Inflammatory cytokine expression in Fabry disease: impact of disease phenotype and alterations under enzyme replacement therapy.

Objectives: The aim of this study is to explore the expression of inflammatory cytokines (ICs) in Fabry disease (FD), the correlation between ICs and FD phenotypes, and the impact of enzyme replacement therapy (ERT) on IC expression. Methods: We recruited 67 FD patients and 44 healthy controls (HCs) and detected concentrations of the following ICs: interferon-γ, interleukin (IL)-1ß, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12P70, IL-17A, IL-17F, IL-22, tumor necrosis factor (TNF)-α, and TNF-ß. We also analyzed the impact of ERT on IC expression in FD patients and the relationship between IC expression and sex, genotype, phenotype, disease burden, and biomarkers. Results: Most ICs were significantly higher in FD patients than in HCs. A number of ICs were positively correlated with clinical aspects, including disease burden (Mainz Severity Score Index [MSSI]) and cardiac and renal markers. IL-8 was higher in the high MSSI (P-adj=0.026*) than in the low MSSI. Conclusions: ICs were upregulated in FD patients, indicating the role of the innate immune process in FD etiology. ERT ameliorated FD-related inflammatory activation, at least to some extent. IC expression was positively correlated with disease burden and clinical markers in FD. Our findings indicated that the inflammatory pathway may be a promising therapeutic target for FD.

Mass cytometry reveals atypical immune profile notably impaired maturation of memory CD4 T with Gb3-related CD27 expression in CD4 T cells in Fabry disease.

Fabry disease (FD) is an X-linked disease characterized by an accumulation of glycosphingolipids, notably of globotriaosylceramide (Gb3) and globotriaosylsphingosine (lysoGb3) leading to renal failure, cardiomyopathy, and cerebral strokes. Inflammatory processes are involved in the pathophysiology. We investigated the immunological phenotype of peripheral blood mononuclear cells in Fabry patients depending on the clinical phenotype, treatment, Gb3, and lysoGb3 levels and the presence of anti-drug antibodies (ADA). Leucocytes from 41 male patients and 20 controls were analyzed with mass cytometry using both unsupervised and supervised algorithms. FD patients had an increased expression of CD27 and CD28 in memory CD45- and CD45 + CCR7-CD4 T cells (respectively p < 0.014 and p < 0.02). Percentage of CD45RA-CCR7-CD27 + CD28+ cells in CD4 T cells was correlated with plasma lysoGb3 (r = 0.60; p = 0.0036) and phenotype (p < 0.003). The correlation between Gb3 and CD27 in CD4 T cells almost reached significance (r = 0.33; p = 0.058). There was no immune profile associated with the presence of ADA. Treatment with agalsidase beta was associated with an increased proportion of Natural Killer cells. These findings provide valuable insights for understanding FD, linking Gb3 accumulation to inflammation, and proposing new prognostic biomarkers.

Genetically Modified Cell Transplantation Through Macroencapsulated Spheroids with Scaffolds to Treat Fabry Disease.

Cell transplantation is expected to be another strategy to treat lysosomal diseases, having several advantages compared to enzyme replacement therapy, such as continuous enzyme secretion and one-time treatment to cure diseases. However, cell transplantation for lysosomal diseases holds issues to be resolved for the clinical field. In this study, we developed a new ex vivo gene therapy platform using a transplant pack, which consists of a porous membrane made of ethylene-vinyl alcohol in the pack-type and spheroids with scaffolds. These membranes have countless pores of less than 0.1 µm2 capable of secreting proteins, including alpha-galactosidase enzyme, and segregating the contents from the host immune system. When the packs were subcutaneously transplanted into the backs of green fluorescent protein (GFP) mice, no GFP-positive cells migrated to the transplanted pack in either autogenic or allogenic mice. The transplanted cells in the pack survived for 28 days after transplantation. When cells overexpressing alpha-galactosidase were used as donor cells for the packs and implanted into Fabry disease model mice, the accumulation of the alpha-galactosidase enzyme was also observed in the livers. In this study, we reported a new ex vivo therapeutic strategy combining macroencapsulation and cellular spheroids with scaffolds. This pack, macroencapsulated spheroids with scaffolds, can also be applied to other types of lysosomal diseases by modifying genes of interest.

