Table 1.
Comparison of parental chicken R3bH01 to humanized & affinity optimized B30.
| Name | VH CDR1 | VH CDR2 | VH CDR3 | VL CDR1 | VL CDR2 | VL CDR3 | Apparent KD (nM) |
Kinexa KD (pM) |
pERK IC50 (nM) |
|---|---|---|---|---|---|---|---|---|---|
| R3bH01 | GFDFSSYDMH | GIDDGGSDTYYGSAVKG | SSYDISWNGHVENIDA | SGAGSGYGYG | SNDKRPS | GTYDSTDAGYAI | 34.42 ± 5.43 (n = 3) |
ND | 65.60 (29.90 − 144.30%CI) (n = 7) |
| B30 | GFDFSSYDMH | GIGDYGIETYYGSAVKG | SSYDISWNGHVEHIDS | SGAGSGYGYG | SNDKRPS | GTYVSAYYGYAI | 0.12 ± 0.01 (n = 3) |
1.06 pM (3.28% error) |
0.08 (0.04–0.14%CI) (n = 20) |
CDR definitions used in this manuscript are an expanded definition in comparison with the classical Kabat, including VH positions 26–29 and 49. Mutations in affinity-optimized B30 are highlighted in bold. BIAcore was used to characterize the binding kinetics for the parental antibody, R3bH01 and the affinity-optimized B30. Rate constants were calculated using a 1:1 binding model (T200 Evaluation Software) and the equilibration dissociation constant (KD) was calculated as kd/ka. KinExa was used to further refine the KD measurement for high affinity B30. The pERK assay was used to measure BDNF-TrkB neutralization in the presence of anti-BDNF antibodies. The Cellul’ERK HTRF detection kit was used to quantify downstream phospho-ERK levels and the results are expressed as pERK IC50.