (salmon) and S1-23 (steel blue) epitopes (PDB ID: 6VYB). (C–J) Examples of synergy between nanobodies binding the S1-RBD, or between the S1-RBD and S1-NTD or S2 domains of spike. The layout is as found in (B), but comparing S1-RBD-15 with S1-23 (C), S1-RBD-15 with S1-RBD-23 (D), S1-23 with S1-46 (E), S1-RBD-15 with S1-46 (F), S1-49 with S1-1 (G), S1-49 with S1-RBD-15 (H), S1-23 with S2-10-dimer (I), and S1-RBD-15 with S2-10-dimer (J).

Figure 7—figure supplement 1
Heatmaps of nanobody synergy.

Neutralization of pseudovirus harboring the SARS-CoV-2 spike by pairs of nanobodies. The normalized % neutralization is visualized on a 2D grid of nanobody concentrations where each nanobody has been titrated in the background of the other tested nanobody. Nanobody concentrations are indicated on their respective axes for (A) S1-23 and S1-27, (B) S1-23 and S1-1, (C) S1-RBD-15 and S1-23, (D) S1-RBD-15 and S1-RBD-23, (E) S1-23 and S1-46, (F) S1-RBD-15 and S1-46, (G) S1-49 and S1-1, (H) S1-49 and S1-RBD-15, (I) S1-23 and S2-10-dimer, and (J) S1-RBD-15 and S2-10-dimer.

Author response image 1

Tables

Table 1
S1 nanobody characterization; related to Figures 2 and 4.

Nanobodies against S1 were determined to bind RBD or non-RBD epitopes by their affinity for recombinant full-length S1 and/or S1 RBD protein. Binding kinetics against these two recombinant proteins were determined by surface plasmon resonance (SPR), with on rates, off rates, and KDs determined by Langmuir fits to binding sensorgrams unless otherwise noted. Nanobody melting temperatures (Tm) were determined by differential scanning fluorimetry (DSF). Nanobodies were assayed for neutralization activity against a SARS-CoV-2 spike pseudotyped HIV-1 virus (PSV), with IC50s calculated from neutralization curves. Standard error of the mean (s.e.m.) is reported when available.