An antibody binding-based fluorescent assay for the rapid quantification of globotriaosylceramide levels in human Fabry cells.

Fabry disease is caused by reduced α-GAL A activity and accumulation of globotriaosylceramide (Gb3). Here, we describe a microplate Gb3 assay using fluorophore-tagged antibody and crude cellular lipid extracts. The assay is able to detect higher Gb3 concentrations in human Fabry cells compared to non-diseased cells. This result was verified by immunofluorescence staining that revealed large amounts of Gb3 deposits in Fabry cell lines, demonstrating the accuracy of this method. This assay may provide the basis for detecting Fabry disease by quantifying Gb3 deposits from human biological samples, for example, from urine and blood.

Generation and Characterization of a Polyclonal Human Reference Antibody to Measure Anti-Drug Antibody Titers in Patients with Fabry Disease.

Male patients with Fabry disease (FD) are at high risk for the formation of antibodies to recombinant α-galactosidase A (AGAL), used for enzyme replacement therapy. Due to the rapid disease progression, the identification of patients at risk is highly warranted. However, currently suitable references and standardized protocols for anti-drug antibodies (ADA) determination do not exist. Here we generate a comprehensive patient-derived antibody mixture as a reference, allowing ELISA-based quantification of antibody titers from individual blood samples. Serum samples of 22 male patients with FD and ADAs against AGAL were pooled and purified by immune adsorption. ADA-affinities against agalsidase-α, agalsidase-ß and Moss-AGAL were measured by quartz crystal microbalance with dissipation monitoring (QCM-D). AGAL-specific immune adsorption generated a polyclonal ADA mixture showing a concentration-dependent binding and inhibition of AGAL. Titers in raw sera and from purified total IgGs (r2 = 0.9063 and r2 = 0.8952, both p < 0.0001) correlated with the individual inhibitory capacities of ADAs. QCM-D measurements demonstrated comparable affinities of the reference antibody for agalsidase-α, agalsidase-ß and Moss-AGAL (KD: 1.94 ± 0.11 µM, 2.46 ± 0.21 µM, and 1.33 ± 0.09 µM, respectively). The reference antibody allows the ELISA-based ADA titer determination and quantification of absolute concentrations. Furthermore, ADAs from patients with FD have comparable affinities to agalsidase-α, agalsidase-ß and Moss-AGAL.

The role of Immunity in Fabry Disease and Hypertension: A Review of a Novel Common Pathway.

Fabry disease is a progressive, X-linked inherited lysosomal storage disorder where accumulation of glycosphingolipids increases the risk for early cardiovascular complications, including heart failure, stroke, and end stage renal disease. Besides disease-specific therapy, blood pressure (BP) control is of central importance in Fabry disease to reduce disease progression and improve prognosis. Both Fabry disease and hypertension are characterized by the activation of the innate component of the immune system, with Toll-like receptor 4 (TLR4) as a common trigger to the inflammatory cascade. The renin-angiotensin system (RAS) participates in the establishment of low-grade chronic inflammation and redox unbalance that contribute to organ damage in the long term. Besides exploiting the anti-inflammatory effects of RAS blockade and enzyme replacement therapy, targeted therapies acting on the immune system represent an appealing field of research in these conditions. The aim of this narrative review is to examine the issue of hypertension in the setting of Fabry disease, focusing on the possible determinants of their reciprocal relationship, as well as on the related clinical and therapeutic implications.

The Interaction of Innate and Adaptive Immunity and Stabilization of Mast Cell Activation in Management of Infusion Related Reactions in Patients with Fabry Disease.

