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Immune Response to Enzyme Replacement Therapy in Lysosomal Storage Disorder Patients and Animal Models
Molecular Genetics and Metabolism 68, 268 –275 (1999) Article ID mgme.1999.2894, available online at http://www.idealibrary.com on
MINIREVIEW Immune Response to Enzyme Replacement Therapy in Lysosomal Storage Disorder Patients and Animal Models Doug A. Brooks 1 The Lysosomal Diseases Research Unit, Department of Chemical Pathology, Women’s and Children’s Hospital, North Adelaide, South Australia 5006, Australia Received June 29, 1999, and in revised form July 8, 1999
present with symptoms which may include short stature, kyphosis, coarse facies, dysostosis multiplex, joint stiffness, heart valve problems, hepatosplenomegaly, corneal clouding, and mental retardation. LSD have a prevalence of approximately 1 in 7700 live births in Caucasians (2). The severity of LSD presents a significant burden, not only to the patient, but also to the families involved, their community, and health care systems. Enzyme replacement therapy (ERT) has been developed as a treatment strategy for LSD, has recently been subjected to clinical trials in human Gaucher patients (3), and is now in clinical practice. LSD animal model studies and these human clinical trials have demonstrated that ERT is an effective treatment strategy for avoiding the onset of pathology in LSD patients. However, immune responses to replacement therapies have been reported and present as a potential complication for treatment. It is important to note that not all patients receiving ERT will develop immune responses. This may depend on the nature of the replacement protein, the genetic background of the patient, the route of enzyme administration, the frequency of treatment, structural differences between the infused and defective protein, the presence or absence of residual mutant protein in the patient, and environmental factors. The monitoring of circulating antibody levels in patients may serve to warn of potential hypersensitivity reactions. High-level antibody responses may also reduce the efficacy of treatment by inducing altered enzyme distribution, altered intracellular enzyme traffic,
The lysosomal storage disorders (LSD) are a group of severe multiple pathology disorders characterized by enzyme deficiencies which cause the lysosomal accumulation of undegraded or partially degraded macromolecules. Enzyme replacement therapy (ERT) has been developed as a therapy for LSD patients. However, immune responses to ERT have been reported in some individuals from LSD animal model and LSD human patient studies. Antibodies can have adverse effects during ERT, which include hypersensitivity/anaphylactic reactions, enzyme inactivation, altered targeting, and increased enzyme turnover. The monitoring of antibody production during replacement therapy is an important consideration for patient management, as high-titer antibodies can affect the safety and efficacy of the therapy. © 1999 Academic Press Key Words: lysosomal storage disease; treatment outcome; immune reaction; antibody; epitope reactivity; intracellular targeting; enzyme replacement therapy.
Lysosomal storage disorders (LSD) are a group of genetically inherited disorders with a devastating clinical presentation (1). Affected children can be born with no discernible clinical signs, but, in the severe form, progress in the early years of life to 1 To whom correspondence should be addressed at The Lysosomal Diseases Research Unit, Department of Chemical Pathology, Women’s and Children’s Hospital, North Adelaide, South Australia 5006, Australia. Fax: (61-8) 8204-7100. E-mail: dbrooks@ medicine.adelaide.edu.au.
268 1096-7192/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
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and increased enzyme turnover. Even low-level antibody production may reduce the level of enzyme reaching critical sites of pathology in some patients. This review focuses on the incidence of immune responses to ERT in animal model studies and human clinical trials, the level and nature of antibodies produced in response to ERT, and the clinical relevance of circulating antibodies to a replacement protein. Potential mechanisms for either avoiding antibody production or averting the adverse effects of antibody production are also discussed. LYSOSOMAL STORAGE DISORDER PATIENTS AND ENZYME REPLACEMENT THERAPY In the early 1900s a subset of LSD, the mucopolysaccharidoses (MPS), were first documented, with the description of patients, now believed to have Hurler syndrome (4). However, the term “lysosomal storage disorder” was not recognized until the 1960s, with Hers’ description of Pompe disease and the co-recognition by DeDuve and co-workers of “sac-like” structures which contained a range of acid hydrolases (5–9). Hers predicted that the a-glucosidase deficiency in Pompe disease represented the first of many lysosomal enzyme deficiencies and their associated LSD. There are now more than 40 separate LSD recognized, most leading to clinical syndromes which are both chronic and progressive (10). ERT for the treatment of LSD was originally proposed by De Duve (11), based on the discovery of functional deficiencies in lysosomal degradive enzymes by Hers (12) and the possible endocytic uptake of functional enzyme to correct the defect. The feasibility of ERT for LSD was demonstrated by coculture of fibroblasts from patients with Hurler syndrome and normal control human fibroblasts and resulted in cross-correction by a soluble “genotypespecific protein” (12). Functional enzyme secreted from normal control fibroblasts was later shown to be taken up by receptor-mediated endocytosis and used to correct the defect in LSD-affected cells. This is now known to occur via a series of organelle trafficking events, through the vacuolar network, to reach the lysosomal compartment (see Fig. 1). Numerous examples of in vitro correction of lysosomal storage have now been reported for purified lysosomal enzymes. With the advent of recombinant DNA technology and expression cell lines, the synthesis and secretion of high levels of lysosomal proteins has
