Immunologic considerations for enzyme replacement therapy in the treatment of lysosomal storage disorders

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Immunologic considerations for enzyme replacement therapy in the treatment of lysosomal storage disorders Susan M. Richards Immunology Laboratory, Cell and Protein Therapeutics R&D, Genzyme Corporation, P.O. Box 9322, One Mountain Road, Framingham, MA 01701 USA Received 11 December 2001; received in revised form 29 April 2002; accepted 1 May 2002

Abstract Lysosomal storage disorders are genetic diseases caused by deficient activity of lysosomal enzymes involved in cellular metabolism. Depending on the particular mutation, patients either lack active enzyme or produce enzyme that does not function properly. Consequently there is an accumulation of substrate within the lysosome that cannot be degraded at a sufficient rate. The accumulation of these biochemical intermediates ultimately leads to malfunctioning of cells and tissues that manifest as severe clinical symptoms often leading to premature death. Enzyme replacement therapy has been shown to be an effective treatment strategy for these patients. Monitoring specific antibody production is an important component of patient management. Clinical studies that monitor the immune responses of patients receiving enzyme replacement therapy indicate that a percentage of patients develop antibodies to the therapeutic protein. These antibodies have the potential to cause immune-mediated reactions and impact efficacy of the therapy. Therefore it is important to characterize the nature of the immune response relative to clinical symptoms. It is through this understanding that clinicians can provide optimal patient care. © 2002 Elsevier Science Inc. All rights reserved. Keywords: lysosomal storage disease; immune response; therapeutic protein; antibody; enzyme replacement therapy

Abbreviations: ANR, above normal range; CIC, circulating immune complexes; CNS, central nervous system; CRIM, cross-reacting immunologic material; ELISA, enzyme linked immunosorbent assay; ERT, enzyme replacement therapy; FDA, Food and Drug Administration; GAL, -galactosidase; GAA, acid -glucosidase; GCR, glucocerebrosidase; GL-3, globotriaosylceramide; GSD II, glycogen storage disease type II; HSA, human serum albumin; HRP, horseradish-peroxidase; ITI, immune tolerance induction; LSD, lysosomal storage disorders/disease; MPS, mucopolysaccharide storage; MPS I, mucopolysaccharidosis I; PBS, phosphate-buffered saline; RIA, radioimmunoassay; rhGAA, recombinant human acid -glucosidase; RIP, radioimmunoprecipitation; WNR, within normal range. * Corresponding author. Tel.: 1-508-270-2411; fax: 1-508-872-9080 E-mail address: [email protected] (S.M. Richards). 1529-1049/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII: S1 5 2 9 - 1 0 4 9 ( 0 2 ) 0 0 0 4 9 -1

