Here we demonstrate the long term impact of antibody formation in male Fabry patients treated with ERT on clearance of urinary Gb3 and to a lesser extent on plasma Gb3 and lysoGb3 levels. In addition, it is also demonstrated that in both AB+ as AB− males plasma lysoGb3 levels seem to plateau after an initial decrease in the first year of treatment and do not reach normal values. While in those AB+ patients treated with 0.2 mg/kg/2 weeks a switch to a higher dose of 1.0 mg/kg/2 weeks resulted in a further reduction of lyso(Gb3), these levels still remained higher compared to AB− patients. Finally, change of lysoGb3 and plasma and urinary Gb3 levels correlate with LVMass change in females, in males with decrease of LVmass and development of strokes or white matter lesions in both males and females. While it is difficult to predict the consequences of the presence of these antibodies, a potential negative effect on therapeutic effectiveness is likely 
Few patients in this study developed complete immune tolerance towards aGal A during prolonged treatment. This finding is consistent with a recent report of the postmarketing surveillance database on agalsidase beta. Of the 571 males treated with agalsidase beta 73% developed antibodies towards the infused enzyme, and only 11% of these tolerized, which is similar to our cohort (3/17, 18%) 
. The continuous presence of (high) levels antibodies may also have consequences for future alternative therapies. It is conceivable that circulating anti-agalsidase antibodies may exert an effect on future second generation aGal A enzyme preparations, on treatment with chaperones that aim to promote the stability of the patient's endogenous mutated aGal A or even in gene therapeutic approaches. In other lysosomal storage disorders, most notably Pompe disease, several strategies have been proposed to reduce the levels of anti-enzyme antibodies, such as methotrexate or anti-CD20 
. In Fabry disease similar studies have been done in the mouse-model, but currently no studies have been performed in man 
. The findings reported here strengthen the need to study in more detail the clinical consequences of antibody formation and approaches on treatment of patients with antibodies. One such approach may be the use of higher dosages of agalsidase as a previous study and this report demonstrate additional reduction of plasma Gb3 and urinary Gb3 following higher dosages
. Another possibility may be to develop immune tolerization regimes.
Previously no correlation between symptoms of Fabry disease and plasma or urinary Gb3 levels was found 
. which led some to question its use as a biomarker 
. However, Gb3 is believed to be pivotal in Fabry pathogenesis and clearance of accumulated Gb3 in renal capillary endothelium has been accepted as (surrogate) primary endpoint. In clinical trials reduction of plasma, urinary or organ Gb3 was considered to reflect a positive effect of ERT 
. In addition, recent studies demonstrate that pre-treatment lysoGb3 levels correlated with some of the clinical features of the disease, such as white matter lesions in males and overall clinical severity and left ventricular mass in females 
. This observation is extended as lysoGb3 response during ERT reflects change in LVmass and development of white matter lesions. In addition, there is an effect of dose: the higher dose of agalsidase beta led to more robust biochemical responses. This is in line with a study in which a lower dose of agalsidase beta (0.3 mg/kg) led to increases in Gb3 in a subset of patients 
. A recent observation of switch to a lower dose as a result of a worldwide shortage of agalsidase beta again confirms this finding 
. These findings once more emphasize that the debate on dose and treatment outcomes needs to be elucidated and deserves additional (clinical) studies.
However, even in those patients who do not develop antibodies, ERT resulted in a far from complete reduction to normal levels of plasma lysoGb3. After 6 years of treatment, plasma lysoGb3 in AB− Fabry patients was on average still 40-fold increased compared to normal values. The fact that decrease in lysoGb3, and in plasma and urinary Gb3 correlates with LVmass reduction upon ERT and influences the hazard of developing white matter lesions, suggests that a more robust decline of these biomarkers is probably associated with a more favourable outcome. Both the phase 4 study with agalsidase beta as well as detailed analyses of the Dutch cohort demonstrate disease progression in many patients, suggestive of a very modest effect of ERT in Fabry disease 
. This modest treatment effect as well as the influence of end organ damage to the responsiveness of the disease 
may also explain why this study and that of others 
fail to demonstrate a clear effect of antibodies on treatment effectiveness. Comparing antibody positive and negative patients with regard to clinical outcome may also be hampered by the fact that patients with residual aGal A activity may not only have a lower risk for developing anti-agalsidase antibodies, but may also have a less severe course of the disease as compared to those without residual enzyme activity. The development of anti-agalsidase antibodies may thus reflect a more severe phenotype of the disease with faster disease progression.
In this study we made some additional observations that may be of practical use in the daily care of patients on ERT. None of the females developed antibodies, which is not surprising as females have substantial residual enzyme activity (given the X-linked nature fo the disease) and the infused recombinant human enzyme is unlikely to be regarded as immunogenic. However, recently another study reported 12% of treated females developed antibodies 
. This observed difference in antibody formation may be related to the method of antibody detection (neutralization assay vs ELISA/RIP was used in 
). Our data suggest, that the need to monitor antibodies may be of lesser importance in females, and may be performed less frequently. In addition, in this study all patients who were antibody negative after one year of treatment remained so, irrespective of an increase in dose, or change in enzyme preparation.
Again these observations should be confirmed in larger cohorts, but may be of importance for physicians considering switching preparation or increase the dose.
Our study has some limitations. Despite covering the entire Fabry population of the Netherlands, the total number of analysed individuals remains limited, especially with regard to those on longer duration of treatment (>5 years). Moreover, during the study observation period several patients had their agalsidase dose and/or preparation changed. Our study demonstrated differences in antibody formation between the two treatment preparations, at least when given at their licensed dose. Additional analyses in larger cohorts are necessary to confirm differences between the two agalsidase preparations with regard to antibody formation or effectiveness in reducing Gb3 and lysoGb3. In addition, in this study we used a neutralizing assay to detect antibodies. While a previous study demonstrated that all patients with antibodies had an aGal A neutralizing effect in vitro
it is possible that a patient may develop antibodies that do not exhibit neutralizing capacity.
The lack of a uniform assay to detect and quantify agalsidase antibodies hampers direct comparison with other studies. In most studies on antibody status, antibody analyses was performed by the manufacturer of the enzyme preparation used. Subsequently, claims with respect to antibody formation cannot be extrapolated from one preparation to the other. This emphasizes the need for standardization of anti agalsidase assays and the results of a standardization initiative is eagerly awaited 
In conclusion, we demonstrated a negative effect of anti-agalsidase antibodies in male Fabry patients on Gb3 and lysoGb3 levels in plasma and particularly profoundly that of Gb3 in urine. Change of (lyso)Gb3 is associated with change in certain clinical parameters, such LVmass and development of white matter lesions or stroke. Thus, these results suggest a possible deleterious effect these antibodies on clinical efficacy of ERT, a finding that needs to be confirmed in a larger cohort. This emphasizes the urgent need to characterize the impact of anti-agalsidase antibodies on clinical efficacy of ERT in larger cohorts.