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Blood, Vol. 93 No. 6 (March 15), 1999:
pp. 2081-2088
Immunosurveillance of Alglucerase Enzyme Therapy for Gaucher Patients:
Induction of Humoral Tolerance in Seroconverted Patients After
Repeat Administration
By
Mireille Rosenberg,
Wytske Kingma,
Mary Anne Fitzpatrick, and
Susan M. Richards
From Genzyme Corp, Framingham, MA.
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ABSTRACT |
Alglucerase, a macrophage-targeted enzyme replacement therapy for
Gaucher disease, has been successfully used for several years to
improve clinical symptoms and reverse disease progression. As part of
an immunosurveillance program, 1,122 Gaucher patients were monitored
for antibody response to glucocerebrosidase, the active component of
alglucerase. Seroconversion was detected in 142 patients (12.8%) by
enzyme-linked immunosorbent assay (ELISA) and confirmed by
radioimmunoprecipitation. The majority (75%) of the seroconverted
population had no detectable levels of circulating inhibitory antibody
as assessed by in vitro inhibition of enzymatic activity of the
therapeutic molecule. Of the remaining patients with putative
inhibitory antibodies, the majority had only low levels of serum
inhibitory activity, which was transient. A very small number of
patients were identified as developing true neutralizing antibodies, as
defined by the development of antibodies that impacted clinical
efficacy. Many of the patient antibody responses were also diminished
with time. Eighty-two of the 142 seroconverted patients have stopped
producing antibody to the molecule and appear tolerized. The mean time
for humoral tolerization was 28 months from initiation of therapy. Of
64 seroconverted patients followed for at least 30 months of therapy,
the tolerization rate was 93%. These results show that although 12.8%
of the patients on therapy developed antibodies to the molecule, 90%
of these patients became tolerized over time.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
IN 1882, PHILIP GAUCHER was the first to
describe the finding of unusual large foamy cells in a patient with an
epithelioma of the spleen, now recognized as a hallmark of Gaucher
disease. Subsequently, lipid abnormalities, related enzymology, and
autosomal recessive inheritance were described.1-4 Gaucher
disease is the most prevalent lysosomal storage disease affecting
20,000 to 30,000 individuals worldwide.5 The pathology of
the disease is associated with a marked accumulation of complex lipids,
glucocerebrosides, in tissues in conjunction with a defective activity
of the enzyme glucocerebrosidase (GCR). This results in an accumulation
of glucocerebrosides in cells of the spleen, liver, bone, and
infrequently, the brain, which ultimately impairs organ
function.3 In 1991, the Food and Drug Administration (FDA)
approved alglucerase enzyme replacement therapy for Gaucher disease.
The active component consists of purified placental-derived GCR
modified to expose  -mannose residues on the oligosaccharide. These
modifications improved the efficiency of targeting to the mannose
receptor on macrophages in the reticuloendothelial system.6,7 Imiglucerase, a replacement therapy using a
recombinant form of this enzyme, was subsequently approved.
A safety issue associated with the use of any therapeutic protein is
the immunogenicity of the molecule and potential development of
immune-mediated complications.8 Parameters that impact a molecule's ability to elicit an immune response include the size of
the molecule, the structural or sequence difference from the native
protein, as well as the dose, frequency, and route of administration. An additional consideration for patients with genetic diseases is that
the endogenous molecule may be absent, mutated, or produced in a
conformation that renders it nonfunctional. Consequently, the native
protein could be recognized as foreign and elicit an immune response.
Immunologic responses to both natural and recombinant therapeutic
proteins have been reported. Serum proteins such as insulin,9 growth hormone,10,11 and Factor
VIII,12 as well as cytokines such as interferon
,13 interferon- ,14 interleukin (IL)-2,15 IL-3,16 and granulocyte-macrophage
colony-stimulating factor (GM-CSF)17 have been reported to
induce antibody formation. Some of these proteins also elicited the
development of neutralizing antibodies,11-19 which impaired
the clinical efficacy of the molecule. The incidence of neutralizing
antibodies can be significant. For example, the development of
neutralizing antibodies varied from 30% to 50% of the patient
population treated with interferon.13,20 Immunologic
reactivity is a key determinant in product efficacy.