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been possible, making available the large amounts of recombinant protein required for ERT. In animal models of LSD, the efficacy of ERT has been evaluated as a treatment strategy for human LSD patients. In a mouse model of Sly syndrome (mucopolysaccharidosis type VII, or MPS VII), ERT using b-glucuronidase has demonstrated the successful correction of enzyme activity and the correction of pathology in most tissues (13,14). In this study, treatment from birth was identified as an important strategy, removing storage product prior to the development of irreversible pathology (14). In a canine model of Hurler syndrome (MPS I), ERT demonstrated clearance of storage product in liver, spleen, and kidney; however, brain, heart, and cornea were recognized as problem areas, due to difficulty in clearing storage product (15). Similarly, in a murine model of MPS I (16), ERT has demonstrated correction of pathology (17). In MPS VI cats (Maroteaux–Lamy syndrome) ERT using recombinant human N-acetylgalactosamine 4-sulfatase (4-sulfatase) has demonstrated a dose-dependent correction of pathology, including reduced heart and skeletal pathology (18,19). MPS VI cats treated with 4-sulfatase ERT from birth also had an improved prognosis when compared to MPS VI cats where treatment was started at older ages. ERT in animal models of LSD clearly demonstrated that replacement therapy is an effective in vivo strategy for averting the onset of pathology. Clinical trials of ERT in human patients with Gaucher disease have demonstrated the correction of storage and the ability to prevent the onset of pathology (20 –22). Gaucher patients on long-term ERT demonstrated improved visceral, hematological, and skeletal parameters, with reduced levels of glucocerebroside storage product in macrophages (20,23–25). Recently, a clinical trial of ERT in human MPS I patients has been initiated and is yielding promising results, with patients showing signs of corrected pathology and improved prognosis (26). The animal model studies and these human clinical trials demonstrate the potential efficacy of replacement therapies and establish ERT as an effective treatment strategy for LSD patients. POTENTIAL FOR IMMUNE RESPONSE TO REPLACEMENT THERAPY Is an immune response expected when introducing a naturally occurring lysosomal protein into an LSD patient or an animal model equivalent? For
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LSD patients with either a deletion mutation or a mutation which severely truncates the mutant protein, there will have been no previous exposure to the protein, and the enzyme would therefore be expected to be seen as foreign by the patients’ immune system during ERT. However, immunochemical and biochemical studies demonstrate that many LSD patients have low levels of enzyme activity which are associated with low levels of conformationally altered protein (27). Introducing a structurally different, albeit “normal,” lysosomal protein in high concentration into the circulatory system is likely to result in the development of an immune response, in at least some patients. This is an important factor with regard to treatment regimens which intend to replace an enzyme deficiency with a source of functional and therefore potentially immunogenic protein. It is postulated that a number of enzyme-treated patients will develop circulating antibodies to the replacement protein and that antibody reactivity may in some cases prove to be detrimental to the efficacy of the treatment. The response to ERT may be dependent upon a variety of factors, including the properties of the replacement protein (e.g., size, structural rigidity, glycosylation, sequence similarity with other proteins), the dose and route of administration, the frequency of treatment, the genetic background of the patient, the presence of residual mutant protein in the patient and/or the structural differences between the normal and mutant proteins. Patients with severe LSD who have no detectable mutant protein may be expected to be more immune responsive to ERT than patients with residual mutant protein (e.g., patients with a mutation which, if untreated, would result in a milder clinical presentation). Similarly gene knockout animal models (e.g., MPS I and MPS VI mouse models) and animal models with null mutations (e.g., MPS I
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dog), which produce no detectable protein, may result in enhanced immune reactivity compared to animal models which produce residual levels of mutant protein. BACKGROUND IMMUNE REACTIVITY TO LYSOSOMAL PROTEINS As a preclude to ERT trials in human MPS VI patients, the background antibody titer to the lysosomal protein 4-sulfatase (deficient in MPS VI patients) has been determined in normal control and MPS VI cat and human plasma samples (28,29). In untreated MPS VI, cats (titer 8390 6 7277, n 5 20) and normal control cats (titer 10,343 6 13,846, n 5 20), high levels of antibody reactivity to 4-sulfatase were detected in plasma samples prior to ERT experiments with recombinant human 4-sulfatase (28). These antibodies reacted equally well with either human or feline 4-sulfatase, suggesting that the antibodies are not just recognizing species-specific differences between feline and human protein (28). In normal control humans (titer 5322 6 5392, n 5 12) and MPS VI patients (titer 681 6 668, n 5 7), high background levels of antibody reactivity were also detected to 4-sulfatase (28). In MPS VI cat ERT experiments, infusion of human 4-sulfatase immediately reduced the circulating antibody to 4-sulfatase, demonstrating the specificity of this plasma antibody for 4-sulfatase protein. It could be speculated that this apparent background antibody reactivity to 4-sulfatase is either due to the exposure of 4-sulfatase protein to the immune system or reflects a cross-reactivity with another antigen. The antibody titer to 4-sulfatase detected in an MPS VI patient plasma, which had no detectable 4-sulfatase protein (truncation mutation), indicated that the 4-sulfatase antibodies are probably not produced in
FIG. 1. The endocytic vacuolar network and ERT. During ERT, enzyme is internalized from the cell surface into the cell by receptor-mediated uptake (e.g., mannose 6-phosphate receptor uptake via clathrin-coated pits) and enters a series of organelles called the endocytic network. The enzyme trafficks through different endosome compartments (e.g., early endosome, endosome carrier vesicles, and late endosome/prelysosomal compartment) toward its final destination, the lysosome. Vacuolar network organelles are arranged on microtubules, which facilitate vesicular traffic between organelles. FIG. 2. Proposed traffic of enzyme in the presence and the absence of specific antibody. During effective ERT (in dark blue), enzyme is taken up by the cell and is trafficked to the lysosome. This replaced enzyme can then degrade the accumulated storage product in the endosome–lysosome, effecting the successful correction of an MPS cell. In the presence of antibody (in red), the enzyme may be internalized by a different receptor-mediated uptake process (e.g., antibody Fc receptor-mediated uptake) and be differentially trafficked to the compartment involved in antigen presentation. This compartment has recently been recognized as the MIIC compartment, characterized by the presence of MHC class II molecules in a lysosome-like organelle (39,40). Traffic of enzyme in the presence of antibody may cause altered targeting to the MIIC compartment, resulting in protein degradation and subsequent antigen presentation of the protein’s peptides.