Fabry disease (FD) is an X-linked lysosomal disorder caused by mutations in GLA gene resulting in lack of or faulty α-galactosidase A (α-GalA) enzyme. Enzyme replacement therapy (ERT) with recombinant human α-GalA enzyme (agalsidase) is the standard treatment option for FD. Infusion-related reactions (IRRs), with symptoms ranging from rigors, to fever, pain, vomiting, angioedema and diarrhea, are often seen due to immune response against the exogenous enzyme. To elucidate the mechanisms causing the IRRs in FD, eight patients who developed IRRs were investigated. All, except one, tested negative for agalsidase-specific IgE and had normal tryptase levels. Circulating dendritic cells were drastically reduced during IRRs, suggesting possible sequestration to the sites of inflammation. An increase in NK cells and a decrease in T cells were also observed. Cytokines IL-4, IL-8 and TNF-α showed a significant increase, indicating nonspecific degranulation of mast cells. All IRRs were managed successfully using a combination of standard premedications and mast cell stabilizers without any interruption of therapy. Taken together, the results indicate crosstalk between immune cells resulting in IgE-independent mast-cell-specific allergic inflammation. Mast cell stabilizers could be used to control IRRs and for safe reintroduction of agalsidase in patients previously treated with ERT.

Detailed epitope mapping of neutralizing anti-drug antibodies against recombinant α-galactosidase A in patients with Fabry disease.

BACKGROUND: Fabry disease (FD) is a lysosomal storage disease, treatable by enzyme replacement therapy (ERT) that substitutes deficient α-galactosidase A (AGAL). The formation of neutralizing anti-drug antibodies (ADA) inhibiting AGAL activity is associated with disease progression in affected male patients. In the current study, we performed a detailed epitope mapping of ADAs from antibody-positive males against infused AGAL. METHODS: A detailed epitope mapping for 34 male FD patients with neutralizing ADAs against AGAL was performed. Based on this data, in silico analyses were used to identify potential epitope clusters and mapped surface-located or buried epitopes. ELISA-based assays against α-galactosidase B (NAGA) were performed to identify ADAs that potentially recognize shared epitopes of AGAL and NAGA. A subset of 20 patients was analyzed to assess if NAGA-recognizing ADAs against AGAL might affect long-term outcomes under ERT. RESULTS: Thirty percent of the AGAL active site was recognized by patients' ADAs. No differences between buried and surface-located epitopes were observed. Dependent on the epitopes, ADAs against AGAL were also able to recognize human NAGA. Patients with NAGA recognizing anti-AGAL antibodies presented with lower plasma NAGA activities. The presence of NAGA-recognizing ADAs had no effect on disease progression. CONCLUSION: In conclusion, our current data underline previous reports demonstrating a large variation of antibody epitopes against AGAL. Detailed epitope mapping in affected patients might be the first step for the generation of patient-specific blocking peptides and/or immune adsorption columns for an individually tailored anti-antibody strategy.

Predicting the Development of Anti-Drug Antibodies against Recombinant alpha-Galactosidase A in Male Patients with Classical Fabry Disease.

Fabry Disease (FD) is a rare, X-linked, lysosomal storage disease that mainly causes renal, cardiac and cerebral complications. Enzyme replacement therapy (ERT) with recombinant alpha-galactosidase A is available, but approximately 50% of male patients with classical FD develop inhibiting anti-drug antibodies (iADAs) that lead to reduced biochemical responses and an accelerated loss of renal function. Once immunization has occurred, iADAs tend to persist and tolerization is hard to achieve. Here we developed a pre-treatment prediction model for iADA development in FD using existing data from 120 classical male FD patients from three European centers, treated with ERT. We found that nonsense and frameshift mutations in the α-galactosidase A gene (p = 0.05), higher plasma lysoGb3 at baseline (p < 0.001) and agalsidase beta as first treatment (p = 0.006) were significantly associated with iADA development. Prediction performance of a Random Forest model, using multiple variables (AUC-ROC: 0.77) was compared to a logistic regression (LR) model using the three significantly associated variables (AUC-ROC: 0.77). The LR model can be used to determine iADA risk in individual FD patients prior to treatment initiation. This helps to determine in which patients adjusted treatment and/or immunomodulatory regimes may be considered to minimize iADA development risk.

