Enzyme replacement therapy (ERT) with recombinant AGAL (i.e. agalsidase alpha/beta) has been shown to be effective in reducing intracellular Gb3 load and lyso-Gb3 plasma levels, which is associated with an improvement in clinical disease course. However, intravenous infusion of recombinant AGAL can lead to the formation of anti-drug antibodies (ADA) that can neutralize ERT, which can cause a clinically relevant problem, especially in male FD patients (40-70% of treated males). A positive antibody status is associated with an impaired therapeutic effect on Gb3 reduction and consequently with a poorer clinical disease course in affected patients (Linthorst et al. 2004; Rombach et al. 2012; Lenders et al. 2016).

For almost 10 years, we have been studying the effects of neutralizing ADAs on the clinical efficacy of ERT. To this end, we have developed and adapted various methods to measure ADA titers in patient sera, expressed either as concentrations or as inhibitory capacities. Furthermore, we could show that depending on the epitopes of the antibodies, the cellular uptake of recombinant AGAL can be impaired and that internalized ADA/AGAL complexes do not dissociate in lysosomes. These mechanisms may lead to reduced therapeutic efficacy of ERT in affected patients and thus to disease progression. Nevertheless, we were able to show that not all affected patients suffer from disease progression, which could be explained by a higher infused AGAL dose in the presence of individual antibody titers or their varying degree of inhibitory capacities.

As one of the leading laboratories, we believe we have a responsibility to further elucidate the mechanisms of antibody formation and their respective impact on the various current and future therapeutic options, including but not limited to next-generation ERTs and gene therapy. Finally, we are evaluating the possibility of developing therapeutic protocols and algorithms that could prevent the formation of antibodies against ERT in the future.
 
Current ongoing studies

A) Detailed characterization of anti-PEG antibodies in Fabry disease patients (DaPaF)
Description
This study analyses the presence (frequency) and impact of antibodies against polyethylene glycol (PEG) and pegunigalsidase-alfa in pegunigalsidase-alfa-naïve and pegunigalsidase-alfa-treated patients.

Background and rationale
Pegunigalsidase-alfa was developed to overcome the pitfalls of agalsidase-alfa and agalsidase-beta: prolonged plasma half-life and lower immunogenicity were achieved by PEGylation. Our recent studies have shown that pre-existing ADAs against agalsidase-alfa/-beta have a lower affinity for pegunigalsidase-alfa, probably due to PEGylation-dependent masked epitopes (Lenders et al. 2022; Lenders et al. 2023). However, due to the presence of polyethylene glycol (stabilizer) in cosmetics, food, drugs and COVID-19 mRNA-based vaccines, up to 40% of the population appears to have anti-PEG antibodies. Therefore, the effects of these antibodies on pegunigalsidase-alfa need to be investigated.

Our hypothesis is that a significant proportion of ERT-naïve FD patients may have pre-formed anti-PEG antibodies. The potential importance for the safety and efficacy of treatment with PEGylated drugs is controversially discussed. This could represent an uncertainty for treating physicians when initiating therapy with pegunigalsidase-alfa in Fabry patients. The aim of this study is to analyze the biochemical effects of these antibodies on the function of pegunigalsidase-alfa in order to gain a better understanding of patients treated with pegunigalsidase-alfa and their clinical outcomes.

Study objectives
The project objectives are1) the determination of pre-existing anti-PEG antibodies in ERT-naïve (n=75) and pegunigalsidase-alfa-treated (n=15) FD patients, 2) the determination of affinities and inhibitory capacities of anti-PEG antibodies against pegunigalsidase-alfa, 3) the isotyping (IgA, IgG, IgM, IgE) of anti-PEG antibodies, 4) the determination of the potential impact of anti-PEG antibodies on cellular pegunigalsidase-alfa uptake and intracellular activity, 5) the determination of the impact of anti-PEG antibodies on serum/plasma half-life and clearance of pegunigalsidase-alfa, and 6) the determination of the impact of anti-PEG antibodies on antibody/ pegunigalsidase-alfa complex formation.

Funding
A funding of this study is provided by Chiesi GmbH, Germany.
 
B) Antibody-mediated inhibition of enzyme replacement therapies in Fabry disease (AMORE)
Description
The evaluation of neutralizing antibodies in FD patients on enzyme replacement therapy (ERT) is critical as ADAs significantly limit ERT efficacy. By comparing the inhibitory capacities of ADAs against agalsidase-alfa/agalsidase-beta with pegunigalsidase-alfa, it can be assessed whether existing ADAs inhibit pegunigalsidase-alfa less (lower affinity), which could improve patient outcomes over time.

