In Vitro and In Vivo Amenability to Migalastat in Fabry Disease.

Migalastat (1-deoxygalactonojirimycin) is approved for the treatment of Fabry disease (FD) in patients with an amenable mutation. Currently, there are at least 367 amenable and 711 non-amenable mutations known, based on an in vitro good laboratory practice (GLP) assay. Recent studies demonstrated that in vitro amenability of mutations did not necessarily correspond to in vivo amenability of migalastat-treated patients. This discrepancy might be due to (methodological) limitations of the current GLP-HEK assay. Currently, there are several published comparable cell-based amenability assays, with partially different outcomes for the same tested mutation, leading to concerns in FD-treating physicians. The aim of this review is to elucidate the idea of amenability assays from their beginning, starting with patient-specific primary cells to high-throughput assays based on overexpression. Consequently, we compare methods of current assays, highlighting their similarities, as well as their pros and cons. Finally, we provide a literature-based list of α-galactosidase A mutations, tested by different assays to provide a comprehensive overview of amenable mutations as a good basis for the decision-making by treating physicians. Since in vitro amenability does not always correspond with in vivo amenability, the treating clinician has the responsibility to monitor clinical and laboratory features to verify clinical response.

Detailed epitope mapping of neutralizing anti-drug antibodies against recombinant α-galactosidase A in patients with Fabry disease.

BACKGROUND: Fabry disease (FD) is a lysosomal storage disease, treatable by enzyme replacement therapy (ERT) that substitutes deficient α-galactosidase A (AGAL). The formation of neutralizing anti-drug antibodies (ADA) inhibiting AGAL activity is associated with disease progression in affected male patients. In the current study, we performed a detailed epitope mapping of ADAs from antibody-positive males against infused AGAL. METHODS: A detailed epitope mapping for 34 male FD patients with neutralizing ADAs against AGAL was performed. Based on this data, in silico analyses were used to identify potential epitope clusters and mapped surface-located or buried epitopes. ELISA-based assays against α-galactosidase B (NAGA) were performed to identify ADAs that potentially recognize shared epitopes of AGAL and NAGA. A subset of 20 patients was analyzed to assess if NAGA-recognizing ADAs against AGAL might affect long-term outcomes under ERT. RESULTS: Thirty percent of the AGAL active site was recognized by patients' ADAs. No differences between buried and surface-located epitopes were observed. Dependent on the epitopes, ADAs against AGAL were also able to recognize human NAGA. Patients with NAGA recognizing anti-AGAL antibodies presented with lower plasma NAGA activities. The presence of NAGA-recognizing ADAs had no effect on disease progression. CONCLUSION: In conclusion, our current data underline previous reports demonstrating a large variation of antibody epitopes against AGAL. Detailed epitope mapping in affected patients might be the first step for the generation of patient-specific blocking peptides and/or immune adsorption columns for an individually tailored anti-antibody strategy.

Neutralising anti-drug antibodies in Fabry disease can inhibit endothelial enzyme uptake and activity.

Fabry disease (FD) is a lysosomal storage disease, treatable by enzyme replacement therapy (ERT) that substitutes deficient α-galactosidase A (AGAL). The formation of neutralising anti-drug antibodies (ADA) inhibiting AGAL activity during infusion is associated with disease progression in affected male patients. In this study we analysed if ADAs also inhibit endothelial enzyme uptake as well as intracellular enzyme activity. Therefore, fluorescence-labelled AGAL in combination with ADA-positive sera from FD patients (n = 8) was used to analyse enzyme uptake in endothelial and FD-specific cells. Furthermore, immune adsorption and a comprehensive ADA epitope mapping were performed. Pre-incubation of AGAL with ADAs significantly inhibited intracellular enzyme activity, which was rescued by immune adsorption (both P < .01). ADAs from some patients also inhibited enzyme uptake. ADA epitope mapping identified an epitope at position 121 to 140 aa potentially responsible for uptake inhibition for these patients. Further analyses revealed the presence of stable AGAL/ADA-immune complexes at pH 4.5 and decreased intracellular enzyme activity in endothelial cells (P < .001). Finally, the pre-incubation of AGAL with ADAs resulted in a reduced depletion of intracellular globotriaosylceramide in patient-derived AGAL-deficient cells, demonstrating a direct negative impact of ADAs on intracellular clearance. Neutralising ADAs may not only inhibit infused AGAL activity, but according to their epitopes can also inhibit endothelial AGAL uptake. Indeed, internalised AGAL/ADA-complexes may not dissociate, underlining the importance of novel therapeutic approaches for ADA reduction and prevention to increase therapy efficiency in affected patients.