IDEpitopeS1 Kon(M–1 s–1)S1 Koff(s–1)S1 KD (M)RBD Kon(M–1 s–1)RBD Koff(s–1)RBD KD (M)Tm (°C)SARS-CoV-2 PSV IC50 (s.e.m.)(nM)
S1-1RBD4.14E+052.98E-057.20E-116.50E+055.98E-079.20E-12 66.56.7 (1.0)
S1-2Non-RBD1.59E+061.88E-031.18E-09No interaction detected 66.5NA
S1-3Non-RBD5.08E+054.32E-048.51E-10No interaction detected 641030 (666)
S1-4RBD1.25E+061.06E-048.46E-111.26E+061.26E-041.37E-10 6641.5 (3.7)
S1-5RBD1.33E+051.15E-038.61E-09 65.25NA
S1-6RBD1.02E+065.75E-045.65E-105.92E+053.69E-046.22E-10 6556.1 (20.7)
S1-7Non-RBD7.59E+059.90E-041.30E-09No interaction detected 60.5NA
S1-9Non-RBD9.51E+051.25E+054.28E-071.57E-044.50E-13*1.25E-09 47.5NA
S1-10Non-RBD8.35E+041.82E-032.19E-08 64NA
S1-11Non-RBD 60NA
S1-12RBD2.90E+058.92E-043.07E-092.33E+052.24E-049.63E-10 68NA
S1-14RBD1.08E+061.10E-031.02E-095.37E+057.99E-041.49E-09 57.5135.8 (36.4)
S1-17Non-RBD 651271 (888)
S1-19RBD1.30E+063.55E+048.86E-032.41E-046.81E-09*6.81E-09 64.5139 (9.6)
S1-20RBD1.48E+074.37E-032.95E-10 6951.8 (3.7)
S1-21RBD4.77E+061.58E-043.31E-111.22E+062.45E-042.00E-10 70.5226 (158)
S1-23RBD2.82E+064.91E-051.74E-111.09E+061.07E-049.78E-11 645.7 (2.2)
S1-24Non-RBD6.49E+052.89E-044.45E-10No interaction detected 71.5724 (144)
S1-25Non-RBD2.15E+053.39E-051.57E-10No interaction detected 58NA
S1-27RBD3.15E+064.52E-041.43E-102.89E+066.30E-042.18E-10 5419.5 (4.9)
S1-28RBD1.38E+067.97E-045.76E-101.79E+061.03E-035.77E-10 6666.0 (10.9)
S1-29RBD2.39E+051.01E-034.21E-091.73E+058.89E+045.12E-09 61.5NA
S1-30Non-RBD6.21E+051.48E-032.38E-09No interaction detected 57717 (388)
S1-31RBD2.17E+065.63E-042.59E-101.94E+069.37E-044.84E-10 7278.7 (3.5)
S1-32Non-RBD2.73E+054.66E-041.71E-09No interaction detected 79NA
S1-35RBD2.46E+062.11E-058.60E-122.70E+069.77E-053.62E-11 70.512.5 (0.1)
S1-36RBD2.28E+063.92E-041.72E-107.87E+061.72E-032.18E-10 6348.5 (21.1)
S1-37RBD4.03E+062.75E-046.82E-114.14E+062.09E-045.05E-11 656.8 (0.7)
S1-38RBD5.34E+061.12E-032.10E-10 6466.1 (2.9)
S1-39RBD2.14E+068.11E-043.79E-101.68E+061.06E-036.30E-10 55111 (4.0)
S1-41Non-RBD8.73E+051.38E-031.58E-09No interaction detected 62.5679 (53.4)
S1-46RBD1.68E+052.94E-041.75E-092.22E+051.70E-047.66E-10 68312 (14.0)
S1-48RBD2.61E+066.22E-052.39E-111.66E+061.64E-049.85E-11 60.55.82 (0.5)
S1-49Non-RBD1.94E+063.63E-031.87E-09 49, 74‡356 (32.8)
S1-50Non-RBD3.33E+053.34E-031.39E-023.94E-044.40E-09No interaction detected 6613(11)
S1-51RBD9.28E+044.22E-044.54E-093.77E+062.01E-035.33E-10 56555.8 (52.5)
S1-52RBD4.22E+053.13E-047.74E-094.53E+041.94E-044.36E-09 57.53343 (291)
S1-53RBD1.40E+062.36E+048.46E-032.19E-046.05E-099.27E-09 51.52466 (939)
S1-54RBD1.13E+066.58E-055.84E-112.55E+042.88E-041.13E-08 691699 (1554)
S1-55RBD3.98E+063.53E+045.41E-035.31E-061.36E-09*1.51E-105.03E+051.84E+041.11E-021.82E-042.21E-08*9.89E-09 54.55725 (3372)
S1-56RBD1.46E+044.45E-032.99E-037.90E-053.57E-092.21E+031.05E-044.73E-08 54NA
S1-58Non-RBD5.73E+051.66E-042.90E-10 53.5940 (795)
S1-60Non-RBD3.30E+054.61E+045.24E-063.67E-031.59E-11*9.58E-08 62NA
S1-61RBD9.87E+058.23E+021.81E-021.10E-041.84E-08*1.34E-074.46E+041.88E-044.21E-09 60NA
S1-62RBD2.68E+069.51E-053.54E-113.30E+066.30E-052.08E-11 71.53.3 (0.8)
S1-63RBD1.09E+063.39E+041.12E-021.67E-041.02E-08*4.94E-095.10E+042.23E-044.37E-09 65NA
S1-64Non-RBD6.97E+051.58E-042.26E-10 6616.4 (11.7)
S1-65Non-RBD1.06E+061.67E-041.57E-10 607.3 (6.0)
S1-66Non-RBD4.66E+052.74E-045.87E-10 59NA
S1-RBD-3RBD8.81E+057.36E+041.76E-021.13E-032.00E-08*1.53E-08 72384 (18.7)
S1-RBD-4RBD2.02E+061.64E-048.09E-112.83E+068.16E-042.89E-10 64.517.5 (1.98)
S1-RBD-5RBD1.94E+061.63E-048.38E-117.21E+061.05E-031.45E-10 64174 (3.3)
S1-RBD-6RBD1.55E+061.63E-041.05E-103.48E+061.13E-033.24E-10 66.577.2 (21.8)
S1-RBD-9RBD2.85E+051.23E-044.30E-10 69235 (97.5)
S1-RBD-10RBD –52.9
S1-RBD-11RBD2.22E+072.94E-041.32E-112.06E+074.06E-041.97E-11 6513.5 (5.5)
S1-RBD-12RBD1.10E+043.39E-053.10E-09 67NA
S1-RBD-14RBD1.33E+043.34E-042.51E-08 65NA
S1-RBD-15RBD5.37E+061.50E-042.79E-117.52E+064.95E-046.58E-11 59.5, 804.6 (1.2)
S1-RBD-16RBD1.68E+046.25E-053.73E-09 6179.2 (4.2)
S1-RBD-18RBD2.28E+066.25E-042.74E-104.43E+061.27E-032.87E-10 69.567.2 (1.9)
S1-RBD-19RBD 602124 (1451)
S1-RBD-20RBD2.37E+062.23E-049.43E-113.05E+067.91E-042.59E-10 49, 7012.4 (1.1)
S1-RBD-21RBD3.50E+061.31E-033.73E-103.15E+061.71E-035.45E-10 48.5, 70.517.3 (3.1)
S1-RBD-22RBD9.34E+052.28E-042.44E-109.24E+054.42E-044.78E-10 57.5100 (0.1)
S1-RBD-23RBD2.89E+064.61E-051.59E-11 617.31 (0.4)
S1-RBD-24RBD1.61E+061.40E-038.65E-102.12E+061.22E-035.75E-10 46, 67221 (4)
S1-RBD-25RBD8.41E+041.16E-021.38E-07 –NA
S1-RBD-26RBD1.06E+054.58E-064.32E-112.15E+051.33E-056.19E-11 66241 (81.4)
S1-RBD-27RBD6.19E+061.24E-022.00E-09 71163 (71.4)
S1-RBD-28RBD1.80E+064.27E-042.38E-101.80E+064.27E-042.38E-10 64.532.7 (3.1)
S1-RBD-29RBD5.36E+051.35E-032.51E-09 749.53 (1.0)
S1-RBD-30RBD2.15E+066.66E-053.10E-113.77E+064.82E-041.28E-10 6525.0 (3.6)
S1-RBD-32RBD1.05E+057.90E-037.52E-08 65NA
S1-RBD-34RBD5.71E+044.88E-038.54E-08 64NA
S1-RBD-35RBD8.01E+051.68E-042.10E-101.33E+062.50E-041.88E-10 57, 6812.3 (2.4)
S1-RBD-36RBD 71NA
S1-RBD-37RBD3.60E+058.88E-042.47E-09 71523 (93.4)
S1-RBD-38RBD1.12E+069.84E-048.79E-10 68.584.6 (22.7)
S1-RBD-39RBD4.92E+057.77E-051.58E-10 67.590.4 (8.9)
S1-RBD-40RBD7.47E+052.77E-053.71E-11 7025.6 (5.9)
S1-RBD-41RBD4.37E+051.39E-043.17E-10 –17.0
S1-RBD-43RBD6.21E+051.82E-042.92E-10 6833.6 (1.3)
S1-RBD-44RBD1.91E+056.97E-053.65E-10 57.593.4
S1-RBD-45RBD4.43E+054.14E-059.30E-11 5322.6
S1-RBD-46RBD4.69E+055.79E-041.23E-09 75.548.0
S1-RBD-47RBD2.11E+056.45E-043.06E-09 53.5127 (11.6)
S1-RBD-48RBD1.05E+052.42E-042.30E-09 58, 6368.7 (14.2)
S1-RBD-49RBD3.24E+053.24E-041.00E-093.15E+055.34E-041.69E-09 66.537.6 (5.6)
S1-RBD-51RBD3.77E+062.01E-035.33E-10 52, 6170.9 (29.3)
  1. *Curves were fit to a heterogeneous ligand model. Respective Kon, Koff, and KD values are shown for each component.

