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Comment to: Head-to-head trial of pegunigalsidase alfa versus agalsidase beta in patients with Fabry disease and deteriorating renal function: results from the 2-year randomised phase III BALANCE study—determination of immunogenicity
  1. Malte Lenders,
  2. Eva Brand
  1. Internal Medicine D, Department of Nephrology, Hypertension and Rheumatology, University Hospital Münster, Münster, Germany
  1. Correspondence to Dr Malte Lenders, Internal Medicine D, Department of Nephrology, Hypertension and Rheumatology, University Hospital Münster, 48149 Münster, Germany; malte.lenders{at}

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With great interest, we followed the recent studies by Linhart and colleagues1 as well as Wallace and colleagues.2 Especially the outcomes of the latter study were decisive for approval of pegunigalsidase alfa as a new enzyme replacement therapy in patients with Fabry disease (FD) by the Food and Drug Administration (FDA) and European Medicines Agency (EMA) in May 2023.

In the BRIDGE study, the authors report about the safety and efficacy in patients with FD under pegunigalsidase alfa, previously treated with agalsidase alfa.1 Overall, the annualised slope of the estimated glomerular filtration rate (eGFR) changed from −5.90 mL/min/1.73 m²/year under agalsidase alfa to −1.19 mL/min/1.73 m²/year under pegunigalsidase alfa. As a biochemical response, treatment with pegunigalsidase alfa led to a mean plasma lyso-Gb3 reduction by 31.5%. In terms of immunogenicity, seven (35%) patients were positive for antidrug antibodies (ADA) at least once during the study. In detail, five patients were ADA-negative at baseline and thus showed a de novo ADA formation. Three of five returned ADA negative at the end of the study.1 In the BALANCE study, the authors reported the non-inferiority of pegunigalsidase alfa compared to agalsidase beta after 24 months of treatment.2 In detail, median (95% CI limits) eGFR slopes after 24 months of treatment were −2.51 (−3.79, −1.24) mL/min/1.73 m2/year with pegunigalsidase alfa and −2.16 (−3.81, –0.51) mL/min/1.73 m2/year with agalsidase beta. The difference in median eGFR slope for the intent-to-treat population between arms was −0.36 mL/min/1.73 m2/year (95% CI −2.44, 1.73). Since the lower limit of the CI was above the prespecified non-inferiority margin, the non-inferiority was achieved.2

Since the PEGylation of pegunigalsidase alfa leads to a sustained half life and seems to reduce the affinity of already pre-existing ADAs towards agalsidase alfa and agalsidase beta,3 4 an additional focus of the study was the analysis of the safety (frequency of infusion-associated reactions) and the immunogenicity of pegunigalsidase alfa compared to agalsidase beta. Concerning the presence of neutralising ADAs, the authors reported that the proportion of patients with neutralising ADAs declined over 24 months under pegunigalsidase alfa from 33% to 15% compared with a change from 28% to 26% under agalsidase beta.

Unfortunately, the authors neither provide detailed methods for antibody measurements nor presented individual antibody titres.2 From the descriptive methods, it could be concluded that only free antibodies were measured. Hence, the presentation of immunogenicity must be evaluated in light of the methodological limitations, which have also been addressed in parts previously in another context.5

Due to the prolonged half life of pegunigalsidase alfa (~80 hours), it is conceivable that antibody titres measured by ELISAs, which target free antibodies (as stated within both manuscripts1 2), might not be appropriate (figure 1A). Concerning the ADA evaluation, the authors stated within the supplements2 as follows: ‘Yet, to be cautious, since pegunigalsidase alfa may be present in the blood at the time of ADA sampling, we cannot completely rule out the possibility that part of the drug remains bound to the ADA and therefore interferes with low titer ADA detection.’ In contrast to agalsidase alfa and agalsidase beta, the circulating long-lasting pegunigalsidase alfa may intercept and bind existing antibodies, potentially forming stable antigen/antibody complexes (figure 1A). In case of low or medium antibody titres, this might result in false negative results. Since this effect is likely to be stronger the closer the time of blood collection is to the last infusion, it would be most useful to measure antibody titres immediately before the next infusion. This general methodical problem might explain why in some patients reduced titres or even no free antibodies/no ADA-mediated inhibition was detected in between or at the end of the study (after 24 months). These patients might therefore be declared as antibody- negative. Of note, comparable methodological problems might also arise in patients treated by gene therapy, where the genetic modification results in a continuous AGAL expression and therefore in increased AGAL serum levels.

