3.1 Safety

In total, 13 patients (seven males) with FD were recruited for this study. All patients were naïve to pegunigalsidase alfa at study start. The treatment duration under pegunigalsidase alfa ranged from 6 to 20 weeks (three to ten infusions; Table 1). Thus, in total, 67 infusions with pegunigalsidase alfa were analyzed. Eleven patients were pre-treated with agalsidase alfa (n = 5, three (60.0%) females) or agalsidase beta (n = 6, two (33.3%) females). One male patient was pre-treated with migalastat (123 mg every other day) for 4 months. One female patient was FD-treatment-naïve before therapy with pegunigalsidase alfa. According to individual treatment decisions by the physicians, patients either start with their individual full dose (1.0 mg/kg BW; n = 3) with subsequent reduction of the infusion time, or dosages were increased stepwise starting with 20 mg total infused pegunigalsidase alfa (n = 10) to prevent potential infusion-associated reactions. Antiallergic pre-medication (antihistamines and cortisone) to prevent infusion-associated reactions was only given to three male patients, one of whom had pre-existing neutralizing anti-AGAL antibodies (Table 1). In one patient without neutralizing antibodies, pre-medication could be tapered to zero during the following infusions. None of the recruited patients reported any severe infusion-associated reaction during treatment with pegunigalsidase alfa.

Table 1 Overview of the recruited patients3.2 Pharmacokinetics of Pegunigalsidase Alfa in Patients Without Pre-Existing Neutralizing Anti-AGAL Antibodies

Due to the prolonged half-life of pegunigalsidase alfa, enzymatic AGAL activities should be measurable in plasma or serum of pegunigalsidase alfa-infused patients without neutralizing antibodies even after 14 days [12] (OSM Resource 1). Individual pegunigalsidase alfa serum profiles immediately after infusion and before the next infusion are presented in Figs. 1 and 2. The overall pegunigalsidase alfa plasma peak activities are presented in Fig. 3. In detail, patients without neutralizing anti-AGAL antibodies showed comparable serum profiles as demonstrated by Schiffmann and colleagues for a dose of 1.0 mg/kg BW [12]. Individual data show increasing AGAL peak activities directly after infusions, which were dependent on the total infused amount of pegunigalsidase alfa (Fig. 1A–I). Importantly, patients presented with remaining AGAL activities in sera even after 14 days, immediately before the next infusion, demonstrating the high in vivo half-life of pegunigalsidase alfa. Of note, the patient depicted in Fig. 1F missed the second infusion, but residual AGAL activities were still measurable 28 days after the first infusion with 20 mg.

Fig. 1

Individual pegunigalsidase alfa serum profiles after infusion over time and anti-PEG antibody detection in patients without neutralizing anti-drug antibodies. A–C Male patients (P1 to P3), D–I female patients (P4 to P9). Blood samples were drawn immediately before and after the infusions to allow monitoring α-galactosidase A (AGAL) activities between consecutive visits. Individual total amounts of infused enzyme during visits are provided within brackets. F Patient missed one infusion (V2), which allowed us to measure the residual AGAL activity 28 days after the first infusion (V1). The dotted black lines highlight the upper limit of normal AGAL activity in serum at 10.8 ng/ml serum

Fig. 2

Individual pegunigalsidase alfa serum profiles after infusion over time and anti-PEG antibody detection in patients with pre-existing neutralizing anti-drug antibodies. A–D Male patients (P10 to P13). Blood samples were drawn immediately before and after the infusions allows monitoring α-galactosidase A (AGAL) activities between consecutive visits. Individual total amounts of infused enzyme during visits are provided within brackets. Anti-AGAL antibodies are measured by ELISAs (µg/ml) against agalsidase beta and pegunigalsidase alfa. Inhibition assays, providing the neutralizing antibodies total inhibitory capacities (mg), were measured against pegunigalsidase alfa. The dotted black lines highlight the upper limit of normal AGAL activity in serum at 10.8 ng/ml serum

Fig. 3

Individual and overall pegunigalsidase alfa plasma peak activities. A Individual peak α-galactosidase A (AGAL) activities directly measured after infusions in dependence of the total infused amount of pegunigalsidase alfa. Black-labeled patients are negative for neutralizing anti-AGAL antibodies. Red-labeled patients are positive for neutralizing anti-AGAL antibodies. B Individual remaining AGAL activities directly measured before the next infusions in dependence of the total infused amount of pegunigalsidase alfa. Only patients negative for neutralizing anti-AGAL antibodies are shown, since residual activities in patients with neutralizing anti-AGAL antibodies were not detectable. C Overall peak activities after and directly before the next infusion in patients treated with pegunigalsidase alfa or directly after the infusions with agalsidase alfa or beta. The dotted black lines highlight the upper limit of normal AGAL activity in serum at 10.8 ng/ml serum

Overall, sera from patients without neutralizing anti-AGAL antibodies showed increasing serum AGAL concentrations depending on the total infused AGAL dose directly after infusion (p < 0.0001, r2 = 0.5325) as well as immediately before the next infusion (p = 0.0193, r2 = 0.2157; Fig. 3). Further pharmacokinetics showed that the AUC immediately before the next infusion was still ~ 3% of the initial AUC directly after infusion (AUCafter: 946,145 ± 160,351 ng mg/ml vs. AUCbefore: 27,704 ± 6,798 ng mg/ml; z-score: 5.7; Fig. 4).