  2. †Curves were fit to a two-state reaction model. Respective Kon, Koff, and KD values are shown for each binding state.

  3. ‡Two peaks were observed in the melting curve. Tms for both are reported.

  4. –, not determined; NA, no activity.

Table 2
S2 nanobody characterization; related to Figures 2 and 4.

Binding kinetics of S2 nanobodies were determined by surface plasmon resonance (SPR) using recombinant S2 protein, with on rates, off rates, and KDs determined by Langmuir fits to binding sensorgrams unless otherwise noted. Nanobody melting temperatures (Tm) were determined by differential scanning fluorimetry (DSF). Nanobodies were assayed for neutralization activity against a SARS-CoV-2 or SARS-CoV-1 spike pseudotyped HIV-1 virus (PSV), with IC50s calculated from neutralization curves with standard error of the mean (s.e.m.).

IDKon(M–1 s–1)Koff(s–1)KD (M)Tm (°C)SARS-CoV-2 PSV IC50 (s.e.m.) (nM)
S2-16.32E+043.79E-046.00E-0965.5NA
S2-21.26E+069.35E-057.41E-1164.54460 (901)
S2-32.62E+057.21E-052.76E-10652234 (751)
S2-42.44E+062.62E-041.08E-1056NA
S2-59.35E+052.74E-042.93E-1066NA
S2-661.5NA
S2-71.66E+069.36E-055.62E-1161NA
S2-99.29E+052.32E-042.50E-1064.5NA
S2-109.31E+043.13E-043.37E-0959, 64.5*5269 (1418)
S2-117.94E+061.12E-031.41E-1069.5NA
S2-137.02E+051.05E-041.49E-1064.5NA
S2-143.16E+061.28E-034.07E-1072.5NA
S2-1570NA
S2-181.63E+064.87E-042.99E-1047, 54.5*NA
S2-22NA
S2-264.45E+058.15E-051.83E-1076.5NA
S2-333.68E+055.58E-052.33E-1070NA
S2-352.36E+054.72E-052.00E-1077NA
S2-364.39E+063.69E-048.41E-1174NA
S2-3958NA
S2-405.08E+047.16E-051.41E-0969.51712 (828)
S2-425.12E+053.77E-067.36E-1269NA
S2-473.86E+051.14E-042.96E-1040, 65*NA
S2-572.33E+067.18E-043.08E-1067NA
S2-5937.5, 60*NA
S2-621.65E+061.12E-046.81E-1164, 77.5*7177 (5801)
  1. *Two peaks were observed in the melting curve. Tms for both are reported.

  2. –, not determined; NA, no activity.

Table 3
Nanobody binding activity against spike S1 variants; related to Figure 2.

Binding kinetics against wild-type spike S1 or two variants of concern were determined by surface plasmon resonance (SPR), with on rates, off rates, and KDs determined by Langmuir fits to binding sensorgrams.

IDSpike S1 variantKon(M–1 s–1)Koff(s–1)KD (M)
S1-1WT (Wuhan 2019)4.14E+052.98E-057.20E-11
20I/501Y.V12.71E+051.21E-054.44E-11
20H/501Y.V22.78E+051.25E-054.51E-11
S1-6WT (Wuhan 2019)1.02E+065.75E-045.65E-10
20I/501Y.V13.55E+066.27E-041.77E-10
20H/501Y.V21.03E+063.29E-043.20E-10
S1-23WT (Wuhan 2019)2.82E+064.91E-051.74E-11
20I/501Y.V15.96E+061.36E-032.29E-10
20H/501Y.V2NANANA
S1-RBD-9WT (Wuhan 2019)2.85E+051.23E-044.30E-10
20I/501Y.V14.84E+043.88E-058.01E-10
20H/501Y.V21.34E+059.55E-057.13E-10
S1-RBD-11WT (Wuhan 2019)2.22E+072.94E-041.32E-11
20I/501Y.V13.84E+062.87E-047.46E-11
20H/501Y.V26.85E+061.10E-031.61E-10
S1-RBD-15WT (Wuhan 2019)5.37E+061.50E-042.79E-11
20I/501Y.V12.99E+061.26E-044.22E-11
20H/501Y.V24.02E+062.22E-045.53E-11
S1-RBD-35WT (Wuhan 2019)8.01E+051.68E-042.10E-10
20I/501Y.V11.33E+072.40E-031.80E-10
20H/501Y.V25.94E+062.65E-034.46E-10
  1. NA, no activity.

Table 4
Characterization of oligomerized spike nanobodies; related to Figure 4.

Nanobody oligomers (1–4 nanobody repeats) were assayed for neutralization activity against a SARS-CoV-2 spike pseudotyped HIV-1 virus (PSV), with IC50s calculated from neutralization curves. Standard error of the mean (s.e.m.) is reported where replicates were available. Epitopes were determined by relative affinity for recombinant S1 or S1 RBD protein.

IDEpitopeSARS-CoV-2 PSV IC50 (s.e.m.)(nM)SARS-CoV-1 PSV IC50 (s.e.m.) (nM)
 S1-1RBD6.7 (1.0)8.6 (7.2)
 S1-1dimerRBD4.9 (0.1)
 S1-1trimerRBD5.7 (0.1)
 S1-23RBD5.7 (2.2)
 S1-23dimerRBD0.22 (0.05)NA
 S1-23trimerRBD0.089 (0.019)NA
 S1-RBD-35RBD12.3 (2.4)NA
 S1-RBD-35dimerRBD0.15 (0.11)
 S1-RBD-35trimerRBD0.068 (0.043)NA
 S1-3S1 non-RBD1,030 (666)4161
 S1-3dimerS1 non-RBD429513
 S1-3trimerS1 non-RBD411
 S1-30S1 non-RBD717 (388)
 S1-30dimerS1 non-RBD18.3662
 S1-30trimerS1 non-RBD77.5 (3.6)
 S1-7S1 non-RBDNA
 S1-7dimerS1 non-RBDNA
 S1-7trimerS1 non-RBDNA
 S1-17S1 non-RBD1271 (888)
 S1-17dimerS1 non-RBD2144
 S1-49S1 non-RBD356 (32.8)NA
 S1-49dimerS1 non-RBD9.1 (1.2)NA
 S1-49trimerS1 non-RBD0.87 (0.08)NA
 S2-7S2NANA
 S2-7dimerS22463516
 S2-7trimerS2112
 S2-7tetramerS229.7
 S2-10S25269 (1418)
 S2-10dimerS248.0 (27.5)
 S2-10trimerS234.3
  1. –, not determined; NA, no activity.

