Structure and Functional Characterization of a Humanized Anti-CCL20 Antibody following Exposure to Serum Reveals the Formation of Immune Complex That Leads to Toxicity.

mAbs have revolutionized the treatment of autoimmune disorders. Even though mAbs have shown impressive efficacy in blocking T cell or B cell activation and/or recruitment to sites of inflammation, this group of biologicals are not devoid of adverse effects. The most serious adverse effects include infusion reactions, including the activation of the complement pathway. In this study, we present a detailed structure-function study of an anti-CCL20 humanized IgG1 mAb that neutralizes CCL20 chemokine and prevents the recruitment of Th17 cells to sites of inflammation. We demonstrate that the anti-CCL20 Ab changes significantly following administration to humans and monkeys and exposure to human serum. Analysis of the drug product revealed that the anti-CCL20 Ab has unexpectedly high C1q binding. This high binding was linked to immune complex formation in vivo but not during in vitro serum incubation. The immune complex contained multiple complement components. Anti-CCL20 Ab-mediated, complement-dependent cytotoxicity occurred when the Ab bound to CCL20 tethered to the cell membrane of target cells. Taken together, these results provide a likely cause for the animal toxicity observed. In addition, anti-CCL20 revealed progressive acidification because of N100 (located in CDR) deamidation over time, which did not directly impact Ag binding. Our study demonstrates that the safety profiling of mAbs should include the evaluation of effector functions in addition to typical stressed conditions.

Biotherapeutic Antibody Subunit LC-MS and Peptide Mapping LC-MS Measurements to Study Possible Biotransformation and Critical Quality Attributes In Vivo.

Biotransformation monitoring involves tracking drug modification occurring during in-life studies. Critical Quality Attribute monitoring from forced degraded drug material or in-life sample sets can provide an in-depth assessment of product quality for support in early- or late-stage drug development. For Critical Quality Attribute analysis, biotherapeutic monoclonal antibody (mAb) subunit analysis and peptide mapping liquid chromatography-mass spectrometry (LC-MS) approaches are used, although typically from an in vitro setting (e.g., formulation buffer) not involving biological samples or material. Here, samples from a high-dose rat study (in vivo) are subjected to analysis by ligand binding assay, mAb subunit LC-MS, and peptide mapping by LC-MS. Taken together, data from the 3 analytical approaches provide information regarding drug concentration in circulation, biotransformation, and biotherapeutic drug product quality. The concept of a multitier workflow for preclinical or clinical sample sets can be applied to other biotherapeutic mAb products such as bispecific mAbs, fusions proteins, or antibody-drug conjugates.