Publications

Development and Characterization of an Anti-rituximab Monoclonal Antibody Panel

Tada M, Suzuki T and Ishii-Watabe A, doi: 10.1080/19420862.2018, MAbs, 2018

Administration of mAbs and other therapeutic proteins may lead to the production of anti-drug-antibodies (ADAs), which could inactivate the therapeutic effects of the treatment and may even induce adverse effects. Described herein is the generation of recombinant human-rat chimeric anti-rituximab mAbs and the development and characterization of a panel of ADAs against anti-CD20 rituximab. Bio-Layer Interferometry (BLI) was used to perform an epitope binning assay, ADA detection assay, and to analyze the binding kinetics. A Pall ForteBio Octet RED384 instrument equipped with Streptavidin (SA) Biosensor probes was used to execute all BLI experiments. The epitope binning assay was performed using an in-tandem format to characterize the binding epitopes of anti-rituximab mAbs. Biotinylated rituximab was immobilized onto SA sensor tips and immersed in wells containing an anti-rituximab mAb (saturating Abs: Ab-1) until saturation is observed. Biosensor tips were then moved into wells containing a second anti-rituximab mAb (competing Abs: Ab-2). The binding levels of Ab-2 in the presence of Ab-1 were analyzed. For the binding kinetics analysis, biotinylated rituximab loaded SA sensor tips were dipped in serially diluted anti-rituximab mAb samples. BLI data were fitted using a 1:1 binding model. The kinetics parameters (ka, kd) and the equilibrium dissociation constants (KD) were determined. In the ADA detection assays, biotinylated rituximab loaded SA sensor tips were dipped in serially diluted anti-rituximab mAb samples with or without human serum. Overall results suggest that the panel of ADAs developed in this study may have important implications for future immunogenicity assessment studies.

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