Table 5
Nanobody neutralization activity against spike variants; related to Figure 4.

Nanobodies were assayed for neutralization activity against a pseudotyped HIV-1 virus (PSV) expressing SARS-CoV-1 or SARS-CoV-2 wild-type or variant spike, with IC50s calculated from neutralization curves. Standard error of the mean (s.e.m.) is reported where replicates were available.

IDEpitopeSARS-CoV-2 PSV IC50 (s.e.m.)(nM)SARS-CoV-1 PSV IC50 (s.e.m.) (nM)SARS-CoV-2 20H/501Y.V2 PSV IC50 (s.e.m.) (nM)SARS-CoV-2 20I/501Y.V1 PSV IC50 (s.e.m.) (nM)SARS-CoV-2 20J/501Y.V3 PSV IC50 (s.e.m.) (nM)
S1-1RBD6.7 (1.0)8.6 (6.4)7.2 (1.6)8.5 (4.2)2.9 (0.2)
S1-3Non-RBD1030 (666)3598 (563)
S1-4RBD41.5 (3.7)179
S1-6RBD56.1 (20.7)227 (205)
S1-17Non-RBD1271 (888)NA
S1-20RBD51.8 (3.7)NANA13.5NA
S1-23RBD5.7 (2.2)NANA78.3NA
S1-24Non-RBD868NA
S1-27RBD19.5 (4.9)NA
S1-30Non-RBD717.8 (387)NA
S1-31RBD78.7 (3.5)NA
S1-35RBD12.5 (0.1)386.8 (350)
S1-36RBD48.5 (21.1)NA
S1-37RBD6.8 (0.7)NANA13.5NA
S1-39RBD111 (4.0)22.1 (18.5)
S1-41Non-RBD679 (53.4)NA
S1-48RBD5.82 (0.5)NA20.9 (1.4)7.3 (1.5)14.2 (3.5)
S1-49Non-RBD356 (32.8)NA
S1-51RBD555.8 (52.5)NA
S1-58Non-RBD940 (795)NA
S1-62RBD3.3 (0.8)NA6.4 (4.5)NA
S1-RBD-6RBD77.2 (21.8)89.7 (4.2)75.878.343.5
S1-RBD-9RBD235 (97.5)149.4 (54.1)115.7 (18.9)35.9 (5.1)24.5 (5.8)
S1-RBD-11RBD13.5 (5.50)NA40.4 (2.5)100.6138.6
S1-RBD-15RBD4.6 (1.2)NA3.4 (1.4)5.2 (1.0)1.3 (0.03)
S1-RBD-16RBD79.2 (4.2)1612 (1303)81.2 (10.9)35.7 (23.1)
S1-RBD-20RBD12.4 (1.1)NANA49.6NA
S1-RBD-21RBD17.3 (3.1)NA128.5 (16.8)24.3 (0.7)40.7 (7.0)
S1-RBD-23RBD7.3 (0.4)NA16.3 (1.9)2.85.7
S1-RBD-27RBD163 (71.4)NANA99.8NA
S1-RBD-29RBD9.5 (1.0)NA
S1-RBD-35RBD12.3 (2.4)NA51.2 (4.2)11.5 (1.1)17.7 (4.0)
S1-RBD-37RBD523 (93.4)NANA
S1-RBD-40RBD25.6 (3.4)NA299.8 (200)10.5 (0.2)32.2 (7.1)
S1-RBD-47RBD127 (11.6)NANA206 (123)NA
S1-RBD-48RBD68.7 (14.2)NANA106.6 (65.6)NA
S2-2S24460 (901)NA
S2-3S22234 (751)6277
S2-40S21712 (828)NA
S2-62S27177 (5801)1954 (364)
  1. –, not determined; NA, no activity.

Table 6
Nanobody neutralization activity against SARS-CoV-2; related to Figure 5.

Nanobodies were assayed for neutralization activity against authentic SARS-CoV-2, with IC50s calculated from neutralization curves.

IDEpitopeSARS-CoV-2 IC50(nM)
S1-1RBD1.1
S1-4RBD1310
S1-23RBD0.7
S1-RBD-4RBD5.4
S1-RBD-11RBD3.0
S1-RBD-23RBD6.1
S2-10Non-RBD91.2
LaM2Non-target ctrlNA
  1. NA, no activity.

Table 7
Nanobody neutralization of rVSV/SARS-CoV-2 and selected resistant mutants; related to Figure 6.

Neutralization assays were carried out using rVSV/SARS-CoV-2 and 293T/ACE2cl.22 target cells treated with the denoted nanobodies. Pseudovirus with either wild-type or variant spike (with escape mutants selected using the corresponding nanobody) was used. Escape mutants and IC50s are listed. Amino acid substitutions contributing to loss of neutralization activity are indicated in bold.