Soluble adenylyl cyclase (sAC) regulates calcium signaling in the vascular endothelium.

The vascular endothelium acts as a selective barrier between the bloodstream and extravascular tissues. Intracellular [Ca2+]i signaling is essential for vasoactive agonist-induced stimulation of endothelial cells (ECs), typically including Ca2+ release from the endoplasmic reticulum (ER). Although it is known that interactions of Ca2+ and cAMP as ubiquitous messengers are involved in this process, the individual contribution of cAMP-generating adenylyl cyclases (ACs), including the only soluble AC (sAC; ADCY10), remains less clear. Using life-cell microscopy and plate reader-based [Ca2+]i measurements, we found that human immortalized ECs, primary aortic and cardiac microvascular ECs, and primary vascular smooth muscle cells treated with sAC-specific inhibitor KH7 or anti-sAC-small interfering RNA did not show endogenous or exogenous ATP-induced [Ca2+]i elevation. Of note, a transmembrane AC (tmAC) inhibitor did not prevent ATP-induced [Ca2+]i elevation in ECs. Moreover, l-phenylephrine-dependent constriction of ex vivo mouse aortic ring segments was also reduced by KH7. Analysis of the inositol-1,4,5-trisphosphate (IP3) pathway revealed reduced IP3 receptor phosphorylation after KH7 application, which also prevented [Ca2+]i elevation induced by IP3 receptor agonist adenophostin A. Our results suggest that sAC rather than tmAC controls the agonist-induced ER-dependent Ca2+ response in ECs and may represent a treatment target in arterial hypertension and heart failure.-Mewes, M., Lenders, M., Stappers, F., Scharnetzki, D., Nedele, J., Fels, J., Wedlich-Söldner, R., Brand, S.-M., Schmitz, B., Brand, E. Soluble adenylyl cyclase (sAC) regulates calcium signaling in the vascular endothelium.

Mutation-specific Fabry disease patient-derived cell model to evaluate the amenability to chaperone therapy.

BACKGROUND: Patients with Fabry disease (FD) and amenable mutations can be treated with the chaperone migalastat to restore endogenous α-galactosidase A (AGAL) activity. However, certain amenable mutations do not respond biochemically in vivo as expected. Here, we aimed to establish a patient-specific and mutation-specific cell model to evaluate the amenability to chaperone therapy in FD. METHODS: Since current tests to determine amenability are limited to heterologous mutation expression in HEK293T cells with endogenous AGAL activity, we generated CRISPR/Cas9-mediated AGAL-deficient HEK293T cells as a basis for mutant overexpression. Furthermore, primary urinary cells from patients were isolated and immortalised as a patient-specific cell model system to evaluate the amenability to chaperone therapy. RESULTS: Under treatment (>13 months), carriers of p.N215S (n=6) showed a significant reduction of plasma lyso-Gb3 (p<0.05). Lyso-Gb3 levels in carriers of p.L294S increased (p<0.05) and two patients developed severe albuminuria. Both missense mutations were amenable in wild-type HEK293T cells (p<0.05), but presented different responses in CRISPR/Cas9-mediated AGAL knockouts and immortalised urinary cells. Chaperone incubation resulted in increased AGAL activity (p<0.0001) and intracellular globotriaosylceramide (Gb3) reduction (p<0.05) in immortalised p.N215S cells but not in p.L294S and IVS2+1 G>A cells. CONCLUSION: We conclude that repeated AGAL activity measurements in patients' white blood cells are mandatory to assess the in vivo amenability to migalastat. Plasma lyso-Gb3 might be an appropriate tool to measure the biochemical response to migalastat. Patients with low AGAL activities and increasing lyso-Gb3 levels despite in vitro amenability might not benefit sufficiently from chaperone treatment.