During alglucerase therapy, Gaucher patients generally received
frequent infusions (often every 2 weeks) of milligram quantities of
GCR. It is recommended that patients be monitored for a period of at
least 18 months to assess their immunological response to therapy. The
development of assays and immunosurveillance of 262 patients treated
with alglucerase has been previously reported.21 These
findings indicated that within the first year of therapy 13% of the
patients developed antibodies to the molecule and that a trend toward
humoral unresponsiveness was observed with several patients. This
report describes the updated findings for over 1,100 patients treated
over the course of 5 years.
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MATERIALS AND METHODS |
Patients
Patients with documented Gaucher disease were treated with periodic
infusions of alglucerase (approximately 15 to 60 U/kg body weight,
generally every 2 weeks). Blood was always drawn before the start of
infusion. Samples were obtained from patients before the start of
therapy (baseline) and at different times after initiation of therapy,
generally at 3, 6, 9, 12, and 18 months. Serum samples were collected
by centrifugation and stored at  80°C until analyzed. The
majority of the samples were obtained as part of an immunosurveillance
program (provided by Genzyme Corp).
Eighty-four percent (1,003 of 1,183) of patients initiating therapy
with alglucerase in the Gaucher registry have been tested for antibody
status. Eleven percent (113 of 1,003) of those tested were found to be
antibody positive.
Though participation in the Gaucher Registry is voluntary and
submission of data may be incomplete, it is reasonable to assume that
data included in this analysis are representative of the population of
patients treated for Gaucher disease.
Evaluation Criteria
To be included in this evaluation, patients were required to have at
least one serum sample drawn at least 30 days past the first infusion
of alglucerase. Patients with only a baseline sample, as well as those
treated exclusively with imiglucerase, were excluded from this
analysis. Samples from patients receiving alglucerase who subsequently
were treated with imiglucerase were included in the study up to the
time they changed therapy.
GCR
Clinical grade, unformulated, placental-derived GCR was used in all the
analyses. The enzyme's carbohydrate structure was enzymatically
modified to yield a molecule with a terminal mannose residue for
enhanced macrophage targeting. The GCR was further purified over a
Protein A (Pierce, Rockford, IL) column, as this preparation contained
small amounts of contaminating human IgG that could give false positive
results in the enzyme-linked immunosorbent assay (ELISA).
Patient Immune Response to GCR
ELISA IgG response.
An ELISA developed as a screening assay for the detection of antibodies
in patients' sera has been previously described and validated.21 Briefly, 96-well microtiter plates were coated overnight with GCR followed by 2 hours blocking with human serum albumin (HSA, Baxter Healthcare, Glendale, CA) in phosphate-buffered saline (PBS). Wells were also coated with HSA because it is used as an
excipient in the formulation. Plates were incubated with patient sera
at a 1/100 dilution in PBS-Tween, HSA for 2 hours at 37°C. Plates
were washed followed by a subsequent 1-hour incubation at 37°C with
a horseradish peroxidase-labeled goat antihuman IgG Fc specific
antibody (Jackson Immunoresearch, West Grove, PA). O-phenylenediamine
(OPD; Sigma, St Louis, MO) was used as a substrate and the
color reaction measured at 490 nm. Assay controls included three
different pooled sera prepared from seronegative Gaucher patients,
seroconverted Gaucher patients' sera, and normal human sera. The upper
limit of reactivity to GCR was defined as 2 standard deviations above
the mean value of commercially available pooled human sera
(Scantibodies Laboratory, Santee, CA). All serum samples with values
above the established normal range were subsequently tested for
confirmation of specific antibodies to GCR in the
radioimmunoprecipitation assay.
Radioimmunoprecipitation.
A radioimmunoprecipitation (RIP) assay developed as a confirmatory
assay for antibodies to GCR has been previously
described.21 Briefly, GCR was iodinated using Iodobeads
(Pierce) following the protocol recommended by the manufacturer.