NanobodyEpitoperVSV/SARS-CoV-2 variantIC50 (nM)± s.e.m.
S1-1RBDwt2.63 ± 0.23
Y369N122 ± 3.0
G404E40.8 ± 1.01
S1-6RBDwt13.0 ± 3.47
D574N*, Q792H, Q992H587 ± 31.1
S371P, H66R, N969T202 ± 29.9
S1-23RBDwt0.58 ± 0.02
F490S, E484K, Q493K> 1000
Q493R, G252R> 1000
E484K> 1000
S1-36RBDwt3.69 ± 0.14
W64R,L452F262 ± 10.1
W64R,F490L,I931G870 ± 202
W64R, F490S>1000
S1-37RBDwt1.83 ± 0.59
W64R, F490S>1000
S1-48RBDwt1.75 ± 0.43
Y449H, F490S, Q787R>1000
S494P>1000
S1-62RBDwt0.65 ± 0.16
E484K>1000
S1-3trimerS1 non-RBDwt60.0
W64R, Y170H, V705M>1000
W64R, Y170H, Q787H>1000
S1-30trimerS1 non-RBDwt150
T315I2400
S1-49S1 non-RBDwt146 ± 53.8
S172G>1000
S1-49dimerS1 non-RBDwt3.38 ± 2.44
S172G>1000
S1-49trimerS1 non-RBDwt0.47 ± 0.00
S172G>1000
S2-10S2wt6649 ± 2,545
W64R, S982R>100,000
S2-10dimerS2wt1015 ± 236
W64R, S982R>40,000
S1-RBD-9RBDwt30.2 ± 7.43
T259K, K378Q>1000
W64R, K378Q>1000
K378Q>1000
S1-RBD-11RBDwt1.44 ± 0.53
F486S>1000
T478R>1000
T478I>1000
S1-RBD-15RBDwt1.21 ± 0.06
Y508H549 ± 36.9
S1-RBD-16RBDwt268 ± 162
N354S>1000
S1-RBD-21RBDwt9.61 ± 1.90
F486L>1000
Y489H>1000
S1-RBD-22RBDwt31.5 ± 11.8
K378Q>1000
S1-RBD-23RBDwt14.8 ± 3.55
L452R>1000
H245R, S349P, H1083Y>1000
S1-RBD-24RBDwt58.0 ± 0.00
P384Q>1000
K378Q>1000
S1-RBD-29RBDwt18.0 ± 1.80
E484G>1000
E484K>1000
S1-RBD-35RBDwt1.80 ± 0.15
T478I>1000
F486L306 ± 17.2
Y489H57.4 ± 4.5
S1-RBD-40RBDwt38.9 ± 11.7
W64R, F490S> 500
  1. *

    Residue 574 is outside the structurally covered region of the RBD (residues 333–526) and, therefore, was not used in the Integrative Modeling Platform modeling.

  2. Variant was separately identified by selection against a different nanobody.

Table 8
Cross-linked residues used in integrative modeling; related to Figure 6.

The indicated nanobodies were bound to RBD, NTD, or the spike ectodomain and cross-linked with disuccinimidyl suberate (DSS). Cross-linked complexes were excised from SDS-PAGE gels, reduced, alkylated, and digested with either trypsin or chymotrypsin. Peptides were extracted and analyzed by mass spectrometry. Cross-linked residues (listed) were identified using pLink, and spectra were manually validated to eliminate false positives.

NanobodyNanobody residue #Spike construct7KRQ residue number
S1-4949NTD187
S1-4970NTD187
S1-4970NTD41
S1-4970NTD182
S1-4981NTD97
S1-4981NTD187
S1-4981NTD182
S1-4981NTD41
S1-147RBD458
S1-147RBD462
S1-147RBD417
S1-169RBD458
S1-169RBD444
S1-169RBD417
S1-180RBD417
S1-180RBD386
S1-180RBD444
S1-180RBD458
S1-180RBD417
S1-191RBD444
S1-1105RBD386
S1-2347RBD458
S1-2347RBD444
S1-2347RBD462
S1-2369RBD444
S1-2369RBD462
S1-2391RBD417
S1-2391RBD444
S1-2391RBD417
S1-4647RBD458
S1-4669RBD386
S1-4669RBD458
S1-4680RBD458
RBD-947RBD444
RBD-969RBD386
RBD-969RBD444
RBD-980RBD458
RBD-9114RBD444
RBD-3547RBD458
RBD-3547RBD462
RBD-3562RBD417
RBD-3562RBD458
RBD-3568RBD458
RBD-3568RBD444
S2-1069Spike ectodomain964
S2-1080Spike ectodomain835
S2-10115Spike ectodomain854
S2-10115Spike ectodomain964
S2-4069Spike ectodomain814
S2-4069Spike ectodomain786
S2-4069Spike ectodomain790
Table 9
Nanobody synergy of neutralization activity; related to Figure 7.

Parameters from modeling the synergy observed for the indicated nanobody pairs. Multidimensional synergy of combinations (MuSyC), equivalent dose, and Bivariate Response to Additive Interacting Doses (BRAID) models were used to determine if statistically significant synergy was evident from the neutralization response in a 2D grid of nanobody concentrations.

First nanobodySecond nanobody(Nanobody #1) experimental range(Nanobody #2) experimental rangeh1h2C1 (nM)C2 (nM)alpha12alpha21a12a21Kappa
S1-23S1-274.1 pM to 717 nM, 0 µM3.4 pM to 600 nM, 0 µM1.801.4625.020.0n.s.n.s.n.s.n.s.n.s
S1-1S1-232.2 pM to 387 nM, 0 µM4.1 pM to 717 nM, 0 µM0.931.514.218.621.431.8SynergySynergySynergy
S1-RBD-15S1-233 pM -to 527 nM, 0 µM4.1 pM to 717 nM, 0 µM1.321.423.710.3300.010.2SynergySynergySynergy
S1-RBD-15S1-RBD-233 pM to 527 nM, 0 µM1.8 pM to 325 nM, 0 µM1.441.004.47.95232.9Synergyn.s.Synergy
S1-23S1-464.1 pM to 717 nM, 0 µM7.55 pM to 1.34 µM, 0 µM1.760.753.3273.050.11.0SynergyAntagonismSynergy
S1-RBD-15S1-4639.7 pM to 7.04 µM, 0 µM7.55 pM to 1.34 µM, 0 µM1.100.754.916410.70.9SynergyAntagonismSynergy
S1-23S2-10-dimer4.1 pM to 717 nM, 0 µM5.1 pM to 897 nM, 0 µM1.481.145.445.44232.02.8Synergyn.s.Synergy
S1-49S1-12.1 pM to 367 nM, 0 µM2.2 pM to 387 nM, 0 µM0.771.62152.01.71,14718.1SynergySynergySynergy
S1-49S1-RBD-152.1 pM to 367 nM, 0 µM39.7 pM to 7.04 µM, 0 µM0.851.20629.02.2243.520.3n.s.SynergySynergy
S1-RBD-15S2-10-dimer3 pM to 527 nM, 0 µM5.1 pM to 897 nM, 0 µM1.571.001.9564,1102.2SynergySynergySynergy
  1. n.s., not significant; h1, h2, Hill slope; C1, C2, IC50 (nM);alpha12, alpha21, synergistic/antagonistic fold change of potency from MuSyC model; a12, a21, equivalent dose model synergy/antagonism; kappa, BRAID model synergy/antagonism.

