The development of Nanosota-1 as anti-SARS-CoV-2 nanobody drug candidates
- Department of Veterinary and Biomedical Sciences, University of Minnesota, United States
- Center for Coronavirus Research, University of Minnesota, United States
- Department of Pharmacology, University of Minnesota, United States
- Department of Microbiology and Immunology, University of Iowa, United States
- Institutional Office of Regulated Nonclinical Studies, University of Texas Medical Branch, United States
- Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, United States
- Laboratory of Viral Immunology, Lindsley F. Kimball Research Institute, New York Blood Center, United States
Figures
Figure 1 with 1 supplement
Construction of a camelid nanobody phage display library and use of this library for screening of anti-SARS-CoV-2 nanobodies.
A large-sized (diversity 7.5 x 1010) naive nanobody phage display library was constructed using B cells of over a dozen llamas and alpacas. Phages were screened for their high binding affinity for …
Figure 1—figure supplement 1
Schematic drawings of nanobodies and conventional antibodies.
VH: variable domain of heavy chain; CH: constant domain of heavy chain; VL: variable domain of light chain; CL: constant domain of light chain; VHH: variable domain of heavy-chain-only antibody; …
Figure 2 with 4 supplements
Crystal structure of SARS-CoV-2 RBD complexed with Nanosota-1C.
(A) Structure of SARS-CoV-2 receptor-binding domain (RBD) complexed with Nanosota-1C, viewed at two different angles. Nanosota-1C is in red, the core structure of RBD is in cyan, and the …
Figure 2—figure supplement 1
Footprint of Nanosota-1C on SARS-CoV-2 RBD.
Receptor-binding domain (RBD) residues that bind to Nanosota-1C are labeled in red, those that bind to angiotensin-converting enzyme 2 (ACE2) are labeled in green, and those that bind to both Nanosot…
Figure 2—figure supplement 2
The binding of Nanosota-1C to SARS-CoV-2 spike protein in different conformations.
(A) The binding of Nanosota-1C to the spike protein in the closed conformation. The structures of the RBD/Nanosota-1C complex and SARS-CoV-2 spike protein in the closed conformation (PDB: 6ZWV) were …
Figure 2—figure supplement 3
Measurement of the binding affinities between Nanosota-1 and SARS-CoV-2 RBD by surface plasmon resonance assay using Biacore.
Purified recombinant SARS-CoV-2 receptor-binding domain (RBD) was covalently immobilized on a sensor chip through its amine groups. Purified recombinant nanobodies (A, B, C, D) flowed over the RBD …
Figure 2—figure supplement 4
Binding interactions between Nanosota-1 and SARS-CoV-2 RBD.
(A) Binding interactions among Nanosota-1C, angiotensin-converting enzyme 2 (ACE2), and SARS-CoV-2 receptor-binding domain (RBD) as evaluated using a protein pull-down assay. Various concentrations …
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Figure 2—figure supplement 4—source data 1
Raw images for Figure 2—figure supplement 4.
- https://cdn.elifesciences.org/articles/64815/elife-64815-fig2-figsupp4-data1-v2.zip
Figure 3 with 2 supplements
Efficacy of Nanosota-1 in neutralizing SARS-CoV-2 infections in vitro.
(A) Neutralization of SARS-CoV-2 pseudovirus entry into target cells by one of three inhibitors: Nanosota-1C-Fc, Nanosota-1C, and recombinant human angiotensin-converting enzyme 2 (ACE2). …
Figure 3—figure supplement 1
Neutralization of SARS-CoV-2 pseudovirus, which contains the D614G mutation in the spike protein, by Nanosota-1.
The procedure was the same as described in Figure 3A, except that the mutant spike protein replaced the wild-type spike protein. The assay was repeated three times (biological replication: new …
Figure 3—figure supplement 2
Detailed data on the neutralization of live SARS-CoV-2 infection of target cells by Nanosota-1.
Data are the mean ± SEM (n = 3). Nonlinear regression was performed using a log (inhibitor) versus normalized response curve and a variable slope model (R2>0.95 for all curves). The assay was …
Figure 4
Efficacy of Nanosota-1 in protecting both hamsters and mice from SARS-CoV-2 infections.
