Utility of Cellular Measurements of Non-Specific Endocytosis to Assess the Target-Independent Clearance of Monoclonal Antibodies

Past studies have demonstrated higher clearance for monoclonal antibodies possessing increased rates of non-specific endocytosis. However, this metric is oftentimes evaluated indirectly using biophysical techniques or cell surface binding studies that may not provide insight into the specific rates of cellular turnover. Furthermore, few examples evaluating non-specific endocytosis have been reported for a therapeutic antibody that reached clinical assessment. In the current report, we evaluated a therapeutic human immunoglobulin G2 monoclonal antibody targeted against the interleukin-4 receptor alpha chain (IL-4R) that exhibited elevated target independent clearance in previous Phase 1 and 2 studies. We confirmed high non-specific clearance of the anti-IL-4R antibody as compared to a reference antibody during pharmacokinetic assessments in wild type mice where target-mediated disposition was absent. We then developed a cell-based method capable of measuring cellular protein endocytosis and demonstrated the anti-IL-4R antibody exhibited marked non-specific uptake relative to the reference compound. Antibody homology modeling identified the anti-IL-4R antibody possessed positive charge patches whose removal via targeted mutations substantially reduced its non-specific endocytosis. We then expanded the scope of the study by evaluating a panel of consisting of both preclinical and clinical monoclonal antibodies and demonstrate those with the highest rates of non-specific uptake in vitro exhibit elevated target independent clearance, low subcutaneous bioavailability, or both. Our results support the observation that high non-specific endocytosis is a negative attribute in monoclonal antibody development and demonstrate the utility of a generic cell-based screen as a quantitative tool to measure non-specific endocytosis of protein therapeutics at the single-cell level. Highlights- Developed a novel, reproducible cellular assay to directly quantify non-specific endocytosis of therapeutic proteins. - A previous clinical candidate monoclonal antibody with rapid target-independent clearance in mice and humans possessed extensive non-specific endocytosis that was due to exposed positive charge features. - Demonstration of distinct rates of endocytosis into mammalian cells for disparate monoclonal antibodies, even those with common specificity for targets or isoelectric points. - Cell-based assay to quantify the potential impact of non-specific endocytosis on target-independent clearance and/or subcutaneous bioavailability of monoclonal antibodies.