Appendix 1—key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Strain, strain background (Escherichia coli)ArticExpress(DE3)AgilentCat# 230192Competent cells, enabling efficient high-level expression of heterologous proteins.
Strain, strain background (vesicular stomatitis virus)rVSV/SARS-CoV-2/GFP; WT2E1Schmidt et al., 2020Recombinant chimeric VSV/SARS-CoV-2 reporter virus.Inquiries should be addressed to P. Bieniasz.
Strain, strain background (vesicular stomatitis virus)2E1-Y369NThis studyMutant rVSV/SARS-CoV-2/GFP derivative.Inquiries should be addressed to P. Bieniasz.
Strain, strain background (vesicular stomatitis virus)2E1-G404EThis studyMutant rVSV/SARS-CoV-2/GFP derivative.Inquiries should be addressed to P. Bieniasz.
Strain, strain background (vesicular stomatitis virus)2E1-D574N, E484K, Q493KThis studyMutant rVSV/SARS-CoV-2/GFP derivative.Inquiries should be addressed to P. Bieniasz.
Strain, strain background (vesicular stomatitis virus)2E1-S371P, H66R, N969TThis studyMutant rVSV/SARS-CoV-2/GFP derivative.Inquiries should be addressed to P. Bieniasz.
Strain, strain background (vesicular stomatitis virus)2E1-F490S, E484K, Q493KThis studyMutant rVSV/SARS-CoV-2/GFP derivative.Inquiries should be addressed to P. Bieniasz.
Strain, strain background (vesicular stomatitis virus)2E1-Q493R, G252RThis studyMutant rVSV/SARS-CoV-2/GFP derivative.Inquiries should be addressed to P. Bieniasz.
Strain, strain background (vesicular stomatitis virus)2E1-W64R, L452FThis studyMutant rVSV/SARS-CoV-2/GFP derivative.Inquiries should be addressed to P. Bieniasz.
Strain, strain background (vesicular stomatitis virus)2E1-H245R, H1083YThis studyMutant rVSV/SARS-CoV-2/GFP derivative.Inquiries should be addressed to P. Bieniasz.
Strain, strain background (vesicular stomatitis virus)2E1-W64R, F490L, I931GThis studyMutant rVSV/SARS-CoV-2/GFP derivative.Inquiries should be addressed to P. Bieniasz.
Strain, strain background (vesicular stomatitis virus)2E1-W64R, F490SThis studyMutant rVSV/SARS-CoV-2/GFP derivative.Inquiries should be addressed to P. Bieniasz.
Strain, strain background (vesicular stomatitis virus)2E1-Y449H, F490S, Q787RThis studyMutant rVSV/SARS-CoV-2/GFP derivative.Inquiries should be addressed to P. Bieniasz.
Strain, strain background (vesicular stomatitis virus)2E1-S494PThis studyMutant rVSV/SARS-CoV-2/GFP derivative.Inquiries should be addressed to P. Bieniasz.
Strain, strain background (vesicular stomatitis virus)2E1-S172GThis studyMutant rVSV/SARS-CoV-2/GFP derivative.Inquiries should be addressed to P. Bieniasz.
Strain, strain background (vesicular stomatitis virus)2E1-E484KSchmidt et al., 2020Mutant rVSV/SARS-CoV-2/GFP derivative.Inquiries should be addressed to P. Bieniasz.
Strain, strain background (vesicular stomatitis virus)2E1-W64R, S982RThis studyMutant rVSV/SARS-CoV-2/GFP derivative.Inquiries should be addressed to P. Bieniasz.
Strain, strain background (vesicular stomatitis virus)2E1-T259K, K378QThis studyMutant rVSV/SARS-CoV-2/GFP derivative.Inquiries should be addressed to P. Bieniasz.
Strain, strain background (vesicular stomatitis virus)2E1-W64R, K378QThis studyMutant rVSV/SARS-CoV-2/GFP derivative.Inquiries should be addressed to P. Bieniasz.
Strain, strain background (vesicular stomatitis virus)2E1-F486SThis studyMutant rVSV/SARS-CoV-2/GFP derivative.Inquiries should be addressed to P. Bieniasz.
Strain, strain background (vesicular stomatitis virus)2E1-T478RThis studyMutant rVSV/SARS-CoV-2/GFP derivative.Inquiries should be addressed to P. Bieniasz.
Strain, strain background (vesicular stomatitis virus)2E1-T478IThis studyMutant rVSV/SARS-CoV-2/GFP derivative.Inquiries should be addressed to P. Bieniasz.
Strain, strain background (vesicular stomatitis virus)2E1-Y508HThis studyMutant rVSV/SARS-CoV-2/GFP derivative.Inquiries should be addressed to P. Bieniasz.
Strain, strain background (vesicular stomatitis virus)2E1-N354SThis studyMutant rVSV/SARS-CoV-2/GFP derivative.Inquiries should be addressed to P. Bieniasz.
Strain, strain background (vesicular stomatitis virus)2E1-F486LThis studyMutant rVSV/SARS-CoV-2/GFP derivative.Inquiries should be addressed to P. Bieniasz.
Strain, strain background (vesicular stomatitis virus)2E1-Y489HThis studyMutant rVSV/SARS-CoV-2/GFP derivative.Inquiries should be addressed to P. Bieniasz.
Strain, strain background (vesicular stomatitis virus)2E1-K378QThis studyMutant rVSV/SARS-CoV-2/GFP derivative.Inquiries should be addressed to P. Bieniasz.