Neutralising anti-drug antibodies in Fabry disease can inhibit endothelial enzyme uptake and activity.

Fabry disease (FD) is a lysosomal storage disease, treatable by enzyme replacement therapy (ERT) that substitutes deficient α-galactosidase A (AGAL). The formation of neutralising anti-drug antibodies (ADA) inhibiting AGAL activity during infusion is associated with disease progression in affected male patients. In this study we analysed if ADAs also inhibit endothelial enzyme uptake as well as intracellular enzyme activity. Therefore, fluorescence-labelled AGAL in combination with ADA-positive sera from FD patients (n = 8) was used to analyse enzyme uptake in endothelial and FD-specific cells. Furthermore, immune adsorption and a comprehensive ADA epitope mapping were performed. Pre-incubation of AGAL with ADAs significantly inhibited intracellular enzyme activity, which was rescued by immune adsorption (both P < .01). ADAs from some patients also inhibited enzyme uptake. ADA epitope mapping identified an epitope at position 121 to 140 aa potentially responsible for uptake inhibition for these patients. Further analyses revealed the presence of stable AGAL/ADA-immune complexes at pH 4.5 and decreased intracellular enzyme activity in endothelial cells (P < .001). Finally, the pre-incubation of AGAL with ADAs resulted in a reduced depletion of intracellular globotriaosylceramide in patient-derived AGAL-deficient cells, demonstrating a direct negative impact of ADAs on intracellular clearance. Neutralising ADAs may not only inhibit infused AGAL activity, but according to their epitopes can also inhibit endothelial AGAL uptake. Indeed, internalised AGAL/ADA-complexes may not dissociate, underlining the importance of novel therapeutic approaches for ADA reduction and prevention to increase therapy efficiency in affected patients.

Fabry disease and immunoglobulin A nephropathy presenting with Alport syndrome-like findings: A case report.

RATIONALE: Fabry's disease is an X-linked inherited syndrome. Herein, we presented an unusual case of Fabry disease coexisting with immunoglobulin A nephropathy (IgAN) presenting with Alport syndrome-like pathological findings. PATIENT CONCERNS: We report a 30-year-old male who presented with proteinuria and elevated serum creatinine and for whom the initial pathologic diagnosis supported Alport syndrome. DIAGNOSES: A diagnosis of Fabry disease with immunoglobulin A nephropathy (IgAN) was finally made after further examination. INTERVENTIONS: After the initial diagnosis the patient was treated with herbal medications and mecobalamin. OUTCOMES: The patient was discharged 1 week later. He was maintained on these treatments and received regular follow-up in our hospital. LESSONS SUBSECTIONS AS PER STYLE: FD coexisting with IgAN is rare and may have nonspecific clinical presentations. Laboratory examination and genetic diagnosis is needed for confirmation. Timely diagnosis and reproductive intervention is needed for therapy.

Tumor necrosis factor-α links heat and inflammation with Fabry pain.

Fabry disease (FD) is an X-linked lysosomal storage disorder associated with pain triggered by heat or febrile infections. We modelled this condition by measuring the cytokine expression of peripheral blood mononuclear cells (PBMC) from FD patients in vitro upon stimulation with heat and lipopolysaccharide (LPS). We enrolled 67 FD patients and 37 healthy controls. We isolated PBMC, assessed their gene expression of selected pro- and anti-inflammatory cytokines, incubated them with heat, LPS, globotriaosylceramide (Gb3), and tumor necrosis factor-α (TNF), and measured TNF secretion in the supernatant and intracellular Gb3 accumulation, respectively. We found increased TNF, interleukin (IL-)1ß, and toll-like receptor 4 (TLR4) gene expression in FD men (p < .05 to p < .01). TNF and IL-10 were higher, and IL-4 was lower in the subgroup of FD men with pain compared to controls (p < .05 to p < .01). Hereby, TNF was only increased in FD men with pain and classical mutations (p < .05) compared to those without pain. PBMC from FD patients secreted more TNF upon stimulation with LPS (p < .01) than control PBMC. Incubation with Gb3 and an additional α-galactosidase A inhibitor did not further increase TNF secretion, but incubation with TNF greatly increased the Gb3 load in FD PBMC compared to controls (p < .01). Also, LPS incubation and heat challenge (40 °C) increased Gb3 accumulation in PBMC of patients compared to baseline (p < .05 each), while no alterations were observed in control PBMC. Our data show that TNF holds a crucial role in the pathophysiology of FD associated pain, which may open a novel perspective for analgesic treatment in FD pain.