Patient sera was incubated with [I125] GCR overnight at
4°C with constant mixing. The following day Sepharose bound Protein
A beads (Sigma) were added to the mixture and incubated for 1 hour.
After extensive washing, the immune complex was precipitated by
centrifugation, the pellet resuspended in sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer
(sepraSol, ISS, Natick, MA), boiled, and the samples separated through
a 10% separating gel (Biorad, Hercules, CA). After drying the gel, the
radioimmunoprecipitate was visualized via autoradiography. Pooled sera
from seroconverted patients and nonseroconverted patients, as well as
pooled normal human sera, were used for controls. Once a patient had
been shown to be positive for antibody to GCR by RIP, all subsequent
samples from that patient were evaluated by RIP regardless of the ELISA results.
In vitro inhibition assay.
An assay to measure inhibition of in vitro enzymatic activity of the
GCR enzyme was developed and qualified. Serum samples were diluted 1/10
in substrate buffer (0.01 mol/L phosphate buffer, 2.3 mmol/L sodium
taurocholate, 0.15% Triton X-100, 0.02% sodium azide, pH 5.9). A
twofold dilution series of the sera was incubated overnight at 4°C
with GCR (17.5 mU diluted from the stock solution in substrate buffer;
one unit is defined as the amount of enzyme required to hydrolyze 1 µmol of substrate in 1 minute at 37°C). The GCR activity was
determined by subsequently incubating a 1/10 dilution of the mixture
(in substrate buffer) with a 6.6-mmol/L solution of
pNP--glucopyranoside (pNP, Sigma) in substrate buffer for 2 hours at
37°C with shaking. Enzymatic activity was interpolated from a
standard curve (3.5 to 0.3 mU) of GCR. Serum inhibition was expressed
as percentage of activity remaining relative to the control GCR
incubated without serum. Pooled normal human sera, pooled sera from
Gaucher patients with inhibitory activity, and pooled sera from Gaucher
patients without inhibitory activity were included as controls. During
qualification of this assay, it was established that 10% of normal
human sera and pretreatment Gaucher patients' sera inhibited 10% to
20% of enzyme activity. Therefore, levels of up to 20% of inhibition
were considered nonspecific.
Adverse event testing.
In addition to the methods described above, patients who experienced
hypersensitivity symptoms during treatment had their sera further
analyzed for the presence of serum tryptase, C3 products, and
development of IgE to GCR. These assays were performed as described
previously.21
Clinical Response Parameters
Four clinical response parameters: platelet counts, hemoglobin, liver
volume, and spleen volume (expressed as multiple of normal organ
volumes) were examined to identify statistically significant
differences in response between patients with different antibody status.
Statistical Analysis
Test of differences in frequency of category of disease type, genotype,
and dose frequency were performed by Pearson  2 test.
Comparison of response trends were conducted for patients who were
antibody negative versus those who were antibody positive and those who
had inhibitory antibodies. Using a weighted analysis of variance,
groups were compared to determine whether there were differences in
responses achieved over time on enzyme therapy in any of the four
clinical parameters. The group comparisons included antibody positive
versus antibody negative patients, patients with inhibitory antibodies
versus those without inhibitory antibodies, and patients with
inhibitory antibodies versus those without antibodies. Patients
included in these group analyses had intact spleen at the start of
therapy. In addition, evaluation time points were excluded following
periods of interruption in enzyme therapy of greater than 3 months.
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RESULTS |
Seroconversion
A patient was considered to have developed a circulating immune
response to GCR (ie, seroconverted) if both the ELISA and the RIP assay
were positive. GCR-specific circulating antibodies were not detected in
any of the patients before the start of therapy. Of the 1,122 evaluable
patients (Table 1), a total of 142 patients (12.8%) seroconverted while on therapy. These patients' sera all had
above normal range ELISA values, and the presence of GCR-specific antibody was subsequently confirmed by RIP. The time course of seroconversion is shown in Fig 1. Most
patients (109 of 142) seroconverted within 3 to 9 months after
initiation of therapy. The median time to seroconversion was 6 months
with a mean time of 8.2 months. The majority (87.5%) of the patients
seroconverted within the first year of therapy. Seroconversion
occurring more than 12 months after initiation of therapy may actually
have occurred earlier, as serum samples drawn within the first year of
therapy were not always available for analysis. None of the patients in
this study developed antibodies to HSA, an excipient used in the
alglucerase formulation.
