Inter-assay variability influences migalastat amenability assessments among Fabry disease variants.

Fabry disease is a lysosomal storage disorder caused by mutations in the GLA gene that encodes for the lysosomal enzyme α-galactosidase A (α-Gal A). Reduced or absent α-Gal A activity leads to substrate accumulation and deleterious effects in multiple organs. Migalastat is a pharmacological chaperone that may stabilize the enzyme in specific GLA variants, considered amenable, assisting enzyme trafficking to lysosomes and thus increasing enzyme activity. Using a good laboratory practice (GLP)-validated human embryonic kidney cell (HEK)-based (GLP-HEK) amenability assay established during the clinical development of migalastat, approximately one-third of GLA variants are reported to be amenable to migalastat. On the basis of this biochemical amenability, migalastat is approved for use in patients with specific GLA variants. In this study, the reproducibility of the amenability assay was assessed by evaluation of 59 GLA variants for α-Gal A activity in the presence and absence of migalastat. As for the GLP-HEK assay, variants were considered amenable when there was both an absolute increase in enzyme activity of ≥3% wild-type and a relative increase in enzyme activity ≥1.2 fold over baseline following incubation with migalastat. Six of the 59 variants tested here did not match the classification of amenability reported using the GLP-HEK assay. Linear regression and Bland-Altman analyses, comparing data from all variants with and without migalastat, provided additional evidence for a lack of assay reproducibility. Data from the GLP-HEK assay (and the resulting classification of amenability) can determine treatment strategy and, ultimately, patient outcomes, so discrepancies between amenability assay data could be a cause for concern for physicians managing patients with Fabry disease.

Development of a panel of highly sensitive, equivalent assays for detection of antibody responses to velaglucerase alfa or imiglucerase enzyme replacement therapy in patients with Gaucher disease.

Anti-drug antibodies are elicited by virtually all therapeutic proteins, and standardized assays are required for clinical monitoring of patients as well as for comparing antibody response to different therapeutic proteins in clinical trials. Velaglucerase alfa and imiglucerase are enzyme replacement therapies for the long-term treatment of type 1 Gaucher disease, a lysosomal storage disease resulting from an inherited deficiency of the enzyme glucocerebrosidase. We used state-of-the-art tools to develop a panel of assays for detection and characterization of antibody responses to velaglucerase alfa and imiglucerase. Highly-sensitive, direct bridging electrochemiluminescence screening assays were developed using samples from treatment-naïve individuals with type 1 Gaucher disease to set cut points. A mouse anti-glucocerebrosidase monoclonal antibody used as a calibrator was shown to have similar affinity and binding kinetics for anti-velaglucerase alfa and anti-imiglucerase antibodies. A quantitative radioimmunoprecipitation assay for IgG antibodies was developed to eliminate false-positives from the highly sensitive screening assay. Using 59 samples from treatment-naïve individuals with type 1 Gaucher disease, the confirmatory cut points were calculated to be 1.42 ng/mL for anti-velaglucerase alfa antibodies and 3.23 ng/mL for anti-imiglucerase antibodies. Isotype-specific indirect electrochemiluminescence assays were developed for IgE, IgA, and IgM subclasses. The IgE subclass assay was shown to be more sensitive than the confirmatory assay using sheep anti-glucocerebrosidase polyclonal antibody cross-linked with fragments specific to human IgE, with cut points for anti-velaglucerase alfa or anti-imiglucerase antibodies determined to be 0.53 and 0.55 ng/mL, respectively. An assay that detects inhibition in vitro of velaglucerase alfa and imiglucerase hydrolysis of a synthetic substrate in the presence of antibodies was developed to test for neutralizing antibodies. Using 52 individual healthy human donor samples and 35 samples from treatment-naïve individuals with type 1 Gaucher disease, cut points for the velaglucerase alfa and imiglucerase neutralizing antibody assays were determined to be 20%, such that a sample with greater than 20% inhibition of enzyme activity in the presence of antibodies was considered positive for neutralizing antibodies. In conclusion, highly sensitive and equivalent methods were developed and validated to directly compare antibody response to velaglucerase alfa and imiglucerase treatments in patients with Gaucher disease, and may contribute to future internationally standardized assays for antibody detection in patients with Gaucher disease.