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.).
| ID | Kon(M–1 s–1) | Koff(s–1) | KD (M) | Tm (°C) | SARS-CoV-2 PSV IC50 (s.e.m.) (nM) |
|---|---|---|---|---|---|
| S2-1 | 6.32E+04 | 3.79E-04 | 6.00E-09 | 65.5 | NA |
| S2-2 | 1.26E+06 | 9.35E-05 | 7.41E-11 | 64.5 | 4460 (901) |
| S2-3 | 2.62E+05 | 7.21E-05 | 2.76E-10 | 65 | 2234 (751) |
| S2-4 | 2.44E+06 | 2.62E-04 | 1.08E-10 | 56 | NA |
| S2-5 | 9.35E+05 | 2.74E-04 | 2.93E-10 | 66 | NA |
| S2-6 | – | – | – | 61.5 | NA |
| S2-7 | 1.66E+06 | 9.36E-05 | 5.62E-11 | 61 | NA |
| S2-9 | 9.29E+05 | 2.32E-04 | 2.50E-10 | 64.5 | NA |
| S2-10 | 9.31E+04 | 3.13E-04 | 3.37E-09 | 59, 64.5* | 5269 (1418) |
| S2-11 | 7.94E+06 | 1.12E-03 | 1.41E-10 | 69.5 | NA |
| S2-13 | 7.02E+05 | 1.05E-04 | 1.49E-10 | 64.5 | NA |
| S2-14 | 3.16E+06 | 1.28E-03 | 4.07E-10 | 72.5 | NA |
| S2-15 | – | – | – | 70 | NA |
| S2-18 | 1.63E+06 | 4.87E-04 | 2.99E-10 | 47, 54.5* | NA |
| S2-22 | – | – | – | – | NA |
| S2-26 | 4.45E+05 | 8.15E-05 | 1.83E-10 | 76.5 | NA |
| S2-33 | 3.68E+05 | 5.58E-05 | 2.33E-10 | 70 | NA |
| S2-35 | 2.36E+05 | 4.72E-05 | 2.00E-10 | 77 | NA |
| S2-36 | 4.39E+06 | 3.69E-04 | 8.41E-11 | 74 | NA |
| S2-39 | – | – | – | 58 | NA |
| S2-40 | 5.08E+04 | 7.16E-05 | 1.41E-09 | 69.5 | 1712 (828) |
| S2-42 | 5.12E+05 | 3.77E-06 | 7.36E-12 | 69 | NA |
| S2-47 | 3.86E+05 | 1.14E-04 | 2.96E-10 | 40, 65* | NA |
| S2-57 | 2.33E+06 | 7.18E-04 | 3.08E-10 | 67 | NA |
| S2-59 | – | – | – | 37.5, 60* | NA |
| S2-62 | 1.65E+06 | 1.12E-04 | 6.81E-11 | 64, 77.5* | 7177 (5801) |
*Two peaks were observed in the melting curve. Tms for both are reported.
–, not determined; NA, no activity.