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1. Introduction Lysosomal storage disorders (LSD) consist of a group of distinct genetic diseases characterized by a genetic defect in one or more specific lysosomal enzymes resulting in disrupted lysosomal function. The consequence of this defect is a progressive accumulation of biochemical intermediates of degradation pathways inside the lysosome. This accumulation causes lysosomes to enlarge and leads to cell degeneration. The accumulation of these biochemical intermediates in various tissues and organs of the body impacts their function, leading to a variety of clinical syndromes that are both chronic and progressive [1]. In most LSD, the pathology of the disease is not apparent at birth and manifests itself in the first few years of life. Diagnosis of LSD is based on clinical presentation along with the demonstrated deficiency of a specific enzyme activity. Currently diagnosis is a complex process involving assays to measure enzyme activity or substrate accumulation in plasma, leukocytes, urine, or skin fibroblasts. Early diagnosis is key in identifying patients before the onset of irreversible pathology. Most of the genes for the proteins involved in the lysosomal system have been cloned, allowing for mutational analysis of individual cases. Although individual LSDs are classified as rare genetic disorders, collectively LSDs are more common, with an incidence of approximately 1 in 7,000–8,000 live births [2]. A number of treatment strategies for these patients have been proposed. These include bone marrow transplantation, enzyme replacement therapy (ERT), and gene therapy. ERT has been demonstrated to be an effective treatment strategy for certain LSD. With this type of therapy, patients are infused with a recombinant form of the human enzyme over a course of a few hours every 1–2 weeks. Receptor-mediated uptake via endocytosis provides a mechanism for delivery of exogenous enzyme to target cells. Since all lysosomal enzymes are glycoproteins, it is possible to utilize carbohydrate-recognizing receptors to enhance uptake of circulating enzyme and target the enzyme to different cells. For example, the N-linked oligosaccharides on recombinant glucocerebrosidase are modified to terminate in mannose, which is recognized by the mannose receptor on macrophages, the major target cell. Lysosomal enzymes also have mannose-6-phosphate that can be used to target the enzyme to the mannose-6-phosphate receptor present on the surface of a wide variety of cells. It is also through this lysosomal-recognition marker that the enzyme is transferred to the lysosomal compartment in the cell. This receptor-mediated uptake process is the molecular basis of ERT strategies, which have been developed for the treatment of patients with LSDs. Preclinical studies, as well as in vitro uptake studies using patient cells, have validated the feasibility of this approach. 2. Enzyme Replacement Therapy ERT is a life-long therapy where the therapeutic enzyme serves to replace the function of subnormal or inactive enzyme. The clinical use of ERT was first implemented in type I Gaucher Disease patients with the approval of alglucerase in 1991. The active component consists of purified placental-derived glucocerebrosidase (GCR), which was enzymatically modified to improve efficacy of targeting to the mannose receptor on macrophages of the reticuloendothelial system [3,4]. Imiglucerase enzyme replacement therapy, the recombinant

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form of the enzyme, was subsequently approved for marketing in the United States and Europe. Since that time, ERT has been successfully developed for several other LSD. The disorders where this approach is currently used or under clinical investigation are shown in Table 1 and summarized below. Once these clinical trials are completed and therapies approved for use, it is estimated that approximately 50% of LSD patients will have treatment available [5]. Gaucher Disease is the most prevalent LSD affecting 20,000 to 30,000 individuals worldwide [6]. The pathology of the disease is characterized by marked accumulation of complex lipids, glucocerebrosides, in tissues due to defective activity of the enzyme GCR. Three types of Gaucher disease have been described based on the degree of central nervous system (CNS) involvement [7]. The disease has a broad spectrum of severity. The clinical manifestations of type I Gaucher disease (non-CNS) results from lipid-engorged macrophages causing hepatosplenomegaly, impaired liver and spleen function, displacement of bone marrow cells, and skeletal abnormalities. Bleeding is a common presenting symptom with thrombocytopenia being the most common peripheral blood abnormality. Fabry disease (also referred to as Anderson-Fabry disease) is an X-linked inborn error of the glycosphingolipid catabolism characterized by subnormal or absent activity of lysosomal -galactosidase A (GAL). Deficiency of GAL leads to progressive accumulation of glycosphingolipids, predominately globotriaosylceramide (GL-3) in the lysosomes of endothelial and smooth-muscle cells of blood vessels. GL-3 accumulation also occurs in ganglion cells of the autonomic nervous system, epithelial cells of glomeruli and tubules in the kidney, cardiomyocytes of the heart, and epithelial cells of the cornea [8]. The clinical manifestations of Fabry disease result primarily from the progressive deposition of GL-3 in the vascular endothelium. Fabry disease progresses to a microvascular disease with key organ dysfunction Table I Enzyme replacement therapy for lysosomal storage diseases Substrate accumulation

Disease

Metabolic disorder

Defective enzyme

Gaucher Disease

Defective hydrolysis of glucosphingolipids

acid -glucosidase glucosyl(glucocerebrosidase) ceramide

Fabry Disease

Defective hydrolysis of glycosphingolipids

-galactosidase A

globotriaosylceramide

-L-iduronidase Mucopolysaccharidosis I Defective catabolism of glycosaminoglycans Pompe Disease/GSD II Defective acid -glucosidase hydrolysis of glycogen Niemann-Pick B Defective sphingomyelinase Disease hydolysis of sphingomyelin

dermatan and heparan sulfates glycogen

Enzyme replacement therapy Alglucerase (Ceredase®) Imiglucerase (Cerezyme®) Agalsidase beta (Fabrazyme®) Agalsidase alfa (Replagal®) Iduronidase (AldurazymeTM ) Clinical trials