Dose-Dependent Effect of Enzyme Replacement Therapy on Neutralizing Antidrug Antibody Titers and Clinical Outcome in Patients with Fabry Disease.

BACKGROUND: Use of enzyme replacement therapy (ERT) to treat Fabry disease, caused by deficient lysosomal α-galactosidase A activity, can lead to formation of neutralizing antidrug antibodies (ADAs). These antibodies are associated with increased accumulation of plasma globotriaosylceramide (Gb3) and disease progression. Because agalsidase ERT can saturate ADA-binding sites during infusions (achieving agalsidase/antibody equilibrium), we investigated in this open cohort study whether saturated patients (who have excess agalsidase after infusions) experience better clinical outcomes compared with not saturated patients (who have excess ADAs after infusions). METHODS: We isolated ADAs from sera of 26 men with Fabry disease receiving ERT (for a median of 94 months) and determined the amount of agalsidase necessary for antibody saturation. Clinical and biochemical outcomes included measurements of eGFR, interventricular septum thickness, and lyso-Gb3. RESULTS: ADA titers decreased significantly in all patients during infusion. Agalsidase-α and agalsidase-ß had similar ADA-binding capacity and comparable ADA saturation frequency. Fourteen patients with saturated ADAs presented with mild (but significant) loss of eGFR, stable septum thickness, and significantly decreased lyso-Gb3 levels. The 12 not saturated patients had a more pronounced and significant loss of eGFR, increased septum thickness, and a smaller, nonsignificant reduction in lyso-Gb3, over time. In three patients, dose escalation resulted in partially elevated ADA titers, but importantly, also in reduced lyso-Gb3 levels. CONCLUSIONS: A not saturated ADA status during infusion is associated with progressive loss of eGFR and ongoing cardiac hypertrophy. Dose escalation can result in saturation of ADAs and decreasing lyso-Gb3 levels, but may lead to increased ADA titers.

Immune-Mediated Myocarditis in Fabry Disease Cardiomyopathy.

Background Glycosphingolipid accumulation in Fabry cells generates a proinflammatory response that may influence disease evolution and responsiveness to enzyme replacement therapy. This study evaluated incidence, mechanism, and impact of myocarditis in Fabry disease cardiomyopathy ( FDCM ). Methods and Results Myocarditis, defined as CD 3+ T lymphocytes >7/mm2 associated with necrosis of glycolipid-laden myocardiocytes, was retrospectively evaluated in endomyocardial biopsies from 78 patients with FDCM : 13 with maximal wall thickness (MWT) <11 mm (group 1), 17 with MWT 11 to 15 mm (group 2), 30 with MWT 16 to 20 mm (group 3), and 18 with MWT >20 mm (group 4). Myocarditis was investigated by polymerase chain reaction for cardiotropic viruses, by serum antiheart and antimyosin antibodies, and by cardiac magnetic resonance. Myocarditis was recognized at histology in 48 of 78 patients with FDCM (38% of group 1, 41% of group 2, 66% of group 3, and 72% of group 4). Myocarditis was characterized by positive antiheart and antimyosin antibodies and negative polymerase chain reaction for viral genomes. CD 3+ cells/mm2 correlated with myocyte necrosis, antimyosin autoantibody titer, and MWT ( P<0.001, r=0.79; P<0.001, r=0.84; P<0.001, r=0.61, respectively). Cardiac magnetic resonance showed myocardial edema in 24 of 78 patients (31%): 0% of group 1, 23% of group 2, 37% of group 3, and 50% of group 4. Conclusions Myocarditis is detectable at histology in up to 56% of patients with FDCM . It is immune mediated and correlates with disease severity. It can be disclosed by antiheart/antimyosin autoantibodies and in the advanced phase by cardiac magnetic resonance. It may contribute to progression of FDCM and resistance to enzyme replacement therapy.