Fig. 4

Comparison of area under the curve values dependent on the total amount of infused α-galactosidase A (AGAL). ADA neutralizing anti-AGAL anti-drug antibody neutralizing anti-AGAL

3.3 Pharmacokinetics of Pegunigalsidase Alfa in Patients with Pre-Existing Neutralizing Anti-AGAL Antibodies

Four patients with pre-existing neutralizing anti-AGAL antibodies also presented with individual peak activities, which were dependent to the total infused amount of AGAL (Fig. 2A–D). However, the overall peak activities did not correlate significantly with the amount of total infused doses (p = 0.5022; Fig. 3). In addition, the patients’ peak activities directly after infusions were lower compared to those without neutralizing anti-AGAL antibodies. Importantly, in patients with neutralizing anti-AGAL antibodies, no remaining serum activities were measured immediately before the next infusion (Figs. 2, 3B), pointing towards a reduced half-life of pegunigalsidase alfa due to antibodies in these patients. The AUC directly after infusions in patients with pre-existing neutralizing anti-AGAL antibodies was ten times lower compared to patients without neutralizing anti-AGAL antibodies (AUCADA-negative: 946,145 ± 160,351 ng mg/ml vs. AUCADA-positive: 90,930 ± 37,991 ng mg/ml; z-score: 5.2; Fig. 4).

For a categorization of the measured serum activities for pegunigalsidase alfa, we additionally measured the peak activities in patients (n = 40) treated with agalsidase alfa or agalsidase beta directly after infusion. In patients without pre-existing anti-drug antibodies (n = 30, ten (33.3%) males), a concentration-dependent serum activity was observed (p < 0.001, r2 = 0.5022; AUCADA-negative: 137,266 ± 14,320 ng mg/ml; Fig. 3C). Interestingly, the AUC directly after infusion was 6.9 times lower compared to pegunigalsidase alfa (AUCpegunigalsidase: 946,145 ± 160,351 ng mg/ml vs. AUCalfa/beta: 137,266 ± 14,320 ng mg/ml; z-score: 5.0; Fig. 4). In patients with neutralizing antibodies (n = 10, all males) receiving agalsidase alfa or beta, peak activities were severely reduced, also showing no significant correlations with the amount of infused AGAL (p = 0.3544; Fig. 3C). Furthermore, in these patients AUC was 2.5-fold lower compared to patients without neutralizing antibodies receiving agalsidase alfa or beta (AUCADA-negative: 137,266 ± 14,320 ng mg/ml vs. AUCADA-positive: 55,157 ± 15,411 ng mg/ml; z-score: 3.9; Fig. 4). Interestingly, patients with pre-existing neutralizing anti-AGAL antibodies receiving pegunigalsidase alfa tended to show a slightly higher AUC compared to those receiving agalsidase alfa or agalsidase beta (AUCpegunigaslidase: 90,930 ± 37,991 ng mg/ml vs. AUCalfa/beta: 55,157 ± 15,411 ng mg/ml; z-score: 0.8; Fig. 4), which might be due to the previously reported lower affinity and thus inhibition of pre-existing anti-AGAL antibodies towards pegunigalsidase alfa [13].

3.4 Antibody Measurements

Next, we measured anti-AGAL antibodies and anti-PEG antibodies in all available serum samples using different ELISA-based approaches (OSM Resource 3). Among patients without pre-existing neutralizing anti-AGAL antibodies we identified six (66.6%) patients as being positive for anti-PEG antibodies (2x IgG (1x male, 1x female), 4x IgM (females)) (Fig. 1). In patients with pre-existing neutralizing anti-AGAL antibodies, three (75.0%) patients were positive for anti-PEG antibodies (1x IgG, 2x IgM; all males) (Fig. 2). During the observational period, we observed no distinct de novo formations or boosts of free antibodies, neither for anti-AGAL, nor for anti-PEG antibodies in patients without pre-existing anti-AGAL antibodies (Fig. 1). In three (75.0%) patients (all males) with pre-existing neutralizing anti-AGAL antibodies, an increase of the neutralizing capacities and thus IgG titers was observed over time, while anti-PEG titers seemed to remain stable (Fig. 2A, B D). In the other male patient with pre-existing neutralizing anti-AGAL antibodies, the inhibitory capacities and thus titers decreased (Fig. 2C).

3.5 Detection of AGAL/Antibody-Complexes in Patients’ Sera

Most methods which are used to detect antibodies in sera are aiming for free (unbound) antibodies. While these methods work well for measurements in patients under agalsidase alfa and beta due to the drugs’ short half-lives, this can lead to technical problems in patients receiving pegunigalsidase alfa [15]. To assess the potential formation of AGAL-antibody complexes, a modified ELISA-based assay was performed (OSM Resource 3). In the serum samples directly drawn before the next infusion, we detected in all patients with pre-existing neutralizing anti-AGAL antibodies measurable antibody-AGAL complexes at least at one visit (Fig. 5).

Fig. 5

Immune complex detection in serum samples from patients with pre-existing neutralizing anti- α-galactosidase A (AGAL) antibodies. Serum samples were drawn immediately before infusions