sphingomeylin Clinical development

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leading to renal failure, cardiac disease, and strokes. Recombinant GAL A replacement therapy [9,10], which is an investigational product under review at the Food and Drug Administration (FDA), has been employed in clinical trials for Fabry disease patients. Studies investigated reverse accumulation of microvascular endothelial deposits of globotriaosylceramide in the kidneys, heart, and skin [9] and patient neuropathic pain symptoms [10]. Continued treatment may be required to reduce deposition of glycosphingolipids in other types of cells. Mucopolysaccharide storage (MPS) disorders are caused by deficiencies of specific lysosomal enzymes required for the catabolism of glycosaminoglycans [11]. Deficiency of -L-iduronidase results in the accumulation of dermatan and heparan sulfate in many tissues and a chronic progressive disorder known as mucopolysaccharidosis I (MPS I). Patients with MPS I are commonly classified into three clinical syndromes (Hurler, Hurler-Scheie, or Scheie) based on their presentation and the severity of their symptoms. Patients present with cardiomyopathy, skeletal deformities, ophthalmologic manifestations, and obstructive airway disease. Respiratory infections and cardiac complications are the usual cause of death. ERT utilizing a recombinant form of -L-iduronidase has been evaluated in clinical trials [12] and is under review by regulatory agencies. Glycogen storage disease type II (GSD II) or Pompe Disease is a rare autosomal recessive disease caused by deficiency or absent activity of acid -glucosidase (GAA), a lysosomal hydrolase that degrades glycogen to glucose [13]. Accumulation of glycogen in cardiac and skeletal muscle account for progressive muscle weakness, including impaired respiratory function. The infantile form is particularly severe with patients developing cardiomegaly, hepatomegaly, and death due to respiratory failure usually before 2 years of age. Clinical studies were completed that examined the safety and efficacy of recombinant human acid GAA for the treatment of infantile Pompe disease [14–16]. Additional studies are in progress [17]. 3. Assessment of patient immune response to therapeutic proteins Clinical experience with several therapeutic proteins suggests that the development of an antibody response in a percentage of patients is to be expected [18]. Consequently, an evaluation program and testing scheme should be established to determine whether patients develop an immune response to any newly introduced recombinant enzyme replacement therapy. A testing scheme would consist of two categories. First Tier assays that determine whether a patient has made an antibody response and therefore seroconverted, and Second Tier assays that are used to evaluate the clinical significance of these antibodies. First Tier assays generally assess for the presence of binding antibodies using an immunoassay format [e.g., enzyme linked immunosorbent assay (ELISA), Biomolecular Interaction Analysis (BIAcore), radioimmunoassay (RIA), etc.]. The specificity of the antibody response can be further characterized in a confirmatory assay such as a radioimmunoprecipitation (RIP) assay or by western blot. Second Tier assays evaluate the potential clinical significance of these antibodies. A prototype immunosurveillance program has been described for patients with Gaucher disease receiving ERT. Patient serum is initially evaluated for the presence of antibody to the recombinant protein using a screening ELISA, followed by confirmation of the specificity of the response by RIP [19]. Briefly, wells of a microtiter plate were coated with GCR followed by blocking any unreacted sites on the polystyrene wells with human serum albumin (HSA).

