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    Generation and Characterization of a Polyclonal Human Reference Antibody to Measure Anti-Drug Antibody Titers in Patients with Fabry Disease

    Year: 2021

    Journal: Int. J. Mol. Sci., Volume 22, MAR

    Authors: Lenders, Malte; Scharnetzki, David; Heidari, Ali; Di Iorio, Daniele; Wegner, Seraphine Valeska; Brand, Eva

    Organizations: University of Muenster Medical School [LE221801]

    Keywords: Fabry disease; anti-drug antibodies; AGAL; affinities; ELISA

    Male patients with Fabry disease (FD) are at high risk for the formation of antibodies to recombinant alpha-galactosidase A (AGAL), used for enzyme replacement therapy. Due to the rapid disease progression, the identification of patients at risk is highly warranted. However, currently suitable references and standardized protocols for anti-drug antibodies (ADA) determination do not exist. Here we generate a comprehensive patient-derived antibody mixture as a reference, allowing ELISA-based quantification of antibody titers from individual blood samples. Serum samples of 22 male patients with FD and ADAs against AGAL were pooled and purified by immune adsorption. ADA-affinities against agalsidase-alpha, agalsidase-beta and Moss-AGAL were measured by quartz crystal microbalance with dissipation monitoring (QCM-D). AGAL-specific immune adsorption generated a polyclonal ADA mixture showing a concentration-dependent binding and inhibition of AGAL. Titers in raw sera and from purified total IgGs (r(2) = 0.9063 and r(2) = 0.8952, both p < 0.0001) correlated with the individual inhibitory capacities of ADAs. QCM-D measurements demonstrated comparable affinities of the reference antibody for agalsidase-alpha, agalsidase-beta and Moss-AGAL (KD: 1.94 +/- 0.11 mu M, 2.46 +/- 0.21 mu M, and 1.33 +/- 0.09 mu M, respectively). The reference antibody allows the ELISA-based ADA titer determination and quantification of absolute concentrations. Furthermore, ADAs from patients with FD have comparable affinities to agalsidase-alpha, agalsidase-beta and Moss-AGAL.
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