Deep characterization of the anti-drug antibodies developed in Fabry disease patients, a prospective analysis from the French multicenter cohort FFABRY.

BACKGROUND: Fabry disease (OMIM #301500) is an X-linked disorder caused by alpha-galactosidase A deficiency with two major clinical phenotypes: classic and non-classic of different prognosis. From 2001, enzyme replacement therapies (ERT) have been available. We aimed to determine the epidemiology and the functional characteristics of anti-drug antibodies. Patients from the French multicenter cohort FFABRY (n = 103 patients, 53 males) were prospectively screened for total anti-agalsidase IgG and IgG subclasses with a home-made enzyme-linked immunosorbent assay (ELISA), enzyme-inhibition assessed with neutralization assays and lysoGb3 plasma levels, and compared for clinical outcomes. RESULTS: Among the patients exposed to agalsidase, 40% of men (n = 18/45) and 8% of women (n = 2/25) had antibodies with a complete cross-reactivity towards both ERTs. Antibodies developed preferentially in men with non-missense GLA mutations (relative risk 2.88, p = 0.006) and classic phenotype (58.6% (17/29) vs 6.7% (1/16), p = 0.0005). Specific anti-agalsidase IgG1 were the most frequently observed (16/18 men), but the highest concentrations were observed for IgG4 (median 1.89 µg/ml, interquartile range (IQR) [0.41-12.24]). In the men exposed to agalsidase, inhibition was correlated with the total IgG titer (r = 0.67, p < 0.0001), especially IgG4 (r = 0.75, p = 0.0005) and IgG2 (r = 0.72, p = 0.001). Inhibition was confirmed intracellularly in Fabry patient leucocytes cultured with IgG-positive versus negative serum (median: 42.0 vs 75.6%, p = 0.04), which was correlated with IgG2 (r = 0.67, p = 0.017, n = 12) and IgG4 levels (r = 0.59, p = 0.041, n = 12). Plasma LysoGb3 levels were correlated with total IgG (r = 0.66, p = 0.001), IgG2 (r = 0.72, p = 0.004), IgG4 (r = 0.58, p = 0.03) and IgG1 (r = 0.55, p = 0.04) titers. Within the classic group, no clinical difference was observed but lysoGb3 levels were higher in antibody-positive patients (median 33.2 ng/ml [IQR 20.6-55.6] vs 12.5 [10.1-24.0], p = 0.005). CONCLUSION: Anti-agalsidase antibodies preferentially develop in the severe classic Fabry phenotype. They are frequently associated with enzyme inhibition and higher lysoGb3 levels. As such, they could be considered as a hallmark of severity associated with the classic phenotype. The distinction of the clinical phenotypes should now be mandatory in studies dealing with Fabry disease and its current and future therapies.

Corneal confocal microscopy detects corneal nerve damage and increased dendritic cells in Fabry disease.