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Patient sera were diluted 1/100 in dilution buffer (phosphate-buffered saline [PBS], 0.05% Tween-20, 0.01% HSA) and 100L was added to each well and allowed to incubate for 1 hour at 37C. The plates were washed with PBS-Tween followed by subsequent incubation with horseradish-peroxidase (HRP)-conjugated goat anti-human IgG Fc specific antibody. The presence of IgG antibodies was detected using substrate and the color reaction was measured by reading absorbance using a microtiter plate reader. In the screening ELISA, patients are assessed as being above normal range (ANR) or within normal range (WNR) based on an assay cut-off established from a normal serum distribution study. A large panel of normal human sera (e.g., 100 normal human serum samples) are evaluated in the ELISA and an absorbance cut-off set at a 95% confidence interval. Establishing the ELISA cut-off in this manner maximizes the sensitivity of the screening assay with minimal false positive results. All serum samples with values above the established absorbance cut-off are subsequently evaluated in the confirmatory RIP assay for specific antibodies to the therapeutic protein [19]. The presence of specific antibody is identified by visualization of immunoprecipitated bands at the appropriate size compared to positive control serum and radiolabeled enzyme. With this testing scheme, a patient is considered to have developed an immune response (i.e., seroconverted) if both the screening and confirmatory assays are positive. Patients who have developed an antibody response can be further titered in the ELISA described above. The ELISA format is useful to assess antibody responses since many samples can be readily analyzed over a short period of time. In addition, establishing serum antibody titers by ELISA can be helpful in monitoring patient responses over the course of their treatment. The RIP assay provides several advantages as a confirmatory assay. This method verifies the specificity of the patient antibody response by demonstrating reactivity to a protein of the appropriate size. The method is sensitive, utilizes small sample volume, can be used to identify time of seroconversion, and provides evidence of development of immune tolerance by patients. The advantage of performing a RIP assay rather than western blot is that the RIP identifies reactivity to conformational epitopes and minimizes cross-reactivity that can occur when irrelevant antibodies bind to denatured epitopes on the enzyme. An easily overlooked, but important consideration with ERT is the timing of blood sampling. Since the therapeutic protein is generally administered by infusion over a few hours, it is critical that serum samples be drawn before infusion since the presence of circulating therapeutic protein can interfere with antibody assessments. Currently the assays available to evaluate immunogenicity are developed by different laboratories and are not standardized. Therefore when comparing patient data, it is important to recognize that the results obtained from assays may differ from laboratory to laboratory depending on how well the assay is optimized, how sensitive it is and the overall robustness of the method. The performance characteristics of these assays also need to be determined in a validation study. Assessment parameters that could be considered include intra-assay precision, inter-assay precision, operator variability, specificity, microtiter plate homogeneity (beginning-to-end variation), and sample stability. Positive and negative serum controls should also be established with reference ranges. Once it is determined that a patient has developed an immune response, Tier 2 assays are used to characterize the nature of this response and whether the antibody response may impact

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efficacy. One of these assessments is whether the patient has developed an inhibitory antibody that is to say antibody specific for the therapeutic protein that may interfere with its enzymatic function. Additional assessments could include evaluation for development of antibodies that interfere with uptake, IgG subclasses, and presence of circulating immune complexes (CIC) or complement activation products. 4. Antibody response to enzyme replacement therapy Antibody response to ERT has been studied with the most detail in type I Gaucher Disease patients. As part of an immunosurveillance program, an initial study evaluated 339 patients who were repeatedly infused with alglucerase and monitored for a period of at least 18 months. Patient serum samples obtained at 3-month intervals were screened by ELISA and the presence of IgG antibody specific to GCR was confirmed by radioimmunoprecipitation. Using these methods, the development of GCR-specific IgG was confirmed in 12.4% (42/ 339) of the patients [19]. All patients that seroconverted did so within the first year of therapy. This seroconversion rate was confirmed in a follow-up study performed in a larger patient population. Of the 1122 evaluatable patients, a total of 142 patients (12.6%) seroconverted while on therapy [20]. The median time to seroconversion was 6 months. Again, the majority of patients (88%) seroconverted within the first year of therapy. These findings were similar for imiglucerase where a 15% incidence of seroconversion has been reported in patients naïve to the recombinant GCR therapy [21]. Evaluation of patients for clinical consequences of seroconversion indicated that most patients who develop an antibody response to GCR do not experience any deleterious effect due to these antibodies. The development of in vitro inhibitory antibodies has been reported in a small number of Gaucher patients receiving ERT [20,22]. The presence of inhibitory antibodies, as determined by the in vitro assay, did not always correlate with a decrease in clinical efficacy. This may have been due to patients producing antibody of sufficient titer or reactivity to an epitope near the enzyme active site such that it causes steric hindrance of the enzyme’s ability to act on its substrate in vitro. Alternatively, the therapeutic dose given to these patients could absorb out these antibodies and still leave sufficient enzyme for clinical efficacy. In addition, very few patients required a specific change in therapy due to the presence of inhibitory antibodies and therefore did not develop a clinically relevant immune response [20]. Three patients have been described that were shown to develop circulating antibodies to GCR that were associated with plateaued improvement [23,24]. Although rare (3 of 2462 patients receiving alglucerase or imiglucerase), the presence of clinically relevant enzyme inhibition by antibody should be considered when there is evidence that a patient is responding poorly to an expected therapeutic regimen [7]. Some antibody positive patients had clinical symptoms associated with infusion related reactions. Patients were treated with immunoprophylactic measures and were able to continue therapy (imiglucerase). Severe anaphylactic reactions appear to be a very rare occurrence. Patient immune responses to ERT for other LSD are under investigation. Immunogenicity was evaluated during clinical trials using two different sources of recombinant human GAL with patients having Fabry disease. Seroconversion was observed in the majority of Fabry patients in these two studies [9,10]. Differences were reported in the frequency of serocon-