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response to endogenous 4-sulfatase protein, but more likely represent a background level of 4-sulfatase antibodies produced in response to a cross-reacting antigen (28). However, a higher antibody titer was observed in normal control human plasma when compared to human MPS VI patient plasma, suggesting that the level of antibodies can be increased by exposure to endogenous 4-sulfatase protein (28). Together, these data suggested that antibodies to 4-sulfatase are normally found in circulation, an observation which may be important when considering the infusion of protein for ERT. It is expected that plasma antibodies may display reactivity to other lysosomal protein. In the author’s laboratory, similar high background levels of antibody to the lysosomal protein a-L-iduronidase, which is deficient in MPS I patients, have been observed (unpublished observations). It is speculated that these antibodies to lysosomal proteins may have a biological role, possibly reflecting a mechanism for removing lysosomal proteins from circulation (e.g., for “mopping up lysosomal proteins released into circulation,” where they may act inappropriately on their natural substrates in the extracellular matrix). High background antibody titers to some lysosomal proteins may increase the likelihood of generating an immune response during treatment by ERT. IMMUNE RESPONSE TO ERT IN ANIMAL MODELS OF LSD Immune responses to ERT have been reported and present as a potential complication for patient treatment. In MPS VI cats treated with human 4-sulfatase ERT there was a significant increase in antibody titer to 4-sulfatase when compared to either untreated normal control or MPS VI cats (28). However, only 30% of ERT-treated MPS VI cats had higher levels of antibody than normal control cats (28). In other ERT experiments 100% of MPS VI mice treated with 4-sulfatase ERT (30) and 100% of MPS I mice treated with a-L-iduronidase ERT exhibited elevated levels of antibodies against replacement proteins following ERT, when compared to normal controls and untreated MPS I mice (unpublished data). Similarly, in MPS I dogs treated with either human or canine a-L-iduronidase, antibody responses to the replacement protein were observed in all animals (31).
IMMUNE RESPONSE TO ERT IN HUMAN LSD PATIENTS In some human Gaucher patients, antibodies to the replacement protein b-glucocerebrosidase have been reported following ERT. In approximately 15% of Gaucher patients, elevated antibody titers have been detected to glucocerebrosidase when compared to sera samples from normal controls and pre-ERT samples (22). In a few of these cases, the antibodies had adverse effects for the patients (32–34). Patients with signs of hypersensitivity have been treated with immunoprophylactic measures and almost all were able to continue therapy without interruption. More recently, longer term follow up of Gaucher patients on ERT showed several patients with either plateaued improvement or disease progression, which has been attributed to a later onset of circulating antibodies to b-glucocerebrosidase (33,34). The antibodies produced to b-glucocerebrosidase in two of these patients reacted specifically with the normal replacement protein, but did not react with the patients’ own mutant forms of the protein (34). Recent data suggest that as few as 12.8% of Gaucher patients (n 5 1122) produced antibody responses to ERT, but moreover, many of these patients became tolerized and had reduced antibody reactivity after repeated enzyme treatment (35). POTENTIAL ADVERSE EFFECTS OF IMMUNE RESPONSE TO ERT The development of immune-mediated complications during ERT treatment of patients is an important consideration for both the well being of the patient and the efficacy of the therapy. Two potential problems have been reported for immune responses to ERT. The first involves hypersensitivity and possible anaphylaxis, either during or immediately after enzyme administration. Hypersensitivity reactions have been reported in some ERT-treated Gaucher patients (22), MPS VI cats (18), and MPS I dogs (31). Notably, severe anaphylactic reactions appear to be a very rare occurrence. An ERT study in immunized high-titer rats demonstrated that infusion of 5 mg/kg 4-sulfatase also resulted in hypersensitivity reactions, but infusion of 1 mg/kg 4sulfatase resulted in no clinical signs of hypersensitivity reactions (29). The hyperreactivity in 5 mg/kgERT-treated high-titer rats appeared to be associated with altered enzyme distribution in plasma and lung, presumably due to the formation of an exten-
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sive antibody–antigen complex within the circulatory system. This study suggested that the dose and level of circulating antibody may be important factors in the onset of hypersensitivity reactions. In MPS VI cats (19) and Gaucher patients (34) hypersensitivity reactions appeared to be controlled by reducing the rate of enzyme infusion. Again, this suggested a relationship among the level of enzyme circulating, the level of antibody, and the development of hypersensitivity reactions. The second potential immune-mediated complication during ERT involves the effect of circulating antibodies to replacement protein on the efficacy of treatment. Several Gaucher patients receiving longterm ERT have developed circulating antibodies to the replacement protein which was associated with a decrease in clinical improvement (33,34). This was attributed to the enzyme neutralizing capacity of the antibody, indicating that inhibitory antibodies can have a direct effect on treatment outcome. The production of neutralizing antibodies appears to be relatively infrequent, but can be expected to be associated with a poor prognosis, if untreated. In a hightiter immunized rat model (relating to MPS VI), evidence of altered enzyme distribution, altered subcellular targeting, and rapid degradation of infused 4-sulfatase has been reported (28,29). In subcellular fractionation experiments it was clear that antibody impeded the traffic of enzyme to the lysosomal compartment, probably due to rerouting of the enzymebound antibody to the MIIC compartment (which has been shown to be involved in antigen presentation; see Fig. 2). Antibody, therefore has the potential to alter the fate of infused enzyme, which has important implications for the efficacy of treatment. Normal animals have been shown to have antibody titers to some lysosomal proteins (e.g., 28). The level at which antibodies will have a significant impact on the distribution and fate of an infused protein remains to be defined. However, preliminary evidence form the author’s laboratory suggests that only animals developing either high-affinity or very high titer antibody responses (e.g., .64,000 for a-Liduronidase) will have an effect on treatment outcome (unpublished observations). CHARACTERIZATION OF EPITOPE REACTIVITY OF ANTIBODIES PRODUCED IN RESPONSE TO ERT In MPS VI cats which produced high-titer antibodies in response to human 4-sulfatase ERT, the