Strain, strain background (vesicular stomatitis virus)2E1-L452RThis studyMutant rVSV/SARS-CoV-2/GFP derivative.Inquiries should be addressed to P. Bieniasz.
Strain, strain background (vesicular stomatitis virus)2E1-H245R, S349P, H1083YThis studyMutant rVSV/SARS-CoV-2/GFP derivative.Inquiries should be addressed to P. Bieniasz.
Strain, strain background (vesicular stomatitis virus)2E1-P384QThis studyMutant rVSV/SARS-CoV-2/GFP derivative.Inquiries should be addressed to P. Bieniasz.
Strain, strain background (vesicular stomatitis virus)2E1-E484GThis studyMutant rVSV/SARS-CoV-2/GFP derivative.Inquiries should be addressed to P. Bieniasz.
Strain, strain background (vesicular stomatitis virus)2E1-W64R, Y170H, V705MThis studyMutant rVSV/SARS-CoV-2/GFP derivative.Inquiries should be addressed to P. Bieniasz.
Strain, strain background (vesicular stomatitis virus)2E1-W64R, Y170H, Q787HThis studyMutant rVSV/SARS-CoV-2/GFP derivative.Inquiries should be addressed to P. Bieniasz.
Strain, strain background (vesicular stomatitis virus)2E1-T315IThis studyMutant rVSV/SARS-CoV-2/GFP derivative.Inquiries should be addressed to P. Bieniasz.
Strain, strain background (betacoronavirus)SARS-CoV-2, Isolate USA-WA1/2020BEI ResourcesNR-52281Wild-type SARS-CoV-2.
Strain, strain background (betacoronavirus)SARS-CoV-2, Isolate USA-WA (B.1.617.2)R.ColerDeltaSARS-CoV-2 delta variant of concern.
Biological sample (Lama glama)Bone marrow aspiratesCapralogicsFrom two male llamas immunized with SARS-CoV-2 spike S1, RBD, and S2.
Biological sample (L. glama)SeraCapralogicsFrom two male llamas immunized with SARS-CoV-2 spike S1, RBD, and S2.
Cell line (Homo sapiens)293T/17ATCCCRL-11268Human kidney epithelial cells.
Cell line (H. sapiens)293/ACE2cl.22Schmidt et al., 2020293T cells expressing human ACE2 (single-cell clone).
Cell line (H. sapiens)293T-ACE2Cawford et al., 2020BEI NR-52511293T cells expressing human ACE2 (single-cell clone).
Cell line (H. sapiens)Primary human airway epithelial cellsThis studyAir-liquid interface culture system.Inquiries should be addressed to J. Debley.
Cell line (Cercopithecus aethiops)VERO C1008 [Vero 76, clone E6, Vero E6]ATCCCRL-1586Monkey kidney epithelial cells.
Cell line (C. aethiops)TMPRSS2+ Vero E6R.ColerVero E6 cells expressing human TMPRSS2.
AntibodyAnti-COVID-19 and SARS-CoV S glycoprotein [CR3022] (human monoclonal)Absolute AntibodyCat# Ab01680-10.0Flow cytometry (1:1000).
AntibodyAnti-human IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (goat polyclonal)InvitrogenCat# A-11013Flow cytometry (1:2000).
Recombinant DNA reagentpET21-pelBFridy et al., 2014Expression plasmid for expressing nanobodies.
Recombinant DNA reagentpET21-pelB-SARS-CoV-2 NanobodyThis study;
Fridy et al., 2014
See Table 1 and Table 2SARS-CoV-2 nanobody expression plasmids.Inquiries should be addressed to M. Rout.
Recombinant DNA reagentpcDNA3.1+ SARS-1-S-C9T.GallagherSARS-CoV-1 spike expression plasmid.
Recombinant DNA reagentpcDNA3.1+ SARS-2-S-C9 WUHAN-1T.GallagherSARS-CoV-2 spike expression plasmid.
Recombinant DNA reagentpcDNA3.1+ SARS-2-B.1.1.7NIAIDSARS-CoV-2 spike expression plasmid for alpha variant.
Recombinant DNA reagentpHDM-SARS-CoV-2-Spike-B.1.351L.StamatatosSARS-CoV-2 spike expression plasmid for beta variant.
Recombinant DNA reagentpcDNA3.1+ SARS-2 P.1NIAIDSARS-CoV-2 spike expression plasmid for gamma variant.
Recombinant DNA reagentpsPAX2D.Trono/AddgenePlasmid #12260Second-generation lentiviral packaging plasmid.
Recombinant DNA reagentpHAGE-CMV-Luc2-IRES-ZsGreen-WJ.Bloom /
Cawford et al., 2020
NR-52516Lentiviral backbone plasmid that uses a CMV promoter to express luciferase followed by an IRES and ZsGreen.
Sequence-based reagent (primer)6N_CALL001This studyPCR and sequencing primerNNNNNNGTCCTGGCTGCTCTTCTACAAGG
Sequence-based reagent (primer)6N_CALL001BThis studyPCR and sequencing primerNNNNNNGTCCTGGCTGCTCTTTTACAAGG
Sequence-based reagent (primer)6N_VHH_SH_revThis studyPCR and sequencing primerNNNNNNCTGGGGTCTTCGCTGTGGTGC
Sequence-based reagent (primer)6N_VHH_LH_revThis studyPCR and sequencing primerNNNNNNGTGGTTGTGGTTTTGGTGTCTTGGG
Peptide, recombinant proteinSpike S1 (Wuhan Str.)Sino BiologicalCat# 40591-V08HFor determining KDs.
Peptide, recombinant proteinSpike RBDSino BiologicalCat# 40592-VNAHFor determining KDs.
Peptide, recombinant proteinSARS-CoV-2 (2019-nCoV) Spike RBD-mFc Recombinant ProteinSino BiologicalCat# 40592-V05HFor epitope mapping.
Peptide, recombinant proteinSpike S2Sino BiologicalCat# 40590-V08BFor immunization and for determining KDs.