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version in these patients, however these differences were most likely influenced by the different therapeutic dosages, study durations, and sensitivity in the analytical methods used. There was little evidence for the development of inhibitory antibodies or IgE antibody in either study. The development of an antibody response has also been reported in the majority of patients receiving ERT for GSD II (Pompe disease) [14–17]. Circulating antibodies had no apparent effect on the enzymatic activity of the product [14,16]. Seroconversion has also been reported in MPS-1 patients [12]. Although not all patients receiving ERT develop an antibody response, the ability to elicit an IgG response occurs to many therapeutic proteins [18]. Seroconversion has also been reported with protein replacement therapies, which are available for other genetic disorders including factor VIII for hemophilia [25,26], insulin for diabetes [27,28], and growth hormone for short stature [29,30]. 5. Factors influencing immunogenicity Individual patient immune response to ERT is difficult to predict, particularly in a genetically diverse population. However, with respect to LSD patients, the development of an immune response may be expected, to some extent, considering these patients are without normal levels of native enzyme. Several factors can influence immunogenicity of a protein. ERT as well as other protein therapeutics are produced using a variety of recombinant DNA expression systems. These have included bacterial, mammalian, and transgenic expression systems. Structural similarity is a key factor influencing immunogenicity of a molecule. The degree of sequence homology and post-translational modification (such as completeness and nature of glycosylation, sialation, oxidation, and deamidation) influence the molecular structure of a protein and consequently delineates its foreignness to the patient. Alterations in the confirmation of the molecule such as denaturing the molecule during purification or causing aggregation can make a protein more immunogenic [31,32]. Process impurities or other protein contaminants can elicit a specific antibody response to the impurity [29,33] or cause an adjuvant effect. Other factors that can influence the host immune response include the dose administered, route of administration, and frequency of treatment. Host factors may be of particular relevance when using therapeutic proteins to treat genetic diseases. The genetic basis for the defect in LSD patients is heterogeneous and can vary from patient to patient. Partial gene rearrangements, splice-junction defects, exon deletions, frame shifts and point mutations have been identified. This results in a spectrum of outcomes that can be grouped into three categories: a) mutations where an enzyme is produced having altered processing or stability resulting in subnormal enzyme activity; b) missense mutations that result in enzymes that are completely devoid of activity; or c) mutations that result in no enzyme protein being produced. Patients have been assessed for the presence of endogenous enzyme protein using a variety of immunologic methods in which the presence of cross-reactive immunologic material (CRIM) in the patient’s cells is detected by an antibody to the enzyme. Methods that have been used by investigators have included rocket immunoelectrophoresis, ELISA, and western blot. The interest in CRIM status was initially to provide an assessment as to whether a patient produced enzyme protein in comparison to having enzyme activity. With the availability of ERT, attempts have been made to correlate CRIM status with immunogenicity. It

