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

Lenders M, Scharnetzki D, Heidari A, Di Iorio D, Wegner SV, Brand E

Zusammenfassung

Male patients with Fabry disease (FD) are at high risk for the formation of antibodies to recombinant -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-, agalsidase- 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 (r2 = 0.9063 and r2 = 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-, agalsidase- and Moss- AGAL (KD: 1.94 0.11 M, 2.46 0.21 M, and 1.33 0.09 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-, agalsidase- and Moss-AGAL.