(A, B) Hamsters (six per group) were injected with a single dose of Nanosota-1C-Fc at the indicated time point and the indicated dosage. At day 0, all groups (experimental and control) were …
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Figure 4—source data 1
Raw images for Figure 4.
- https://cdn.elifesciences.org/articles/64815/elife-64815-fig4-data1-v2.zip
Figure 5 with 1 supplement
Analysis of expression, purification, and pharmacokinetics of Nanosota-1C-Fc.
(A) Purification of Nanosota-1C-Fc from bacteria. The protein was nearly 100% pure after gel filtration chromatography, as demonstrated by its elution profile and sodium dodecyl …
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Figure 5—source data 1
Raw images for Figure 5.
- https://cdn.elifesciences.org/articles/64815/elife-64815-fig5-data1-v2.zip
Figure 5—figure supplement 1
Pharmacokinetics of Nanosota-1C.
Tables
Table 1
Binding affinities between Nanosota-1 and SARS-CoV-2 RBD as measured using surface plasmon resonance.
The previously determined binding affinity between human ACE2 and RBD is shown as a comparison (Shang et al., 2020a).
| Kd with SARS-CoV-2 RBD (M) | koff (s−1) | kon (M−1s−1) | |
|---|---|---|---|
| Nanosota-1A (before affinity maturation) | 2.28 x 10−7 | 9.35 x 10−3 | 4.10 x 104 |
| Nanosota-1B (after first round of affinity maturation) | 6.08 x 10−8 | 7.19 x 10−3 | 1.18 x 105 |
| Nanosota-1C (after second round of affinity maturation) | 1.42 x 10−8 | 2.96 x 10−3 | 2.09 x 105 |
| Nanosota-1C-Fc (after second round of affinity maturation; containing a C-terminal human Fc tag) | 1.57 x 10−11 | 9.68 x 10−5 | 6.15 x 106 |
| ACE2 | 4.42 x 10−8 | 7.75 x 10−3 | 1.75 x 105 |
Table 2
X-ray data collection and structure refinement statistics (SARS-CoV-2 RBD/Nanosota-1C complex).
| Data collection | |
|---|---|
| Wavelength | 0.979 |
| Resolution range | 45.48–3.19 (3.30–3.19) |
| Space group | P 43 21 2 |
| Unit cell | 60.849 60.849 410.701 90 90 90 |
| Total reflections | 64167 (5703) |
| Unique reflections | 13607 (1308) |
| Multiplicity | 4.7 (4.4) |
| Completeness (%) | 96.82 (97.60) |
| Mean I/sigma(I) | 8.41 (1.80) |
| Wilson B-factor | 83.24 |
| R-merge | 0.145 (0.928) |
| R-meas | 0.1638 (1.053) |
| R-pim | 0.07385 (0.4858) |
| CC1/2 | 0.995 (0.861) |
| CC* | 0.999 (0.962) |
| Refinement | |
| Reflections used in refinement | 13567 (1301) |
| Reflections used for R-free | 674 (62) |
| R-work | 0.2483 (0.3521) |
| R-free | 0.2959 (0.4153) |
| CC (work) | 0.963 (0.819) |
| CC (free) | 0.909 (0.615) |
| Number of non-hydrogen atoms | 4890 |
| Macromolecules | 4833 |
| Ligands | 57 |
| Protein residues | 621 |
| RMS (bonds) | 0.002 |
| RMS (angles) | 0.45 |
| Ramachandran favored (%) | 93.11 |
| Ramachandran allowed (%) | 6.89 |
| Ramachandran outliers (%) | 0.00 |
| Rotamer outliers (%) | 3.23 |
| Clashscore | 5.25 |
| Average B-factor | 90.29 |
| Macromolecules | 89.84 |
| Ligands | 127.91 |
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Statistics for the highest-resolution shell are shown in parentheses.
Additional files
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Source data 1
Raw data for figures and figure supplements.
- https://cdn.elifesciences.org/articles/64815/elife-64815-data1-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/64815/elife-64815-transrepform-v2.docx
