MO017THERAPEUTICAL POTENTIAL OF ENZYME REPLACEMENT: NEW INSIGHTS AND PERSPECTIVES IN HUMAN ENDOTHELIAL CELLS TREATED WITH CHLOROQUINE

Background and Aims COVID-19 is a pandemic with no end in sight. There is only one approved antiviral agent but global stocks are deemed insufficient. Despite in vitro antiviral activity, clinical trials of chloroquine and hydroxychloroquine were disappointing, and they may even impair outcomes. Chloroquine causes zebroid deposits reminiscent of Fabry disease (α-galactosidase A deficiency) and endothelial cells are key targets of COVID-19. The study aims to investigate in vitro the effect of enzyme replacement therapy (ERT) in chloroquine-induced endothelial dysfunction. Method We have explored the effect of chloroquine on cultured endothelial cells and its modulation by recombinant α-galactosidase A (agalsidase-β). Following dose-response studies, 0.5 μg/mL chloroquine was added to cultured human endothelial cells. Neutral red and Lysotracker were used to assess lysosomes. Cytotoxicity was evaluated by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) - MTT assay and cell stress by assessing reactive oxygen species (ROS) and nitric oxide (NO). In endothelial cells, chloroquine induced dose-dependent cytotoxicity at in vitro test concentrations for COVID-19 therapy. Results Chloroquine significantly induced the accumulation of acid organelles (P<0.05), increased ROS levels, and decreased NO production (P<0.05), in vitro. These adverse effects of chloroquine on endothelial cell biology were decreased by agalsidase-β (P<0.05). Conclusion Chloroquine-induced endothelial cell cytotoxicity and stress is attenuated by agalsidase-β treatment. This suggests that endothelial cell injury may contribute to the failure of chloroquine as therapy for COVID-19 and may be at least in part related to causing dysfunction of the lysosomal enzyme α-galactosidase A.

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

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