Background and rationale
Our recent study has shown that pre-existing ADAs against agalsidase-alfa/-beta have a lower affinity for pegunigalsidase-alfa, which is likely due to PEGylation-dependent masked epitopes [Lenders et al. 2022]. Furthermore, it is not yet known whether de novo formed ADAs against pegunigalsidase-alfa have similar biochemical properties as existing ADAs against agalsidase-alfa and agalsidase-beta. Therefore, the effect of these antibodies on pegunigalsidase-alfa needs to be investigated and the measurement needs to be offered to interested FD centers and physicians in D-A-CH (Germany, Austria and Switzerland) to improve patient care.
Our rationale is that FD-treating physicians should know the ADA status of their patients in order to be able to make a safe therapy decision (possibly switching to pegunigalsidase-alfa).

Study objectives
The aim of the project is to determine the neutralizing ADA status in ERT-treated male and female Fabry patients from Fabry centers in Germany, Austria and Switzerland (D-A-CH) in order to 1) identify affected risk patients, 2) analyze whether the individually infused ERT dose is sufficient to supersaturate ADA titers, by determining individual inhibitory capacities, 3) to determine the half-life of agalsidase-beta and pegunigalsidase-alfa in individual serum samples, and 4) to analyze whether the neutralizing properties of antibodies against agalsidase-beta are similar to those against pegunigalsidase-alfa (and vice versa), to analyze whether theoretically an approved switch (from agalsidase-alfa [0. 2 mg/kg] or agalsidase-beta [1.0 mg/kg] to pegunigalsidase-alfa [1 mg/kg] will theoretically lead to a higher activity of pegunigalsidase-alfa compared to other ERTs.

Funding
A comprehensive funding of this study is provided by Chiesi GmbH, Germany.

New therapies for the treatment of Fabry disease are being developed and approved. One main focus of our clinical research is to monitor the therapeutic effect of these new drugs after their approval. To this end, we conduct non-interventional observational studies in a so-called "real-world design", as this type of study best reflects the course of the disease in patients in everyday clinical practice.
 
German observational multicenter study of patients with Fabry disease under enzyme replacement therapy with pegunigalsidase-alfa (GoPEG)
Description
A prospective Germany-wide multicenter observational study
Investigational sites

• Fabry disease center Münster, University Hospital Münster (principal investigator)
• Fabry disease center Würzburg, University Hospital Würzburg
• Fabry disease center Berlin - Charité - Universitätsmedizin Berlin, Campus Mitte (CCM)
• Fabry disease center Cologne, University Hospital Cologne
• Fabry disease center Hamburg, University Hospital Hamburg
• Fabry disease center Hannover, University Hospital Hannover
• Fabry disease center Mainz, University Medical Center Mainz
• Internal medicine practice, Müllheim

Background and rationale
Pegunigalsidase-alfa, a recently approved PEGylated, covalently cross-linked form of α-galactosidase A developed as an enzyme replacement therapy (ERT) for Fabry disease (FD), has been designed to increase plasma half-life and decrease immunogenicity, thereby potentially improving efficacy compared to available ERT products. The rationale of the current project is that disease progression in patients with Fabry disease underpegunigalsidase-alfa can be stabilized comparable to patients on current ERT, leading to validation of phase 3 clinical trials and translation of these previous results to a nationwide study in Germany.

Study objectives
Pegunigalsidase-alfa could represent an advance in ERT for FD due to its unique pharmacokinetics and apparently low immunogenicity. The aim of the study is to document long-term data on treatment with pegunigalsidase-alfa under "real-life" conditions. 60 patients with FD (treatment-naive or pre-treated with agalsidase-alfa or agalsidase-beta) will be recruited in 8 German Fabry centers. The treatment duration will be 2 years. All patients will be followed up by the Fabry expert centers listed above.

Course of the study
The European guidelines and recommendations for the treatment of patients with FD recommend a consistent examination of affected patients every 12 months (Biegstraaten et al. 2015). As treatment with pegunigalsidase-alfa is a recently approved therapy option and the current literature is scarce, the examination interval should be shortened once from 12 to six months in the first year of treatment. For this reason, five visits of the included patients to the participating centers will be documented during the study period:
Visit 1: T-1= retrospective evaluation of the patient’s disease state (including cerebral MRI investigation, and measurements of α-Gal A-activity and lyso-Gb3) at -6 months,
Visit 2: T0= baseline (including cerebral MRI investigation), therapy initiation/switch
Visit 3: T1 = 6 months follow-up,
Visit 4: T2 = 12 months follow-up,
Visit 5: T3 = 24 months follow-up (including cerebral MRI investigation)

Echocardiographic analyses will be performed by a cardiological core center. MRI analyses will be performed by a core neuroimaging working group. Compact discs of the echocardiographic imaging and MRI data from all participating centers will be collected.

