TABLE 1.
Binding affinity and analytical data for the parental and humanized sequence variants of VHH1 and VHH2.
| KD (nM) | K on (1/Ms) | K off (1/s) | Emax100nM (nm) | R 2 | Yield (mg/L) | SEC purity (%) | |
|---|---|---|---|---|---|---|---|
| VHH1‐v1.0 | 0.2 | 1.1E+06 | 2.7E‐04 | 0.13 | 0.99 | 66.5 | 87.2 |
| VHH1‐v1.1 | 0.3 | 1.0E+06 | 2.9E‐04 | 0.13 | 0.99 | 58.8 | 89.5 |
| VHH1‐v1.2 | 0.3 | 1.1E+06 | 3.2E‐04 | 0.12 | 1.00 | 41.9 | 85.3 |
| VHH1‐v1.3 | 1.1 | 8.8E+05 | 9.4E‐04 | 0.12 | 0.99 | 38.3 | 85.4 |
| VHH1‐v1.4 | 0.5 | 1.1E+06 | 6.2E‐04 | 0.12 | 0.99 | 86.3 | 76.8 |
| VHH1‐v1.5 | 0.9 | 1.1E+06 | 9.7E‐04 | 0.10 | 0.98 | 87.3 | 73.0 |
| VHH1‐v1.6 | 0.8 | 1.1E+06 | 8.7E‐04 | 0.14 | 0.99 | 74.1 | 63.8 |
| VHH1‐v1.7 | 0.5 | 1.0E+06 | 5.5E‐04 | 0.09 | 0.99 | 80.7 | 65.0 |
| VHH1‐v1.8 | 1.1 | 1.1E+06 | 1.3E‐03 | 0.10 | 0.99 | 70.0 | 73.0 |
| VHH1‐v1.9 | 1.1 | 8.7E+05 | 9.3E‐04 | 0.10 | 0.98 | 68.4 | 69.8 |
| VHH1‐v1.10 | nb | nb | nb | nb | nb | 85.2 | 64.4 |
| VHH2‐v1.0 | 2.9 | 2.3E+05 | 6.6E‐04 | 0.17 | 1.00 | 98.2 | 89.1 |
| VHH2‐v1.1 | 2.9 | 2.7E+05 | 7.9E‐04 | 0.18 | 1.00 | 104.7 | 89.5 |
| VHH2‐v1.2 | 2.1 | 3.5E+05 | 7.4E‐04 | 0.15 | 1.00 | 120.2 | 89.5 |
| VHH2‐v1.3 | 10.0 | 9.3E+04 | 9.3E‐04 | 0.13 | 1.00 | 43.1 | 97.3 |
| VHH2‐v1.4 | nb | nb | nb | nb | nb | 26.1 | 100.0 |
| VHH2‐v1.5 | 2.9 | 3.8E+05 | 1.1E‐03 | 0.11 | 0.99 | 86.6 | 95.7 |
| VHH2‐v1.6 | 27.6 | 4.6E+04 | 1.3E‐03 | 0.07 | 0.98 | 77.6 | 94.0 |
| VHH2‐v1.7 | 9.8 | 1.1E+05 | 1.1E‐03 | 0.10 | 0.99 | 63.1 | 96.9 |
| VHH2‐v1.8 | 6.6 | 1.3E+05 | 8.4E‐04 | 0.10 | 0.99 | 109.9 | 94.4 |
| VHH2‐v1.9 | nb | nb | nb | nb | nb | 37.5 | 98.1 |
| VHH2‐v1.10 | nb | nb | nb | nb | nb | 42.3 | 97.9 |
| VHH2‐v1.11 | 4.7 | 1.6E+05 | 7.4E‐04 | 0.16 | 0.99 | 100.1 | 93.5 |
| VHH2‐v1.12 | 4.2 | 1.3E+05 | 5.5E‐04 | 0.14 | 0.99 | 118.6 | 93.3 |
| VHH2‐v1.13 | nb | nb | nb | nb | nb | 98.6 | 95.8 |
| VHH2‐v1.14 | nb | nb | nb | nb | nb | 82.1 | 95.9 |
| VHH2‐v1.15 | 12.3 | 5.8E+04 | 7.2E‐04 | 0.07 | 0.99 | 21.5 | 99.1 |
| VHH2‐v1.16 | 4.7 | 8.7E+04 | 4.1E‐04 | 0.11 | 0.99 | 86.1 | 97.8 |
| VHH2‐v1.17 | nb | nb | nb | nb | nb | 18.0 | 100.0 |
| VHH2‐v1.18 | nb | nb | nb | nb | nb | 91.9 | 97.1 |
Note: K on is the rate constant of association, while K off is the rate constant of dissociation. Emax is the maximum interference pattern shift that was observed for a sample in the experiment, reflecting the maximum binding capacity. R 2: fit quality to the experimental BLI data.