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linear sequence epitope reactivity of the antibodies and the structural nature of the reactive epitopes have been defined. The latter study aimed to elucidate the possible origin of the immune reactivity to 4-sulfatase and the effect of ERT on this reactivity. As most of the immune reactivity was shown to be to denatured protein in these MPS VI cats, the antibody reactivity was amenable to characterization by epitope mapping, using synthetic 4-sulfatase linear sequence peptides and ELISA reactivity (36). For MPS VI cat plasma, antibody reactivity was observed prior to ERT, with distinct regions of 4-sulfatase linear sequence displaying low-affinity antibody reactivity. There was an increase in antibody titer to 4-sulfatase for MPS VI cats post-ERT and an increase in the level of reactivity to linear sequence epitopes. One cat with a high titer (1,024,000) on prolonged ERT (.1 year, treated from 9 months of age) demonstrated several high-affinity antibodyreactive epitopes. The sites for these high-affinity antibody epitopes were reactive in all other cats tested (albeit at lower affinity), suggesting that they may represent the source of immune reactivity to 4-sulfatase. Some MPS VI cats from short-term ERT experiments (3 months, treated from birth) showed high titers to 4-sulfatase, but evidence of only lowaffinity antibody reactivity. This study suggested that prolonged exposure to the replacement protein may be required for high-affinity antibody production and that specific protein epitopes may be involved. POTENTIAL STRATEGIES FOR AVERTING THE ADVERSE EFFECTS OF ANTIBODY PRODUCTION In Gaucher patients producing neutralizing antibodies, strategies for either immunosuppression or tolerance induction have been trialed to prevent disease progression (33,34). High doses of enzyme to induce tolerance, plasma exchange to reduce the concentration of circulating antibody, infusion of intravenous IgG to inhibit immune complex formation and control immune reactivity (e.g., anti-idiotypic antibody production), and cyclophosphamide to kill reactive B cells are all strategies that have been used in various combinations to manage reactive antibodies. Interestingly, a recent study on glucocerebrosidase ERT indicated that 12.8% of patients (142 of 1122) responded with antibody production, but the majority of these seroconverted patients were tolerized by subsequent repeated infusions of
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enzyme, as part of the normal treatment regimen (35). The recent promising result with immunomodulation reagents, such as either antibody to the CD4 antigen on T-cells or FK506, may also have a valuable role in the control of immune reactivity to replacement proteins (37,38). If the reactive epitopes on the replacement protein have been defined, as discussed above, it may be possible to induce tolerance to specific epitopes, even before therapy is initiated. Similarly, the treatment of patients at birth, particularly with high-dose therapy, may take advantage of the early window in immunity status, where the immune system is more amenable to tolerance induction.
ACKNOWLEDGMENTS D. A. Brooks is supported as a Senior Research Fellow, by an NH&MRC Program Grant in Australia. Additional funding for work, in the author’s laboratory, on immune responses to ERT has been supported by the Women’s and Children’s Hospital Research Foundation, Adelaide, Australia, and the Rebecca L. Cooper Medical Research Foundation. The author thanks Professor John J. Hopwood and Chris T. Turner for their assistance in the preparation of the manuscript.
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CONCLUSIONS There has been considerable progress in establishing ERT as a viable first-line treatment strategy for LSD patients. The potential complication of immune reactivity to the replacement protein is likely in some patients, but the number of responsive patients may vary considerably depending on the nature of the replacement protein and its baseline immune reactivity. Current experience with patients having Gaucher disease and more recently MPS I suggest that on the order of 10 –30% of patients may be expected to demonstrate immune reactivity to the replacement protein. In most patients with Gaucher disease this reactive antibody does not appear to significantly impact on treatment outcome, except in a few individuals. However, MPS VI animal model studies suggest that there may be effects on the efficacy of treatment caused by enzyme redistribution, altered subcellular targeting, and increased enzyme turnover. The latter effects may only be subtle, unless high-titer high-affinity antibody responses are generated. If the immune response to the replacement protein results in inhibitory antibody formation, then evidence suggests that there may be a marked effect on treatment outcome, resulting in disease progression. The monitoring of antibody production is therefore an important component of patient management. In the near future, it is likely that the current aggressive strategies for removing reactive antibodies in patients may be improved by using either more sophisticated (e.g., anti-CD4 antibody administration with ERT) or less invasive (e.g., high dose tolerance at birth) approaches.
