There is growing interest in the fast and robust engineering of protein pH-sensitivity that aims to reduce binding at acidic pH, compared to neutral pH. binding at pH 7.4 in the sub-nanomolar range was retained. Furthermore, binding to huFcRn and thermal stability were not affected by histidine substitutions. Overall, this study emphasizes a generalizable strategy for engineering pH-switch functions potentially applicable to a variety of antibodies and further proteins-based therapeutics. biolayer interferometry (BLI) (data not shown). Three clones (PSV#1, PSV#2 and PSV#3) were selected according to their differently pronounced pH-sensitive binding profiles and subjected to detailed binding kinetic analysis at pH 7.4 and pH 6.0 compared to commercially available adalimumab (Fig. 4). Results indicate single-digit picomolar binding affinity for wild-type adalimumab at pH 7.4, which corresponds to the affinity determined by Kaymakcalan et?al.35 (KD: 30.4 pM), considering the KD detection limit of BLI at approximately KD: 100 pM.36 Due to the very slow off-rate of adalimumab, fittings are susceptible to little variances within the blank measurement, and that resulted in an approximately 10-fold enhanced KD due to a 9-fold decreased dissociation rate constant compared to the previously published data. Selected antibody variants PSV#1, PSV#2 and PSV#3 showed approximately 10-, 17- and 24-fold reduced affinities compared to adalimumab, mainly driven by increased off-rates (Fig. 4, left panel; Table 1, Table 2). pH-sensitivity was resolved by measuring dissociation at pH 6.0 after association at pH 7.4 (Fig. 4, right panel; Table 1). All 3 selected variants showed distinct rapid AZD6482 antigen release when dissociation was done at pH 6.0. To determine off-rates of the selected variants, a local partial fitting was applied in which the assumption of complete dissociation within the measured time-frame was made. As adalimumab showed only slow rhTNF release within the measured time-window, accurate Kd-values were determined by using a global fitting (association and dissociation). Table 1. Binding kinetics and Tm of adalimumab and pH-dependent variants binding to rhTNF Figure 4. BLI sensorgrams of kinetic analyses of rhTNF binding to immobilized adalimumab and pH-dependent binding variants (PSV#1, PSV#2 and PSV#3). Association with rhTNF at indicated concentrations was measured for 300?s at pH 7.4. Dissociation was performed … The results demonstrate that the dissociation rate constants of PSV#1, PSV#2 and PSV#3 at pH 6.0 were increased by 157-, 1527- and 2293-fold compared to adalimumab. Ratios of kd-values determined at pH 6.0 and pH 7.4 (kd at pH 6.0 / kd at pH 7.4) for PSV#1, PSV#2 and PSV#3 revealed a 231-, 785- and 505-fold enhanced release of antigen, whereas adalimumab showed only a ratio by factor 9 (Table Rabbit Polyclonal to RFA2. 1). In comparison to adalimumab, this indicates that all engineered variants showed a significantly enhanced pH-dependence with only slightly weakened binding affinities at pH 7.4. To analyze whether the thermal stability of the variants was affected by the histidine substitutions, thermal shift assay measurements were performed at pH 7.4 and pH 6.0 (Table 1; Fig. S4). All variants exhibited high thermal stabilities at physiological pH with the lowest Tm value for PSV#3 (67.2C) and the highest Tm for adalimumab (69.8C) indicating only a minor decreases in thermal stability due to the substitution with histidines. In contrast, low thermal stabilities at pH 6.0 were indicated by values that ranged from 61.7C to 63.4C for all 3 variants and wild-type adalimumab, indicating that structural modifications that account for pH-sensitivity seem to have only minor effects on the thermal stability. Reversible pH-dependent antigen binding To investigate AZD6482 reversible association (pH 7.4) and dissociation (pH 6.0) of rhTNF by pH-sensitive variants PSV#1, PSV#2 and PSV#3, 2 consecutive binding cycles were performed in which antigen association to all 4 immobilized antibodies was carried out at pH 7.4, followed by dissociation at pH 6.0 for 400?s (Fig. 5). In all sensorgrams, the binding curves were aligned to the baselines that were measured at pH 7.4 after every dissociation step. All engineered antibodies showed rapid release of AZD6482 rhTNF during the first dissociation step at pH 6.0. This is in accordance with respective kd-values AZD6482 determined before (Table 1). PSV#2 and PSV#3 showed a complete loss of antigen during dissociation. Therefore, similar binding signals compared to initial binding signals were acquired throughout the second association step. Figure 5. Functional analyses AZD6482 of reversible pH-dependent antigen binding of PSV#1, PSV#2, PSV#3 and adalimumab. Two cycles of association to 13?nM rhTNF at pH 7.4 for 300?s and dissociation at pH 6.0 for 400?s were.