Table 2.
Binding kinetics of immobilized CD70 wild-type and mutants with wild-type CD27 (glycosylated) as analytes at the temperature of 37 °C
| Sample | n | ka (104 M−1s−1) | kd (10−3 s−1) | KD (nM) | Result |
|---|---|---|---|---|---|
| CD70 (WT) | 6 | 3.66 (0.20) | 4.41 (0.16) | 121 (8) | Homogeneous kinetics |
| CD70-Q61A | 6 | 3.30 (0.38) | 6.76 (0.27) | 205 (25) | |
| CD70-A80F | 6 | 1.44 (0.33) | 9.27 (0.44) | 643 (151) | |
| CD70-A80R | 6 | 1.66 (0.21) | 2.70 (0.29) | 163 (24) | |
| CD70-S137A | 6 | 3.80 (0.54) | 5.91 (0.10) | 156 (22) | |
| CD70-S137E | 6 | 2.83 (0.04) | 14.90 (0.59) | 526 (22) | |
| CD70-S137K | 6 | 2.92 (0.18) | 3.93 (0.16) | 135 (10) | |
| CD70-S146A | 6 | 3.59 (0.06) | 4.17 (0.08) | 116 (3) | |
| CD70-H148A | 6 | 3.93 (0.07) | 9.28 (0.29) | 236 (9) | |
| CD70-H148E | 4 | 5.37 (2.23) | 20.40 (7.03) | 380 (205) | |
| CD70-H148D | 6 | 3.41 (0.30) | 16.30 (1.81) | 477 (68) | |
| CD70-A80L | 2 | -- | -- | -- | Altered binding kinetics compared with CD70-WTa |
| CD70-N180A | 2 | -- | -- | -- | |
| CD70-A80V | 2 | -- | -- | -- | No bindingb |
| CD70-R83A | 2 | -- | -- | -- | |
| CD70-R83E | 2 | -- | -- | -- | |
| CD70-R83K | 2 | -- | -- | -- | |
| CD70-C115AC151A | 2 | -- | -- | -- | |
| CD70-R144A | 2 | -- | -- | -- | |
| CD70-R144E | 2 | -- | -- | -- | |
| CD70-S146D | 5 | -- | -- | -- | |
| CD70-N170Q | 2 | -- | -- | -- | |
| CD70-S178A | 2 | -- | -- | -- | |
| CD70-N180R | 2 | -- | -- | -- | |
| CD70-N180W | 2 | -- | -- | -- | |
| CD70-C133AC168A | 2 | -- | -- | -- | Low expressionc |
| CD70-N63Q | 2 | -- | -- | -- | |
| CD70-T65A | 2 | -- | -- | -- | |
| CD70-T172A | 2 | -- | -- | -- | |
| CD70-D165R | 3 | -- | -- | -- | |
| CD70-D165A | 2 | -- | -- | -- | |
| CD70-P135A | 2 | -- | -- | -- |
Avi-tagged Human CD70 wild-type and mutants were expressed in Expi293 cells and cell culture supernatants were captured onto anti-Avi surfaces. At least four independent expression batches were tested for CD70 mutants with homogeneous kinetic profiles. The number of replicate measurements is listed as n. Standard deviations are shown in parenthesis.
a
Kinetics and affinity parameters were not reported due to weak binding.
b
Expression was satisfactory (CD70 ligand capture of >15 RU), but no CD27 binding was observed.
c
Expression levels were too low (CD70 ligand capture of <5 RU) to accurately assess CD27 binding.