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| Fig 1.
Time to seroconversion for Gaucher patients treated with
alglucerase. Sera from patients with at least one sample beyond
initiation of therapy were analyzed by ELISA and RIP. A patient was
considered seroconverted if a positive ELISA result was confirmed by
RIP.
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A statistical analysis was performed to determine whether the disease
type, genotype, or dose frequency had any impact on seroconversion. The
majority of both antibody positive and antibody negative groups
reported type I disease (935 patients). The distribution of disease
types did not differ between those with antibodies and those without
(2, P = .37).
Patients who developed antibodies were as likely to have the N370S
allele as those who did not develop antibodies (2,
P = .71). The same was true for the L444P allele
(2, P = .23). Additionally, N370S homozygoity
was equally prevalent in both the antibody positive and antibody
negative groups (2, P = .28).
Regarding dose frequency, every 2 weeks was the most common initial
dose frequency (81%) among registry patients who were tested for
antibodies. Those who developed antibodies were as likely to be treated
at this frequency as any other (2, P = .60).
Inhibitory Antibodies
The presence of inhibitory antibodies was evaluated by the ability of
patient's serum to interfere with the in vitro enzymatic activity of
GCR using pNP as a substrate. The patient's sera were incubated
overnight with GCR having a known enzymatic activity. The percent
inhibition represents the loss of activity due to antibody binding.
This assay does not discriminate between inhibitory and neutralizing
antibodies, ie, an antibody binding the catalytic site of GCR
and/or an antibody causing steric hindrance versus an antibody
that interferes with the clinical effect of the therapeutic molecule.
Validation of this assay demonstrated that the sera of normal
individuals, as well as pretreatment sera of the Gaucher population,
inhibited GCR enzymatic activity at levels ranging from 0% to 20%.
Only patients who were found to have seroconverted were tested for
potential presence of inhibitory antibodies.
No inhibitory activity was detected in sera of most seroconverted
patients (75%). Sera from an additional 15% of the seroconverted patients inhibited GCR enzymatic activity by <50%. This low level of
inhibition was generally transient in these patients. This finding is
illustrated in Fig 2A. Sera from one
patient representative of the group with low levels of inhibition were
serially diluted (1/10 to 1/80) and analyzed for inhibition of GCR
activity. A small level of inhibition was detected at 13 months after
initiation of therapy. By 15 months, it was below 20% and not
detectable at 18 months. Only 10% of the patients (1.2% of the total
tested population) who seroconverted were found to have antibodies that could inhibit more than 50% of the in vitro activity of the enzyme. Figure 2B illustrates inhibition results for a patient representative of that group. It is significant to note that the presence of inhibitory antibodies, as determined by the in vitro assay, did not
always correlate with a decrease in clinical efficacy. A review of
these individual cases indicated that antibodies may have had an impact
on less than 1% of the treated population. Very few patients required
a specific change in therapy due to the presence of inhibitory
antibodies and therefore developed true neutralizing antibodies. One of
these patients had type III Gaucher disease. Three patients with
neutralizing antibodies have been recently described.22,23
Interestingly, analysis of a recent serum sample of one of the
described patients showed a decrease in reactivity to the molecule and
disappearance of neutralizing antibodies.
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| Fig 2.
Titration of in vitro inhibition of GCR enzymatic
activity by sera of seroconverted Gaucher patients. (A) Representative
patient sera showing <50% in vitro inhibitory activity. Transient
inhibition was detected when pretreatment, 13, 15, and 18 months
posttreatment sera were compared. (B) Patient sera showing >50%
inhibition, comparison to normal human serum (NHS) and different time
points in therapy.
