Pre-existing Reactivity to an IgG4 Fc-Epitope: Characterization and Mitigation of Interference in a Bridging Anti-drug Antibody Assay.

Twenty percent of baseline patient samples exhibited a pre-existing response in a bridging anti-drug antibody (ADA) assay for a human IgG4 monoclonal antibody (mAb) therapeutic. In some cases, assay signals were more than 100-fold higher than background, potentially confounding detection of true treatment-emergent ADA responses. The pre-existing reactivity was mapped by competitive inhibition experiments using recombinant proteins or chimeric human mAbs with IgG4 heavy chain regions swapped for IgG1 sequences. These experiments demonstrated that the majority of the samples had reactivity to an epitope containing leucine 445 in the CH3 domain of human IgG4. The pre-existing reactivity in baseline patient samples was mitigated by replacing the ADA assay capture reagent with a version of the drug containing a wild type IgG1 proline substitution at residue 445 without impacting detection of drug-specific, treatment-emergent ADA. Finally, purification on Protein G or anti-human IgG (H + L) columns indicated the pre-existing response was likely due to immunoglobulins in patient samples.