Development of NanoLuc bridging immunoassay for detection of anti-drug antibodies.

Anti-drug antibodies (ADAs) are generated in-vivo as an immune response to therapeutic antibody drugs and can significantly affect the efficacy and safety of the drugs. Hence, detection of ADAs is recommended by regulatory agencies during drug development process. A widely accepted method for measuring ADAs is "bridging" immunoassay and is frequently performed using enzyme-linked immunosorbent assay (ELISA) or electrochemiluminescence (ECL) platform developed by Meso Scale Discovery (MSD). ELISA is preferable due to widely available reagents and instruments and broad familiarity with the technology; however, MSD platform has gained wide acceptability due to a simpler workflow, higher sensitivity, and a broad dynamic range but requires proprietary reagents and instruments. We describe the development of a new bridging immunoassay where a small (19kDa) but ultra-bright NanoLuc luciferase enzyme is used as an antibody label and signal is luminescence. The method combines the convenience of ELISA format with assay performance similar to that of the MSD platform. Advantages of the NanoLuc bridging immunoassay are highlighted by using Trastuzumab and Cetuximab as model drugs and developing assays for detection of anti-Trastuzumab antibodies (ATA) and anti-Cetuximab antibodies (ACA). During development of the assay several aspects of the method were optimized including: (a) two different approaches for labeling drugs with NanoLuc; (b) sensitivity and dynamic range; and (c) compatibility with the acid dissociation step for improved drug tolerance. Assays showed high sensitivity of at least 1.0ng/mL, dynamic range of greater than four log orders, and drug tolerance of >500.