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Clinical Analysis
Clinical data regarding hematologic and organ volume response were
available for review through the Gaucher Registry for a substantial
number of patients who had demonstrated in vitro inhibition of enzyme.
Fifteen patients who exhibited >50% in vitro inhibition were
examined. Three of these patients were receiving immunosuppressive therapy to manage the inhibitory antibodies and were clearly thought by
their physicians to have had either inadequate clinical responses or
deterioration of clinical responses. Eight of the remaining patients
had sufficient data for evaluation (Table
2). For these patients, the mean time to detection of inhibitory
antibodies was 11 months with a range of 3 to 19 months. The mean time
to detection of initial IgG antibodies was 6 months with a range of 3 to 12 months. Three of these eight patients tolerized after 59, 17, and
17 months, respectively. Two of the eight patients showed a
deterioration of clinical response in the parameters available. One of
these patients experienced a decreased response in platelet count.
However, the resulting value was only slightly below normal. Of
important note, this patient's dose was reduced 50% 2 to 3 months
after development of inhibitory antibodies; this dose reduction may
have also played a role in the decreased response. The second patient
who showed a deterioration in response had only platelet counts
available for assessment. This patient's platelet count decreased 31%
to 97,000/mm3. The patient's dose of enzyme was reported
to have been increased after the development of inhibitory antibodies
and the platelet count subsequently improved.
Of the 22 patients who had low levels (<50%) of inhibitory
antibodies detected, there were 12 for whom sufficient data was available to evaluate the impact of these antibodies on clinical response. The mean time to detection of inhibitory antibodies for the
12 evaluable patients was 9 months with a range of 3 to 17 months
(median, 8 months). Nine of these 12 patients tolerized after a mean
time of 24 months on therapy with a range of 7 to 53 months (median, 20 months). Only one of the 12 patients evaluated appeared to have clearly
not maintained responses in the clinical parameters after the
development of inhibitory antibodies. Two additional patients had
variable clinical responses, which are somewhat difficult to interpret.
Four clinical response parameters, two laboratory parameters (percent
change in platelet count [mm3]) and absolute change in
hemoglobin [g/dL]), and two visceral organ parameters (percent change
in liver volume [multiples of normal (MN)] and percent change in
spleen volume [MN]) were examined to identify statistically
significant differences in clinical response between patients with
different antibody status. Registry patients on alglucerase therapy
with an intact spleen at baseline and known antibody status were
included in the analysis. Data was restricted to that collected while
on alglucerase therapy and prior to any interruption in therapy of more
than 3 months. Organ volume multiples of normal refer to normal volume
predicted for body weight,24 approximately 2.5% and 0.2%
of body weight for liver and spleen, respectively.
Two comparisons were performed: antibody negative patients (n = 406)
versus those who were antibody positive (n = 60) and antibody negative
patients (n = 406) versus those antibody positive patients with
inhibitory antibodies (n = 20). Two sample t-test was used to
compare baseline values of clinical response parameters. To compare
response over time in each of the clinical response parameters, the
slope of change from baseline for each patient was determined by simple
linear regression. Both percent and absolute changes were examined. The
differences between the slopes of groups differing in antibody status
were assessed by weighted analysis of variance (ANOVA), weighted by the
number of observations within patient contributing to the slope.
There were no statistically significant differences in any of the four
clinical response parameters at baseline within either the two
groupings (two-sample t-test, all P > .05).
Comparisons of clinical responses of patients with antibodies (n = 60)
to patients without antibodies (n = 406) showed no significant
differences in platelet count response (% change mm3;
P = .13), hemoglobin response (absolute change g/dL;
P = .58), liver volume (% change MN; P = .48), spleen volume (% change MN; P = .32). Clinical responses
over time for patients with inhibitory antibodies (n = 20) were not
different compared with those patients without antibodies (n = 406) in
any of the four parameters: platelet count response (% change
MN; P = .22), hemoglobin response (absolute change
g/dL; P = .78), liver volume (% change MN; P = .75) spleen volume (% change MN; P = .59). No meaningful
differences were observed in mean clinical responses between antibody
positive and antibody negative patients when examined as groups.