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MINIREVIEW Immune Response to Enzyme Replacement Therapy in Lysosomal Storage Disorder Patients and Animal Models Doug A. Brooks 1 The Lysosomal Diseases Research Unit, Department of Chemical Pathology, Women’s and Children’s Hospital, North Adelaide, South Australia 5006, Australia Received June 29, 1999, and in revised form July 8, 1999
present with symptoms which may include short stature, kyphosis, coarse facies, dysostosis multiplex, joint stiffness, heart valve problems, hepatosplenomegaly, corneal clouding, and mental retardation. LSD have a prevalence of approximately 1 in 7700 live births in Caucasians (2). The severity of LSD presents a significant burden, not only to the patient, but also to the families involved, their community, and health care systems. Enzyme replacement therapy (ERT) has been developed as a treatment strategy for LSD, has recently been subjected to clinical trials in human Gaucher patients (3), and is now in clinical practice. LSD animal model studies and these human clinical trials have demonstrated that ERT is an effective treatment strategy for avoiding the onset of pathology in LSD patients. However, immune responses to replacement therapies have been reported and present as a potential complication for treatment. It is important to note that not all patients receiving ERT will develop immune responses. This may depend on the nature of the replacement protein, the genetic background of the patient, the route of enzyme administration, the frequency of treatment, structural differences between the infused and defective protein, the presence or absence of residual mutant protein in the patient, and environmental factors. The monitoring of circulating antibody levels in patients may serve to warn of potential hypersensitivity reactions. High-level antibody responses may also reduce the efficacy of treatment by inducing altered enzyme distribution, altered intracellular enzyme traffic,
The lysosomal storage disorders (LSD) are a group of severe multiple pathology disorders characterized by enzyme deficiencies which cause the lysosomal accumulation of undegraded or partially degraded macromolecules. Enzyme replacement therapy (ERT) has been developed as a therapy for LSD patients. However, immune responses to ERT have been reported in some individuals from LSD animal model and LSD human patient studies. Antibodies can have adverse effects during ERT, which include hypersensitivity/anaphylactic reactions, enzyme inactivation, altered targeting, and increased enzyme turnover. The monitoring of antibody production during replacement therapy is an important consideration for patient management, as high-titer antibodies can affect the safety and efficacy of the therapy. © 1999 Academic Press Key Words: lysosomal storage disease; treatment outcome; immune reaction; antibody; epitope reactivity; intracellular targeting; enzyme replacement therapy.
Lysosomal storage disorders (LSD) are a group of genetically inherited disorders with a devastating clinical presentation (1). Affected children can be born with no discernible clinical signs, but, in the severe form, progress in the early years of life to 1 To whom correspondence should be addressed at The Lysosomal Diseases Research Unit, Department of Chemical Pathology, Women’s and Children’s Hospital, North Adelaide, South Australia 5006, Australia. Fax: (61-8) 8204-7100. E-mail: dbrooks@ medicine.adelaide.edu.au.
268 1096-7192/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
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and increased enzyme turnover. Even low-level antibody production may reduce the level of enzyme reaching critical sites of pathology in some patients. This review focuses on the incidence of immune responses to ERT in animal model studies and human clinical trials, the level and nature of antibodies produced in response to ERT, and the clinical relevance of circulating antibodies to a replacement protein. Potential mechanisms for either avoiding antibody production or averting the adverse effects of antibody production are also discussed. LYSOSOMAL STORAGE DISORDER PATIENTS AND ENZYME REPLACEMENT THERAPY In the early 1900s a subset of LSD, the mucopolysaccharidoses (MPS), were first documented, with the description of patients, now believed to have Hurler syndrome (4). However, the term “lysosomal storage disorder” was not recognized until the 1960s, with Hers’ description of Pompe disease and the co-recognition by DeDuve and co-workers of “sac-like” structures which contained a range of acid hydrolases (5–9). Hers predicted that the a-glucosidase deficiency in Pompe disease represented the first of many lysosomal enzyme deficiencies and their associated LSD. There are now more than 40 separate LSD recognized, most leading to clinical syndromes which are both chronic and progressive (10). ERT for the treatment of LSD was originally proposed by De Duve (11), based on the discovery of functional deficiencies in lysosomal degradive enzymes by Hers (12) and the possible endocytic uptake of functional enzyme to correct the defect. The feasibility of ERT for LSD was demonstrated by coculture of fibroblasts from patients with Hurler syndrome and normal control human fibroblasts and resulted in cross-correction by a soluble “genotypespecific protein” (12). Functional enzyme secreted from normal control fibroblasts was later shown to be taken up by receptor-mediated endocytosis and used to correct the defect in LSD-affected cells. This is now known to occur via a series of organelle trafficking events, through the vacuolar network, to reach the lysosomal compartment (see Fig. 1). Numerous examples of in vitro correction of lysosomal storage have now been reported for purified lysosomal enzymes. With the advent of recombinant DNA technology and expression cell lines, the synthesis and secretion of high levels of lysosomal proteins has
