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

Abstract

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.

Safety and efficacy results of switch from imiglucerase to velaglucerase alfa treatment in patients with type 1 Gaucher disease

“…An electrochemiluminescence assay was used to screen samples for the presence of anti-drug antibodies and an enzymatic activity neutralizing antibody assay was used to analyze samples that screened positive (methods described previously) [6,10].…”

Velaglucerase alfa enzyme replacement therapy compared with imiglucerase in patients with Gaucher disease

“…Using highly sensitive, equivalent electrochemiluminescence assays [12], anti-velaglucerase alfa and anti-imiglucerase antibodies were evaluated at screening and pre-infusion every 6 weeks until Month 9. Positive samples were titered and tested for in vitro inhibition of enzyme activity.…”

“…research article enzymatic activity) and cross-reacted in the anti-velaglucerase alfa antibody assay (titer 1,598 ng/mL with 35% inhibition of enzymatic activity) [12]. He continued in the study for a further 10 weeks, with an apparent response in hematologic parameters.…”

Competing for the treasure in exceptions

“…Six of 17 patients in the original imiglucerase group had attributed adverse events, including severe chills in one. In neither group was any patient discontinued as a result of these events and apparently no patient receiving velaglucerase alfa developed antibodies to either agent; four patients receiving imiglucerase are reported to have IgG antibodies to imiglucerase-again, as determined in the sponsor's laboratory [23]. In one case, these antibodies reacted with velaglucerase alfa.…”

“…Overall, the findings are compatible with those reported in the velaglucerase alfa phase 3 clinical trial program. Of note, one patient developed a grade 2 anaphylactoid reaction shortly after the first infusion and withdrew; antibodies to velaglucerase alfa were not detected using assays carried out and developed by Shire HGT [23]. Gonzalez et al [17] report a multiethnic trial conducted in 25 treatment na€ ıve anemic patients at least 4 years old randomized to receive velaglucerase alfa at two doses (45 units per kg or 60 units per kg) every two weeks-both groups met the primary endpoint, an increase in hemoglobin concentration (2.4 g/dL).…”