Peptide, recombinant proteinSpike S1 (501Y.V1)Sino BiologicalCat# 40591-V08H12For determining KDs.
Peptide, recombinant proteinSpike S1 (501Y.V2)Sino BiologicalCat# 40591-V08H10For determining KDs.
Peptide, recombinant proteinSARS-CoV-2 Spike S1, Sheep Fc-TagThe Native Antigen Co.Cat# REC31806For immunization.
Peptide, recombinant proteinSARS-CoV-2 Spike S2, Sheep Fc-TagThe Native Antigen Co.Cat# REC31807For immunization.
Peptide, recombinant proteinProtein MGrover et al., 2014Used to deplete light-chain containing IgGs.
Peptide, recombinant proteinThyroglobulinSigma-AldrichCat# A8531-1VUsed to calibrate mass photometer.
Peptide, recombinant proteinBovine serum albuminSigma-AldrichCat# T9145-1VLUsed to calibrate mass photometer.
Peptide, recombinant proteinBeta-amylaseSigma-AldrichCat# A8781-1VLUsed to calibrate mass photometer.
Peptide, recombinant proteinFabRICATOR (IdeS)GenovisCat# A0-FR1-050Protease to cleave VHH domain from the HCAb.
Commercial assay, kitProteOn Amine Coupling KitBio-RadCat# 1762410Used to couple VHH domain to beads.
Commercial assay, kitTruSeq Nano DNA Low Throughput Library Prep KitIlluminaCat# 20015964Used to sequence VHH.
Commercial assay, kitSteady-GLOPromegaCat# E2520Used in pseudovirus assay.
Software, algorithmLlama-MagicFridy et al., 2014https://github.com/FenyoLab/llama-magicFor identifying nanobody sequences.
Software, algorithmIMP, the Integrative Modeling PlatformRussel et al., 2012https://integrativemodeling.orgFor integrative structural modeling.
Software, algorithmUCSF ChimeraXPettersen et al., 2021https://www.rbvi.ucsf.edu/chimerax/download.htmlFor visualizing structural models.
Software, algorithmsynergy v0.4Wooten et al., 2021https://pypi.org/project/synergy/For observing synergy.
Software, algorithmmatplotlib v3.4.1Hunter, 2007https://pypi.org/project/matplotlib/For preparing figures.
Software, algorithmseaborn v0.11.0Waskom, 2021https://seaborn.pydata.org/index.htmlFor preparing figures.
Software, algorithmplotly v4.12.02019 Plotly, Inchttps://plotly.com/python/For preparing figures.
Software, algorithmnumpy v1.19.2Huo et al., 2020ahttps://numpy.orgFor data analysis.
Software, algorithmpandas v1.1.2The pandas development team, 2020https://pandas.pydata.orgFor data analysis.
Software, algorithmscipy v1.5.0Virtanen et al., 2020https://www.scipy.orgFor data analysis.
Software, algorithmscikit-learn v0.23.2Pedregosa, 2011https://scikit-learn.org/stable/For data analysis.
Software, algorithmpython v3.8Van Rossum and Drake, 2009https://www.python.orgFor data analysis and preparing figures.
Software, algorithmPrism 9GraphPadhttps://www.graphpad.comFor data analysis and preparing figures.
Software, algorithmOCTETSartoriusFor data analysis.
OtherProteOn GLC Sensor ChipBio-RadCat# 176-5011For protein interaction analysis.
OtherSeries S Sensor Chip CM5CytivaCat# BR100530For protein interaction analysis.
OtherHard-shell PCR plates, 96-well, thin-wallBio-RadCat# HSP9661For differential scanning fluorimetry.
OtherMicroseal ‘B’ sealBio-RadCat# MSB1001For differential scanning fluorimetry.
OtherPrecision CoverslipsThorlabsCat# CG15KH1For mass photometry.
OtherCultureWell Reusable GasketGrace Bio-LabsCat# 103250For mass photometry.
OtherProtein A Sepharose 4BThermo FisherCat# 101042For protein purification.
OtherRecombinant Protein G Sepharose 4BThermo FisherCat# 101243For protein purification.
OtherCNBr-activated Sepharose 4 Fast FlowCytivaCat# 17098101For nanobody screening.
OtherSuperScript VILO Master MixThermo FisherCat# 11755250For viral escape analysis.
OtherAnti-mouse IgG Fc Capture BiosensorsSartoriusCat# 18-5088For epitope mapping.
OtherKOD Xtreme Hot Start DNA PolymeraseSigma-AldrichCat# 71975For viral escape analysis.

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  1. Fred D Mast
  2. Peter C Fridy
  3. Natalia E Ketaren
  4. Junjie Wang
  5. Erica Y Jacobs
  6. Jean Paul Olivier
  7. Tanmoy Sanyal
  8. Kelly R Molloy
  9. Fabian Schmidt
  10. Magdalena Rutkowska
  11. Yiska Weisblum
  12. Lucille M Rich
  13. Elizabeth R Vanderwall
  14. Nicholas Dambrauskas
  15. Vladimir Vigdorovich
  16. Sarah Keegan
  17. Jacob B Jiler
  18. Milana E Stein
  19. Paul Dominic B Olinares
  20. Louis Herlands
  21. Theodora Hatziioannou
  22. D Noah Sather
  23. Jason S Debley
  24. David Fenyö
  25. Andrej Sali
  26. Paul D Bieniasz
  27. John D Aitchison
  28. Brian T Chait
  29. Michael P Rout
(2021)
Highly synergistic combinations of nanobodies that target SARS-CoV-2 and are resistant to escape
eLife 10:e73027.
https://doi.org/10.7554/eLife.73027