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been possible, making available the large amounts of recombinant protein required for ERT. In animal models of LSD, the efficacy of ERT has been evaluated as a treatment strategy for human LSD patients. In a mouse model of Sly syndrome (mucopolysaccharidosis type VII, or MPS VII), ERT using b-glucuronidase has demonstrated the successful correction of enzyme activity and the correction of pathology in most tissues (13,14). In this study, treatment from birth was identified as an important strategy, removing storage product prior to the development of irreversible pathology (14). In a canine model of Hurler syndrome (MPS I), ERT demonstrated clearance of storage product in liver, spleen, and kidney; however, brain, heart, and cornea were recognized as problem areas, due to difficulty in clearing storage product (15). Similarly, in a murine model of MPS I (16), ERT has demonstrated correction of pathology (17). In MPS VI cats (Maroteaux–Lamy syndrome) ERT using recombinant human N-acetylgalactosamine 4-sulfatase (4-sulfatase) has demonstrated a dose-dependent correction of pathology, including reduced heart and skeletal pathology (18,19). MPS VI cats treated with 4-sulfatase ERT from birth also had an improved prognosis when compared to MPS VI cats where treatment was started at older ages. ERT in animal models of LSD clearly demonstrated that replacement therapy is an effective in vivo strategy for averting the onset of pathology. Clinical trials of ERT in human patients with Gaucher disease have demonstrated the correction of storage and the ability to prevent the onset of pathology (20 –22). Gaucher patients on long-term ERT demonstrated improved visceral, hematological, and skeletal parameters, with reduced levels of glucocerebroside storage product in macrophages (20,23–25). Recently, a clinical trial of ERT in human MPS I patients has been initiated and is yielding promising results, with patients showing signs of corrected pathology and improved prognosis (26). The animal model studies and these human clinical trials demonstrate the potential efficacy of replacement therapies and establish ERT as an effective treatment strategy for LSD patients. POTENTIAL FOR IMMUNE RESPONSE TO REPLACEMENT THERAPY Is an immune response expected when introducing a naturally occurring lysosomal protein into an LSD patient or an animal model equivalent? For
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LSD patients with either a deletion mutation or a mutation which severely truncates the mutant protein, there will have been no previous exposure to the protein, and the enzyme would therefore be expected to be seen as foreign by the patients’ immune system during ERT. However, immunochemical and biochemical studies demonstrate that many LSD patients have low levels of enzyme activity which are associated with low levels of conformationally altered protein (27). Introducing a structurally different, albeit “normal,” lysosomal protein in high concentration into the circulatory system is likely to result in the development of an immune response, in at least some patients. This is an important factor with regard to treatment regimens which intend to replace an enzyme deficiency with a source of functional and therefore potentially immunogenic protein. It is postulated that a number of enzyme-treated patients will develop circulating antibodies to the replacement protein and that antibody reactivity may in some cases prove to be detrimental to the efficacy of the treatment. The response to ERT may be dependent upon a variety of factors, including the properties of the replacement protein (e.g., size, structural rigidity, glycosylation, sequence similarity with other proteins), the dose and route of administration, the frequency of treatment, the genetic background of the patient, the presence of residual mutant protein in the patient and/or the structural differences between the normal and mutant proteins. Patients with severe LSD who have no detectable mutant protein may be expected to be more immune responsive to ERT than patients with residual mutant protein (e.g., patients with a mutation which, if untreated, would result in a milder clinical presentation). Similarly gene knockout animal models (e.g., MPS I and MPS VI mouse models) and animal models with null mutations (e.g., MPS I
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dog), which produce no detectable protein, may result in enhanced immune reactivity compared to animal models which produce residual levels of mutant protein. BACKGROUND IMMUNE REACTIVITY TO LYSOSOMAL PROTEINS As a preclude to ERT trials in human MPS VI patients, the background antibody titer to the lysosomal protein 4-sulfatase (deficient in MPS VI patients) has been determined in normal control and MPS VI cat and human plasma samples (28,29). In untreated MPS VI, cats (titer 8390 6 7277, n 5 20) and normal control cats (titer 10,343 6 13,846, n 5 20), high levels of antibody reactivity to 4-sulfatase were detected in plasma samples prior to ERT experiments with recombinant human 4-sulfatase (28). These antibodies reacted equally well with either human or feline 4-sulfatase, suggesting that the antibodies are not just recognizing species-specific differences between feline and human protein (28). In normal control humans (titer 5322 6 5392, n 5 12) and MPS VI patients (titer 681 6 668, n 5 7), high background levels of antibody reactivity were also detected to 4-sulfatase (28). In MPS VI cat ERT experiments, infusion of human 4-sulfatase immediately reduced the circulating antibody to 4-sulfatase, demonstrating the specificity of this plasma antibody for 4-sulfatase protein. It could be speculated that this apparent background antibody reactivity to 4-sulfatase is either due to the exposure of 4-sulfatase protein to the immune system or reflects a cross-reactivity with another antigen. The antibody titer to 4-sulfatase detected in an MPS VI patient plasma, which had no detectable 4-sulfatase protein (truncation mutation), indicated that the 4-sulfatase antibodies are probably not produced in
FIG. 1. The endocytic vacuolar network and ERT. During ERT, enzyme is internalized from the cell surface into the cell by receptor-mediated uptake (e.g., mannose 6-phosphate receptor uptake via clathrin-coated pits) and enters a series of organelles called the endocytic network. The enzyme trafficks through different endosome compartments (e.g., early endosome, endosome carrier vesicles, and late endosome/prelysosomal compartment) toward its final destination, the lysosome. Vacuolar network organelles are arranged on microtubules, which facilitate vesicular traffic between organelles. FIG. 2. Proposed traffic of enzyme in the presence and the absence of specific antibody. During effective ERT (in dark blue), enzyme is taken up by the cell and is trafficked to the lysosome. This replaced enzyme can then degrade the accumulated storage product in the endosome–lysosome, effecting the successful correction of an MPS cell. In the presence of antibody (in red), the enzyme may be internalized by a different receptor-mediated uptake process (e.g., antibody Fc receptor-mediated uptake) and be differentially trafficked to the compartment involved in antigen presentation. This compartment has recently been recognized as the MIIC compartment, characterized by the presence of MHC class II molecules in a lysosome-like organelle (39,40). Traffic of enzyme in the presence of antibody may cause altered targeting to the MIIC compartment, resulting in protein degradation and subsequent antigen presentation of the protein’s peptides.