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has been hypothesized that CRIM () patients, who do not produce enzyme, would be prone to develop antibodies, which could antagonize the effect of replacement enzyme. CRIM ( ) patients however would produce enzyme and therefore should not develop antibodies to enzyme replacement. It has been the experience of our laboratory that the presence of endogenous enzyme protein or CRIM is not predictive of the development of an antibody response. This finding was observed with both Pompe disease and Fabry disease patients receiving ERT. Based on the above described hypotheses, the CRIM () patient population was chosen for a prospective multinational, multicenter, clinical trial evaluating the safety and efficacy of recombinant human acid -Glucosidase (rhGAA) in CRIM () patients with classical Infantile GSD II. All CRIM () patients have developed serum antibodies to rhGAA during this study [17]. Furthermore, analysis in our laboratory of sera from patients in a Phase I/II clinical trial involving two CRIM () patients and one CRIM () patient indicated that all three patients had developed an antibody response to rhGAA, which is in contrast to the original report indicating that antibodies to rhGAA were not detected in serum from the CRIM () patient [15]. Since different assays were performed, this discrepancy is most likely due to assay sensitivity differences. The relevance of CRIM status was also evaluated in Fabry disease patients. A retrospective analysis of data generated by our laboratory indicated there was no correlation between the presence of endogenous enzyme protein and the development of antibodies (seroconversion) in approximately 60 patients with Fabry disease that were treated with ERT. These findings indicate that immunogenicity is a complex process that is not simply correlated to CRIM status. The presence of residual enzyme in the patient does not indicate that the patient will not mount an antibody response to the therapeutic enzyme. Structural differences between normal and mutant protein may elicit an immune response [23]. Immunochemical and biochemical analysis demonstrated that many LSD patients have low levels of enzyme activity, which are associated with low levels of conformationally altered protein [34]. This would suggest that at least some patients could recognize the normal form of the enzyme as being structurally different from their residual mutant form and cause the development of an immune response. Consequently one may have a higher percentage of seroconversion with patients receiving ERT than interventional protein therapies. Additional studies are needed to understand relationships between genotype, protein expression and activity, seroconversion, and clinical outcome. 6. Consequences of developing an immune response Immunologic reactions have been associated with the administration of most therapeutic proteins [18]. The development of antibodies to replacement enzymes may effect clinical outcome in several ways. These include: a) development of hypersensitivity or anaphylactoid reactions often involving evidence suggesting histamine release such as flushing, urticaria, and less often bronchospasm and hypotension; b) development of febrile reactions associated with infusion; c) the formation of circulating immune complexes that may activate the complement system resulting in a generalized inflammatory response and cytokine release; d) immune complex deposition can ultimately lead to glomerular nephritis or other pa-

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thology; e) direct effects on the enzyme resulting in enzyme inactivation or degradation; and f) altered targeting or pharmacokinetics of the therapeutic protein. These outcomes have been seen in patients to various degrees. It has been reported that some antibody positive patients experience signs of hypersensitivity related to enzyme infusions. Patients experiencing allergic reactions should be evaluated for development of protein-specific IgE antibody. IgE is normally present in very low concentrations in circulation but is elevated in atopic disease and other disorders [35]. Mast cells and basophils have receptors for the Fc region of IgE, and the bridging of two IgE molecules by antigen results in the release of inflammatory mediators causing an allergic reaction. When testing patient samples, it is critical to use IgE (epsilon-chain)-specific conjugates and to evaluate the degree of cross-reactivity of this conjugate with other immunoglobulins, particularly IgG. Positive in vitro IgE results should be verified by an alternative method such as either skin testing or assessing for mediators of mast cell degranulation, such as serum tryptase. The development of IgE antibody as a consequence of ERT has been rare [9,20]. An underlying mechanism for the hypersensitivity response in many patients appears to be immune complex-mediated complement activation [19]. Complement can be activated by binding of C1q to antigen:antibody complexes thereby triggering a series of cleavage reactions that can release peptides such as C3a and C5a, which are considered anaphylatoxins and can mediate inflammation and contribute to hypersensitivity symptoms [36]. Patients experiencing symptoms generally demonstrate evidence of complement activation by the presence of complement activation products in circulation. A plasma sample drawn during the reaction should be evaluated along with a pre-infusion sample, whenever possible. The majority of patients who experienced these symptoms were successfully managed using immunoprophylactic measures (such as antihistamines and slowing infusion rates) and were able to continue therapy. The presence of circulating immune complexes should also be evaluated when clinically appropriate. Evidence of actual immune complex disease (i.e. glomerular nephritis, arthritis, or vasculitis) has been very rare. Commercial diagnostic test kits are available for both complement and circulating immune complex determinations. Inhibitory antibodies can be particularly problematic with proteins administered in relatively low doses, as evidenced with hemophilia patients receiving factor VIII [25,26]. However, the development of inhibitory antibodies by patients receiving ERT may have less of an effect on efficacy due to dose differences. For effective targeting, LSD patients generally receive milligram quantities of protein; consequently the clinical impact of the formation of inhibitory antibodies may be diminished. In addition, the presence of specific antibody is postulated to alter enzyme targeting by resulting in Fc receptor uptake, which then targets the protein to an antigen-presenting pathway. This is intuitively thought as being deleterious. However, in the diseases where the ERT is targeted to macrophages or endothelial cells, the presence of antibody may actually provide an alternative mechanism of targeting the enzyme to the target cell via the Fc receptor on these cells [37]. In many situations, the presence of specific antibody may have little clinical consequence. The effect of antibody may be more related to the specificity of the antibody or its concentration in circulation. The impact antibodies have on altered pharmacokinetics may be primarily influenced by antibody titer.