To analyze the potentially reduced immunogenicity of pegunigalsidase-alfa full spectrum of biochemical analysis will be performed including ELISA-based determination of antibody titers and antibody-enzyme complexes and antibody-mediated ERT inhibition assays and titrations.

Funding
A full funding of this study is provided by Chiesi GmbH, Germany.

Although Fabry disease is inherited X-linked, affected women can be just as severely affected as male patients. Unfortunately, there are no standardized criteria for when an affected female patient should be treated with a Fabry-specific therapy. One focus of our clinical research is to close this knowledge gap so that female patients with Fabry disease in particular can be treated timely before irreversible end organ damage occurs.
 
Multicenter Female Fabry Study (MFFS) – impact of early ERT start on clinical manifestations in females with Fabry disease
Description
A prospective multicenter study of Germany, Austria and Switzerland

Investigational sites

• Fabry disease center Münster, University Hospital Münster (principal investigator)
• Fabry disease center Berlin - Charité - Universitätsmedizin Berlin, Campus Mitte (CCM)
• Fabry disease center Cologne, University Hospital Cologne
• Fabry disease center Hamburg, University Hospital Hamburg
• Fabry disease center Hannover, University Hospital Hannover
• Fabry disease center Mainz, University Medical Center Mainz
• Internal medicine practice, Müllheim
• Clinic and Polyclinic for Internal Medicine, University Hospital Zurich, Switzerland
• Morbus Fabry Center, AKH Wien, Austria

Background and rationale
Fabry disease (FD; OMIM #301500) is an X-linked (Xq22.1) inborn error of glycosphingolipid catabolism resulting from deficient alpha-galactosidase A activity (GLA; 300644) leading to a progressive lysosomal accumulation of glycosphingolipids (mainly globotriaosylceramide [Gb3]) within the vascular endothelium, as well as renal, cardiac, and neuronal cells (Zarate & Hopkin, 2008; Najafian et al. 2001). These accumulations result in a multisystemic disease with early myocardial failure and stroke, end-stage renal disease and severely decreased life expectancy. The enzymatic deficiency is based on GLA gene mutations (n >600) (http://www.hgmd.cf.ac.uk/ac/gene.php?gene=GLA). Enzyme replacement therapy (ERT) is based on infusions of biotechnologically produced GLA enzyme (Agalsidase alfa, Replagal, 0.2 mg/kg BW every other week, Shire Pharmaceuticals, UK, and Agalsidase beta, Fabrazyme, 1 mg/kg BW every other week, Sanofi Genzyme, USA), to compensate for the  loss of endogenous enzyme.

Although X-linked inheritance and X-lionization in females (mosaic pattern) may explain why FD-specific symptoms and manifestations are delayed and often milder in heterozygous affected females, lionization is not a sufficient model to explain the observed bright variability in disease progression in females. Furthermore, less is known about the vascular damage. Furthermore, it is unclear which affected women benefit from ERT and when ERT should be initiated.

Our previous study (Lenders et al. 2016) comprising 261 females with FD (6 German Fabry centers) revealed that the treatment concept for females with FD in Germany is in line with the current European Fabry guidelines (Biegstraaten et al. 2015). However, a relevant number of females remain untreated despite organ involvement, necessitating a careful reevaluation of these females. Furthermore, elevated lyso-Gb3 levels in females seem to be a marker of disease burden, even if the prognostic value of lyso-Gb3 levels for ERT initiation in females needs evaluation in future observational studies.

Study objectives
Evaluation of a large female Fabry cohort (Germany, Austria, Switzerland, 9 centers, ~277 females) with well characterized clinical phenotypes at 3 time points to assess the impact of early ERT start in affected females compared to males (n~60).

Reevaluation of consensus criteria for ERT initiation in females with FD. Analysis of the impact of early ERT start in affected females (compared to males) with special focus on vasoprotection.

Course of the study
To address the studies objectives, 3 groups of females (group 1: “long-term ERT”; group 2: “long-term ERT naïve” (without ERT); group 3: “new on ERT”) will be analyzed at 3 time-points (t-1, t0, t1): In the “new on ERT” group: t0 = last visit of female patients naïve to ERT; t1 = visit of female patients after a 12 month follow-up on ERT. All female patients on ERT or naïve to ERT will be analyzed at t0 and t1 with a 12 month interval. In females on “long-term ERT” an additional time point (t-1) will cover the last naïve status, while t0 covers an actual visit. In “long-term ERT naïve” females t-1 will cover the first visit of females. A group of male patients (n=60) will be included as a control group.

Funding
A comprehensive funding of this study is provided by Takeda (previously Shire), Germany.