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response to endogenous 4-sulfatase protein, but more likely represent a background level of 4-sulfatase antibodies produced in response to a cross-reacting antigen (28). However, a higher antibody titer was observed in normal control human plasma when compared to human MPS VI patient plasma, suggesting that the level of antibodies can be increased by exposure to endogenous 4-sulfatase protein (28). Together, these data suggested that antibodies to 4-sulfatase are normally found in circulation, an observation which may be important when considering the infusion of protein for ERT. It is expected that plasma antibodies may display reactivity to other lysosomal protein. In the author’s laboratory, similar high background levels of antibody to the lysosomal protein a-L-iduronidase, which is deficient in MPS I patients, have been observed (unpublished observations). It is speculated that these antibodies to lysosomal proteins may have a biological role, possibly reflecting a mechanism for removing lysosomal proteins from circulation (e.g., for “mopping up lysosomal proteins released into circulation,” where they may act inappropriately on their natural substrates in the extracellular matrix). High background antibody titers to some lysosomal proteins may increase the likelihood of generating an immune response during treatment by ERT. IMMUNE RESPONSE TO ERT IN ANIMAL MODELS OF LSD Immune responses to ERT have been reported and present as a potential complication for patient treatment. In MPS VI cats treated with human 4-sulfatase ERT there was a significant increase in antibody titer to 4-sulfatase when compared to either untreated normal control or MPS VI cats (28). However, only 30% of ERT-treated MPS VI cats had higher levels of antibody than normal control cats (28). In other ERT experiments 100% of MPS VI mice treated with 4-sulfatase ERT (30) and 100% of MPS I mice treated with a-L-iduronidase ERT exhibited elevated levels of antibodies against replacement proteins following ERT, when compared to normal controls and untreated MPS I mice (unpublished data). Similarly, in MPS I dogs treated with either human or canine a-L-iduronidase, antibody responses to the replacement protein were observed in all animals (31).
IMMUNE RESPONSE TO ERT IN HUMAN LSD PATIENTS In some human Gaucher patients, antibodies to the replacement protein b-glucocerebrosidase have been reported following ERT. In approximately 15% of Gaucher patients, elevated antibody titers have been detected to glucocerebrosidase when compared to sera samples from normal controls and pre-ERT samples (22). In a few of these cases, the antibodies had adverse effects for the patients (32–34). Patients with signs of hypersensitivity have been treated with immunoprophylactic measures and almost all were able to continue therapy without interruption. More recently, longer term follow up of Gaucher patients on ERT showed several patients with either plateaued improvement or disease progression, which has been attributed to a later onset of circulating antibodies to b-glucocerebrosidase (33,34). The antibodies produced to b-glucocerebrosidase in two of these patients reacted specifically with the normal replacement protein, but did not react with the patients’ own mutant forms of the protein (34). Recent data suggest that as few as 12.8% of Gaucher patients (n 5 1122) produced antibody responses to ERT, but moreover, many of these patients became tolerized and had reduced antibody reactivity after repeated enzyme treatment (35). POTENTIAL ADVERSE EFFECTS OF IMMUNE RESPONSE TO ERT The development of immune-mediated complications during ERT treatment of patients is an important consideration for both the well being of the patient and the efficacy of the therapy. Two potential problems have been reported for immune responses to ERT. The first involves hypersensitivity and possible anaphylaxis, either during or immediately after enzyme administration. Hypersensitivity reactions have been reported in some ERT-treated Gaucher patients (22), MPS VI cats (18), and MPS I dogs (31). Notably, severe anaphylactic reactions appear to be a very rare occurrence. An ERT study in immunized high-titer rats demonstrated that infusion of 5 mg/kg 4-sulfatase also resulted in hypersensitivity reactions, but infusion of 1 mg/kg 4sulfatase resulted in no clinical signs of hypersensitivity reactions (29). The hyperreactivity in 5 mg/kgERT-treated high-titer rats appeared to be associated with altered enzyme distribution in plasma and lung, presumably due to the formation of an exten-
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sive antibody–antigen complex within the circulatory system. This study suggested that the dose and level of circulating antibody may be important factors in the onset of hypersensitivity reactions. In MPS VI cats (19) and Gaucher patients (34) hypersensitivity reactions appeared to be controlled by reducing the rate of enzyme infusion. Again, this suggested a relationship among the level of enzyme circulating, the level of antibody, and the development of hypersensitivity reactions. The second potential immune-mediated complication during ERT involves the effect of circulating antibodies to replacement protein on the efficacy of treatment. Several Gaucher patients receiving longterm ERT have developed circulating antibodies to the replacement protein which was associated with a decrease in clinical improvement (33,34). This was attributed to the enzyme neutralizing capacity of the antibody, indicating that inhibitory antibodies can have a direct effect on treatment outcome. The production of neutralizing antibodies appears to be relatively infrequent, but can be expected to be associated with a poor prognosis, if untreated. In a hightiter immunized rat model (relating to MPS VI), evidence of altered enzyme distribution, altered subcellular targeting, and rapid degradation of infused 4-sulfatase has been reported (28,29). In subcellular fractionation experiments it was clear that antibody impeded the traffic of enzyme to the lysosomal compartment, probably due to rerouting of the enzymebound antibody to the MIIC compartment (which has been shown to be involved in antigen presentation; see Fig. 2). Antibody, therefore has the potential to alter the fate of infused enzyme, which has important implications for the efficacy of treatment. Normal animals have been shown to have antibody titers to some lysosomal proteins (e.g., 28). The level at which antibodies will have a significant impact on the distribution and fate of an infused protein remains to be defined. However, preliminary evidence form the author’s laboratory suggests that only animals developing either high-affinity or very high titer antibody responses (e.g., .64,000 for a-Liduronidase) will have an effect on treatment outcome (unpublished observations). CHARACTERIZATION OF EPITOPE REACTIVITY OF ANTIBODIES PRODUCED IN RESPONSE TO ERT In MPS VI cats which produced high-titer antibodies in response to human 4-sulfatase ERT, the
