Antioxidant excipient-mediated stabilization preserves FcγRIIIa binding and ADCC activity of rituximab

Antibody-dependent cell-mediated cytotoxicity (ADCC) is one of the mechanisms of action of therapeutic monoclonal antibodies (mAbs). This mechanism of action is often enhanced either through Fc mutagenesis or glycoengineering. However, certain mAbs exhibit undesirable side effects in patients due to these structural modifications for ADCC enhancement. In this study, instead of modifying the antibody structure through Fc engineering, we explored a formulation-based antioxidant treatment strategy to preserve Fc effector function and possible effects on ADCC activity. The efficacy of this approach was investigated using Rituximab (RmAb) biosimilar. We specifically focused on FcγRIIIa-mediated ADCC of RmAb, as it represents a key effector pathway impacted by Fc stability and post-translational modifications. Our results showed that affinity constants measured using surface plasmon resonance (SPR) exhibited only minor changes in presence of anti-oxidants. An in vitro ADCC reporter assay was used to evaluate FcγRIIIa-mediated effector function, serving as a surrogate assay. The ADCC reporter assay indicated modest within-run shifts in EC₅₀ values, consistent with preservation of FcγRIIIa-mediated signaling under identical assay conditions. The potential of all anti-oxidants was demonstrated using DPPH radical scavenging assay. Further, investigations related to peptide mapping revealed that the addition of anti-oxidants prevented deamidation in asparagine and glutamine residues. We hypothesize that, these effects may be associated with a reduction in pH and the dielectric constant of formulation buffer by the addition of anti-oxidants. Thus, the addition of anti-oxidants to the mAb formulation prevented unfavorable modifications of amino acids and aided in the optimum effector function of Rituximab.