Patients with inhibitory antibodies achieved only slightly lesser
responses at 12 and/or 18 months that were not clinically
different from patients with noninhibitory antibodies or
antibody-negative patients. The slight differences were not observed at
later time intervals when the number of patients with observations was
sufficient for conclusions, ie, greater than three. Using a weighted
ANOVA, no significant differences were observed between responses in
any of the group comparisons (P > .05).
In summary, the presence of inhibitory antibodies in Gaucher patients
treated with alglucerase does not necessarily denote reduced
effectiveness of therapy. Rather inhibition of clinical effectiveness
was restricted to a small fraction of patients, particularly those with
inhibition of >50% in the assay described.
Immunologically Related Adverse Events
Seventy-four patients (about 5% of the total treated population)
reported symptoms suggestive of immediate hypersensitivity during the
time of infusion or shortly thereafter (Table 1). The majority of these
patients had mild cutaneous symptoms, which included urticeria,
flushing, and pruritis. All patients recovered quickly and without
sequalea. Patients were able to continue treatment and their symptoms
were managed by slowing the rate of infusion as well as administration
of antihistamines. Patients were tested for the presence of IgG or IgE
specific to GCR. While 34 of the 74 patients who experienced symptoms
of hypersensitivity developed IgG-specific antibodies to GCR, only one
patient was found to have IgE antibodies (0.06% of the total treated
population). Most of the adverse events occurred during the first year
of therapy (Fig 3). A difference was
observed between the time of onset of an adverse event and patient
antibody status. Patients who seroconverted generally experienced
adverse events within the first 14 months of therapy, with a median
time of 7 months and a mean of 6.6 months (Fig 3A). Nonseroconverted
patients experienced adverse events at different times ranging from the
day of first infusion up to 48 months poststart of therapy with a
median time of 6 months and a mean of 10.9 months (Fig 3B).
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| Fig 3.
Time to adverse event for Gaucher patients treated with
alglucerase in seroconverted patients (A) and in nonseroconverted
patients (B).
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Humoral Tolerization
After seroconversion, many patients exhibited an increase in the levels
of antibodies to GCR, as demonstrated by an increase in ELISA
absorbency values and a sustained or increased RIP signal. However,
with ongoing treatment, a gradual decrease in the assay signals was
observed. Figure 4 is a composite of six
representative patients on therapy. This decrease in reactivity was
initially manifested by a reduction in intensity in the ELISA, followed by a subsequent decrease in intensity of the RIP signal. Over time, the
patients' serologic reactivity to GCR was no longer detectable and the
patients were therefore considered tolerized. Generally, patients were
considered tolerized if after seroconversion, both the ELISA and the
RIP assay were negative for two consecutive samples.
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| Fig 4.
ELISA reactivity and tolerization to GCR in six
representative seroconverted patients. Sera from individual patients
were analyzed during their course of therapy by ELISA and RIP. The
arrows indicate the time of seroconversion (RIP+) and the
time of tolerization (RIP-). A patient was considered
seroconverted if a positive ELISA result was confirmed by RIP and
tolerized if after seroconversion, both ELISA and RIP of subsequent
sera samples were negative.
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Humoral tolerization was evaluated in patients treated for at least 1 year (Table 3). Of the 122 seroconverted
patients followed for at least 1 year from the start of therapy, 82 were found to be subsequently negative in both the ELISA and the RIP
and were therefore considered tolerized. The median time to
tolerization was 24 months, while the mean time was 28 months from
start of therapy. Because not all seroconverted patients had samples
tested for extended periods beyond the initiation of therapy,
seroconverted patients with samples beyond 30 months of therapy (past
the mean time of tolerization) were analyzed. Of the 142 seroconverted patients, 69 had samples beyond 30 months of therapy, and 64 of those
patients (93%) were tolerized to the therapeutic molecule. Five
patients were still showing positive ELISA and RIP reactivity. Two
patients receiving immunochemotherapy were excluded from this analysis
because this treatment inherently affected their ability to produce
antibodies.