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linear sequence epitope reactivity of the antibodies and the structural nature of the reactive epitopes have been defined. The latter study aimed to elucidate the possible origin of the immune reactivity to 4-sulfatase and the effect of ERT on this reactivity. As most of the immune reactivity was shown to be to denatured protein in these MPS VI cats, the antibody reactivity was amenable to characterization by epitope mapping, using synthetic 4-sulfatase linear sequence peptides and ELISA reactivity (36). For MPS VI cat plasma, antibody reactivity was observed prior to ERT, with distinct regions of 4-sulfatase linear sequence displaying low-affinity antibody reactivity. There was an increase in antibody titer to 4-sulfatase for MPS VI cats post-ERT and an increase in the level of reactivity to linear sequence epitopes. One cat with a high titer (1,024,000) on prolonged ERT (.1 year, treated from 9 months of age) demonstrated several high-affinity antibodyreactive epitopes. The sites for these high-affinity antibody epitopes were reactive in all other cats tested (albeit at lower affinity), suggesting that they may represent the source of immune reactivity to 4-sulfatase. Some MPS VI cats from short-term ERT experiments (3 months, treated from birth) showed high titers to 4-sulfatase, but evidence of only lowaffinity antibody reactivity. This study suggested that prolonged exposure to the replacement protein may be required for high-affinity antibody production and that specific protein epitopes may be involved. POTENTIAL STRATEGIES FOR AVERTING THE ADVERSE EFFECTS OF ANTIBODY PRODUCTION In Gaucher patients producing neutralizing antibodies, strategies for either immunosuppression or tolerance induction have been trialed to prevent disease progression (33,34). High doses of enzyme to induce tolerance, plasma exchange to reduce the concentration of circulating antibody, infusion of intravenous IgG to inhibit immune complex formation and control immune reactivity (e.g., anti-idiotypic antibody production), and cyclophosphamide to kill reactive B cells are all strategies that have been used in various combinations to manage reactive antibodies. Interestingly, a recent study on glucocerebrosidase ERT indicated that 12.8% of patients (142 of 1122) responded with antibody production, but the majority of these seroconverted patients were tolerized by subsequent repeated infusions of
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enzyme, as part of the normal treatment regimen (35). The recent promising result with immunomodulation reagents, such as either antibody to the CD4 antigen on T-cells or FK506, may also have a valuable role in the control of immune reactivity to replacement proteins (37,38). If the reactive epitopes on the replacement protein have been defined, as discussed above, it may be possible to induce tolerance to specific epitopes, even before therapy is initiated. Similarly, the treatment of patients at birth, particularly with high-dose therapy, may take advantage of the early window in immunity status, where the immune system is more amenable to tolerance induction.
ACKNOWLEDGMENTS D. A. Brooks is supported as a Senior Research Fellow, by an NH&MRC Program Grant in Australia. Additional funding for work, in the author’s laboratory, on immune responses to ERT has been supported by the Women’s and Children’s Hospital Research Foundation, Adelaide, Australia, and the Rebecca L. Cooper Medical Research Foundation. The author thanks Professor John J. Hopwood and Chris T. Turner for their assistance in the preparation of the manuscript.
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CONCLUSIONS There has been considerable progress in establishing ERT as a viable first-line treatment strategy for LSD patients. The potential complication of immune reactivity to the replacement protein is likely in some patients, but the number of responsive patients may vary considerably depending on the nature of the replacement protein and its baseline immune reactivity. Current experience with patients having Gaucher disease and more recently MPS I suggest that on the order of 10 –30% of patients may be expected to demonstrate immune reactivity to the replacement protein. In most patients with Gaucher disease this reactive antibody does not appear to significantly impact on treatment outcome, except in a few individuals. However, MPS VI animal model studies suggest that there may be effects on the efficacy of treatment caused by enzyme redistribution, altered subcellular targeting, and increased enzyme turnover. The latter effects may only be subtle, unless high-titer high-affinity antibody responses are generated. If the immune response to the replacement protein results in inhibitory antibody formation, then evidence suggests that there may be a marked effect on treatment outcome, resulting in disease progression. The monitoring of antibody production is therefore an important component of patient management. In the near future, it is likely that the current aggressive strategies for removing reactive antibodies in patients may be improved by using either more sophisticated (e.g., anti-CD4 antibody administration with ERT) or less invasive (e.g., high dose tolerance at birth) approaches.
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