Figure 1

Potential methodological pitfalls using standard ELISA techniques to measure free antidrug antibody (ADA) titres in patients treated with enzyme replacement therapy (ERT). (A) Pegunigalsidase alfa with long circulating plasma half life may cause problems if only free ADAs are measured. Pegunigalsidase alfa might bind and catch circulating ADAs, which cannot be measured as free antibodies in subsequent assays. (B) Theoretical plasma α-galactosidase A levels following infusions: green—agalsidase beta for comparison, blue—theoretical plasma activity of pegunigalsidase alfa in a patient without neutralising ADAs, red—theoretical plasma activity of pegunigalsidase alfa in a patient with neutralising ADAs. Depending on the antibody titre pegunigalsidase alfa activity may decrease due to reduced half life of the enzyme. (C) Patients treated with pegunigalsidase alfa could have anti-AGAL antibodies and anti-PEG antibodies, which could consist of additional immunoglobulins (Ig), including IgA and Ig.

To address the above-mentioned methodological challenges, an indication of masked antibodies could be obtained if the AGAL enzyme activity shortly before the next infusion is more reduced than would be expected with a half life of 80 hours. Due to the long half life of pegunigalsidase alfa (80 hours=3.33 days) and in accordance with already published data,6 7 a certain residual enzymatic AGAL activity should be detected in patients’ plasma still after 2 weeks after infusion (figure 1B). Furthermore, if this measured enzymatic activity does not correlate with an ELISA-based AGAL quantification from the same plasma or serum samples, this might be a direct hind for the presence of (inhibitory) ADAs. Interestingly, the authors further stated within the supplemental material, ‘In most patients, the drug levels at the time of ADA sampling were below the assay drug tolerance level.’2 This statement is very curious, since this either means that the applied method was not sensitive enough for this kind of study and thus has changed from previous studies,6 7 or that the plasma half life of pegunigalsidase alfa was reduced in certain patients, which might be due to ADAs. Of note, these methodological pitfalls might only be relevant for patients with low or medium ADA titres, but could explain why some patients showed a transitory ADA response.

Finally, due to the PEGylation of pegunigalsidase alfa, PEG-specific antibodies might also recognise these residues.4 Indeed, three patients in the BALANCE study were identified as (transiently) positive for ADAs recognising PEG. Unfortunately, it is unclear which antibodies exactly were measured. Anti-PEG antibodies seem to show a brighter immunoglobulin (Ig) class variance than (inhibitory) anti-AGAL antibodies, which are mostly limited to IgGs8 (figure 1C). Thus, it is conceivable that depending on the applied method, the most prominent Ig classes including IgA, IgG and IgM9 10 might have been overseen.

Therefore, we conclude that due to the lack of methodological information and resulting data, the presented results concerning the immunogenicity of pegunigalsidase alfa should be interpreted with caution. Due to the unique biochemical characteristic of pegunigalsidase alfa, new methods for the detection of ADAs need to be developed in order to detect and measure ADAs in pegunigalsidase alfa-treated patients correctly.

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  • ML and EB contributed equally.

  • Contributors Both authors contributed equally.

  • Funding The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors.

  • Competing interests ML received speaker honoraria, travel funding and research grants from Amicus Therapeutics, Chiesi, Sanofi Genzyme and Takeda. EB received research grants and speaker honoraria from Amicus Therapeutics, Chiesi, Eleva, Sanofi Genzyme and Takeda.

  • Provenance and peer review Not commissioned; externally peer reviewed.

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