Fabry disease is characterised by neuropathic pain and accelerated vascular disease. This study evaluates the utility of corneal confocal microscopy (CCM) to non-invasively quantify corneal nerve and endothelial cell morphology and dendritic cell (DC) density in relation to disease severity in subjects with Fabry disease. Seventeen consecutive participants with Fabry disease and 17 healthy control subjects were included in this cross-sectional study. Fabry disease severity was measured using the Mainz Severity Score Index (MSSI). Central corneal sensitivity was assessed with a contact corneal esthesiometer. There was a significant reduction in the corneal sensitivity (5.75 [5.25-6.00] vs. 6.00 [6.00-6.00] cm, P = 0.014), nerve fiber density (NFD) (26.4 ± 10.1 vs. 33.7 ± 7.9 fibers/mm2, P = 0.025) and nerve fiber length (NFL) (15.9 ± 3.4 vs. 19.5 ± 4.4 mm/mm2, P = 0.012) and an increase in DC density (38.3 [17.5-97.3] vs. 13.5 [0-29.4] cells/mm2, P = 0.004) in subjects with Fabry disease compared to the healthy control subjects. The total MSSI score correlated with NFD (ρ = -0.686; P = 0.006), NFL (ρ = -0.692; P = 0.006), endothelial cell density (ρ = -0.511; P = 0.036), endothelial cell area (ρ = 0.514; P = 0.036) and α-galactosidase A enzyme activity (ρ = -0.723; P = 0.008). This study demonstrates reduced corneal sensitivity, corneal nerve fiber damage and increased DCs in subjects with Fabry disease.

Antibody Epitope of Human α-Galactosidase†A Revealed by Affinity Mass Spectrometry: A Basis for Reversing Immunoreactivity in Enzyme Replacement Therapy of Fabry Disease.

α-Galactosidase (αGal) is a lysosomal enzyme that hydrolyses the terminal α-galactosyl moiety from glycosphingolipids. Mutations in the encoding genes for αGal lead to defective or misfolded enzyme, which results in substrate accumulation and subsequent organ dysfunction. The metabolic disease caused by a deficiency of human α-galactosidase A is known as Fabry disease or Fabry-Anderson disease, and it belongs to a larger group known as lysosomal storage diseases. An effective treatment for Fabry disease has been developed by enzyme replacement therapy (ERT), which involves infusions of purified recombinant enzyme in order to increase enzyme levels and decrease the amounts of accumulated substrate. However, immunoreactivity and IgG antibody formation are major, therapy-limiting, and eventually life-threatening complications of ERT. The present study focused on the epitope determination of human α-galactosidase A against its antibody formed. Here we report the identification of the epitope of human αGal(309-332) recognized by a human monoclonal anti-αGal antibody, using a combination of proteolytic excision of the immobilized immune complex and surface plasmon resonance biosensing mass spectrometry. The epitope peptide, αGal(309-332), was synthesized by solid-phase peptide synthesis. Determination of its affinity by surface plasmon resonance analysis revealed a high binding affinity for the antibody (KD =39×10-9 m), which is nearly identical to that of the full-length enzyme (KD =16×10-9 m). The proteolytic excision affinity mass spectrometry method is shown here to be an efficient tool for epitope identification of an immunogenic lysosomal enzyme. Because the full-length αGal and the antibody epitope showed similar binding affinities, this provides a basis for reversing immunogenicity upon ERT by: 1) treatment of patients with the epitope peptide to neutralize antibodies, or 2) removal of antibodies by apheresis, and thus significantly improving the response to ERT.

Enzymes as Immunotherapeutics.

Enzymes are attractive as immunotherapeutics because they can catalyze shifts in the local availability of immunostimulatory and immunosuppressive signals. Clinical success of enzyme immunotherapeutics frequently hinges upon achieving sustained biocatalysis over relevant time scales. The time scale and location of biocatalysis are often dictated by the location of the substrate. For example, therapeutic enzymes that convert substrates distributed systemically are typically designed to have a long half-life in circulation, whereas enzymes that convert substrates localized to a specific tissue or cell population can be more effective when designed to accumulate at the target site. This Topical Review surveys approaches to improve enzyme immunotherapeutic efficacy via chemical modification, encapsulation, and immobilization that increases enzyme accumulation at target sites or extends enzyme half-life in circulation. Examples provided illustrate "replacement therapies" to restore deficient enzyme function, as well as "enhancement therapies" that augment native enzyme function via supraphysiologic doses. Existing FDA-approved enzyme immunotherapies are highlighted, followed by discussion of emerging experimental strategies such as those designed to enhance antitumor immunity or resolve inflammation.