Table 2. SPR binding parameters for HER2 constructs binding to the DARPin G3 and trastuzumab.
| DARPin G3 | ||||
| HER2 protein | ka×105 (M−1s−1) | kd×10−3 (s−1) | KD (nM) | fold difference in KD |
| wild-typea | 3.22±0.20 | 1.07±0.01 | 3.3±0.2 | 1.0 |
| Leu525-Ala | 1.49±0.03 | 39.3±4.2 | 265±30 | 79.7 |
| Ser551-Ala | 2.85±0.59 | 3.5±2.1 | 12.8±3.1 | 3.8 |
| Val552-Ala | 1.69±0.16 | 64.0±3.7 | 380±39 | 114.4 |
| Phe555-Ala | 1.84±0.09 | 38.4±1.0 | 210±15 | 63.2 |
| trastuzumab | ||||
| HER2 protein | ka×105 (M−1s−1) | kd×10−4 (s−1) | KD (nM) | fold difference in KD |
| wild-type | 1.19±0.09 | 1.56±0.03 | 1.31±0.13 | 1.0 |
| Leu525-Ala | 1.04±0.01 | 1.32±0.05 | 1.26±0.03 | 1.0 |
| Ser551-Ala | 1.07±0.03 | 1.47±0.01 | 1.38±0.04 | 1.0 |
| Val552-Ala | 0.96±0.02 | 1.28±0.08 | 1.33±0.06 | 1.0 |
| Phe555-Ala | 1.06±0.01 | 1.48±0.02 | 1.39±0.03 | 1.1 |
Note 1: The values given are the average values for three separate measurements and the uncertainties represent one standard deviation.
a
Note 2: About 30-fold higher affinities are obtained when avoiding random amine coupling of this very small DARPin [9,36], (Nagy-Davidescu and Plückthun, unpublished).