Protein Engineering Strategies to Overcome Charge-Driven Clearance Liabilities of Monoclonal Antibodies

The disposition of immunoglobulins (IgGs) is influenced by target-dependent and target-independent mechanisms. Target-independent clearance (CLind) of monoclonal antibodies (mAbs) is primarily determined by the propensity for non-specific endocytosis (NSE) and the efficiency of subsequent pH-dependent cellular recycling mediated by the neonatal Fc receptor (FcRn). These processes were evident in a previous clinical candidate, anti-IL-4Rα mAb, which exhibited high CLind (11 mL/d/kg) in humans. We identified four arginine residues in its variable domain (Fv) causing substantial NSE, impeded FcRn engagement, and rapid CLind. Next, we aimed to expand our understanding of the cellular mechanisms driving CLind of Fc-bearing biologics by determining the extent to which Fc-engineered half-life extension strategies could successfully overcome variable degrees of non-specificity to decrease CLind. To this end, we generated various anti-IL-4Rα mAb point mutants possessing decreasing exposed positive charge with or without the YTE Fc mutation. We demonstrated that reducing the Fv charge significantly decreased the elevated NSE and CLind. Additionally, we observed that targeted Fv mutations to reduce general non-specificity can have a greater impact on mAb CLind when compared to YTE mutations alone. Incorporating both Fv and Fc mutations resulted in better cellular FcRn recycling efficiency scores and reduced CLind.

Learning Objectives: 

1. Discuss the impact of exposed positive charge on monoclonal antibody pharmacokinetics.

2. Explain the utilization of protein engineering to extend half-life of monoclonal antibodies.

3. Summarize how in vitro cellular assays predict in vivo pharmacokinetics of monoclonal antibodies.