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8. Immune tolerance induction The development of antibodies to replacement enzyme during therapy for LSD does not appear to impact clinical efficacy in most cases reported. This is in contrast to other genetic diseases, like hemophilia where the development of inhibitory antibodies to factor VIII occurs in approximately 30–40% of patients with severe hemophilia A [38]. However, development of antibodies does put patients at higher risk for immune-mediated reactions and immune tolerance induction (ITI) therapy should be considered in cases where a seropositive patient is experiencing clinical decline or disease progression. The majority of studies using ITI therapy have been done with hemophilia patients who have developed inhibitor to factor VIII and Factor IX [39,40]. Although there are several established protocols for ITI therapy (generally utilizing plasmaphoresis, cyclophosphamide, daily high doses of therapeutic protein for several days, and intravenous infusion of IgG) the optimal regimen in terms of safety, clinical efficacy, and pharmacoeconomics has yet to be determined. Alternative immunosuppressive and/or anti-inflammatory approaches that have been used for other diseases with varying degrees of success include administration of corticosteroids, cyclophosphamide, methotrexate, high dose intravenous IgG therapy, splenectomy, and bone marrow transplantation. Immunotherapies that regulate B cells or plasma cells also warrant further investigation. CD20 is a B-cell-restricted antigen that is expressed from the pre-B cell to the mature B-cell stage of B-cell differentiation [36]. Rituxan (Rituximab) is a chimeric anti-CD20 monoclonal antibody that targets mature B-cells in most B-cell malignancies [39,41]. Rituxan is currently used as a therapy for recurrent B-cells lymphoma [42]. However, CD20 is expressed on malignant lymphoplasmacytomas from patients with Waldenstrom’s macroglobulinemia and multiple myeloma, as well as a subpopulation of normal donor plasma cells [43]. The use of this immunotherapy in nonmalignant plasma cell disorders is under investigation. Natural immune tolerance has been observed in Gaucher patients receiving ERT. With continued treatment, the serologic response became abrogated despite the fact that these patients continue to be infused with enzyme at the same dose and frequency. Of the 122 seroconverted patients followed for at least 1 year from the start of therapy, 82 (63%) were found to be subsequently negative in both the ELISA and RIP assays and were therefore considered tolerized [20]. The median time to tolerization was 24 months, while the mean was 28 months. In addition, 69 of these patients were evaluated beyond 30 months of therapy, and 64 of those patients (93%) were now tolerized to the therapeutic protein. The development of immune tolerance has also been observed with continuous interferon-alpha treatment administered to patients with hairy cell leukemia [44]. 9. Summary Clinical data have demonstrated that enzyme replacement therapy can be effective in treating lysosomal storage disorders. Monitoring patients for antibody production is an important component of patient management. As with any therapeutic protein, the development of an antibody response in some percentage of patients seems to be inevitable. The presence

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of specific antibody, however, indicates that other immune-mediated reactions can occur. Since LSD patients receive life-long therapy, which is required to ameliorate severe clinical symptoms, efforts to manage these patients through immune-mediated symptoms is warranted. Additional studies are needed to understand the basis for immune reactivity in these patients. Establishing relationships among genotype, phenotype, protein structure, and immunogenicity are key to determine which epitopes on the lysosomal enzyme are effected. Certain epitopes may be more immunogenic than others and elicit higher affinity antibodies. Antibody responses to certain sites on the molecule may promote enzyme degradation or inhibition of enzyme activity, thereby effecting efficacy. Specific subclass responses may influence complement activation, Fc binding, and allergenic activity. By characterizing the specificity of the antibodies produced among a cross-section of patients, common epitopes may emerge that influence the nature of the antibody response and underlying immune-mediated mechanisms. Lastly, although induction of a natural tolerance process may occur, this process often requires several months to years. New strategies for preventing unwanted antibody responses or accelerating tolerance induction will provide physicians new tools to enhance LSD patients well-being and general health. References [1] Hopwood JJ, Brooks DA. An introduction to the basic science and biology of the lysosome and storage disease. In: Applegarth D, Dimmick J, Hall J, editors. Organelle diseases. London: Chapman Hall; 1997. p. 7–35. [2] Meikle PJ, Hopwood JJ, Claque AE, Carey WF. Prevalence of lysosomal storage disorders. JAMA 1999; 281:249–54. [3] Furbish FS, Steer CJ, Barranger JA, Jones EA, Brady RO. The uptake of native and desialylated glucocerebrosidase by rat hepatocytes and kupffer cells. Biochem Biophys Res Commun 1978;81:1047–53. [4] Barton NW, Bradley RO, Dambrosia JM, DiBiseglie AM, Doppelt SH, Hill SC, et al. Replacement therapy for inherited deficiency macrophage targeted glucocerebrosidase for Gaucher’s disease. N Engl J Med 1991; 324:1464–70. [5] Meikle PJ, Ramieri E, Ravenscroft EM, Hua CT, Brooks DA, Hopwood JJ. Newborn screening for lysosomal storage disorders. Southeast Asian J Trop Med Public Health 1999;30:104–10. [6] National Institute of Health Technology Assessement Conference Statement. Gaucher disease: current issues in diagnosis and treatment. Bethesda: NIH, 1995. [7] Beutler E, Grawbowski GA. Gaucher disease. In: Scriver C, Beaudet A, Valle D, Sly W, editors. The metabolic and molecular basis of inherited disease. 8th ed. New York: McGraw-Hill; 2001. p. 3635–68. [8] Desnick RJ, Ioannou YA, Eng CM. -Galactosidase A deficiency: Fabry disease. In: Beaudet A, Valle D, Scriver C, Sly W, editors. The metabolic and molecular basis of inherited disease. 8th ed. New York: McGraw-Hill; 2001. p. 3733–74. [9] Eng CM, Guffon N, Wilcox WR, Germain DP, Lee P, Waldek S, et al. Safety and efficacy of recombinant human -galactosidase a replacement therapy in Fabry’s disease. N Engl J Med 2001;345:9–16. [10] Schiffmann R, Kopp JB, Austin III HA, Sabnis S, Moore DF, Weibel T, et al. Enzyme replacement therapy in Fabry disease. JAMA 2001;285:2743–9. [11] Neufeld EF, Muenzer J. The mucopolysaccharidoses. In: Scriver C, Beaudet A, Valle D, Sly W, editors. The metabolic and molecular basis of inherited disease. 8th ed. New York: McGraw-Hill; 2001. p. 3421–52. [12] Kakkis ED, Muenzer J. Enzyme replacement therapy in mucopolysaccharidosis I. N Engl J Med 2001;344:182–8. [13] Hirschhorn R, Reuser AJ. Glycogen storage disease type II: acid -glucosidase (acid maltase) deficiency. In: Scriver C, Beaudet A, Valle D, Sly W, editors. The metabolic and molecular basis of inherited disease. 8th ed. New York: McGraw-Hill; 2001. p. 3389–20.

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