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DISCUSSION |
The use of alglucerase enzyme therapy for Gaucher disease has proven to
be efficacious in treating this genetic disorder. Visceromegaly or
hematologic abnormalities were arrested in most patients with
concomitant improvement in quality of life. Bone involvement, although
less understood, also can be improved with enzyme
therapy.25 An ongoing concern with any protein therapeutic is the immunologic response to the treatment molecule. We previously reported in a study of 262 patients21 that approximately
13% of the Gaucher patient population developed an antibody response to GCR, which became diminished with time in some patients. This report
describes our immunologic findings from 1,122 treated patients, confirming our previous results that only a relatively small percentage of patients (12.8%) produce antibodies to the therapeutic molecule. This percentage appears to be lower than what has been reported in the
literature for other therapeutic molecules. Interferon alpha given
subcutaneously in chronic hepatitis patients resulted in 50% of the
patients developing antibodies,20 while given intramuscularly to metastatic renal carcinoma patients, it resulted in
63% of the population developing antibodies.13 The general incidence of antibody production for insulin given subcutaneously was
reported as 20%.9 However, a study administering insulin intraperitoneally indicated that 40% of the patients had antibodies in
serum within 1 year of treatment,26 emphasizing the
importance of route of administration. Experience with growth hormone
has been varied primarily due to differences in the source and purity of the molecule. However, an incidence of up to 60% of seroconversion has been documented.27 Recently, it has been reported that
administration of a genetically engineered humanized anti-tumor
necrosis factor (TNF) antibody gave rise to specific antibodies in 30%
of the patients.28
The lower incidence of antibody production to GCR may reflect several
parameters related to immunogenicity. Of significance is the fact that
the enzyme is generally not absent in Gaucher patients, merely present
in a nonactive mutated form. Second, many therapies are given
subcutaneously and thereby come in contact with antigen-presenting
cells. Alglucerase is given intravenously over a period of hours, and
the enzyme gets readily distributed to target tissues. Last, because
the molecule is targeted to the mannose receptor on macrophages, these
cells do not "see" the molecule in the context of major
histocompatability complex (MHC) presentation and may more
readily recognize GCR as a self-antigen.
As more protein therapeutics are becoming available, reports in the
literature citing the development of antibodies that can neutralize the
efficacy of the protein are more common. Some of the first reports of
inhibitory antibodies were for Factor VIII replacement therapy. The
prevalence has been reported at 14%, with the most severely affected
patients developing predominantly IgG4 subclass.12 To
overcome those inhibitory antibodies, immunosuppressive therapy,
combined with a high dose of factor VIII, has been the treatment of
choice. More recently, it has been reported that recombinant
staphylokinase given to patients with myocardial infarctions can
rapidly induce neutralizing antibodies, even within 2 weeks of
treatment.29 The development of neutralizing antibodies
have also been reported in cytokine therapy. Injections of IL-3, given subcutaneously or intravenously, resulted in 50% of the animals developing neutralizing antibodies.16 Similar results have
been reported for IL-2 in humans, although it has been suggested that the use of the natural molecule was not inhibited by the antibodies raised to the recombinant molecule.15 Extensive studies
performed with subcutaneous injections of interferon alpha or beta,
reported that 30% to 50% of the patients develop neutralizing
antibodies.13,18,20
The development of neutralizing antibodies to GCR has been rare, in
contrast, most of the antibodies elicited to GCR were not inhibitory.
Only a small percentage of the seroconverted patients had transient low
inhibitory antibodies, the nature of which is not clear. Of the
patients who did have higher levels of in vitro inhibition, the
majority did not have this inhibitory activity correlate with
deleterious effects on clinical efficacy. These patients may have
developed a polyclonal response that clearly inhibited the molecule's
in vitro enzymatic activity, yet were clinically irrelevant. Because
the enzyme must be active within the lysosomes of macrophages, it may
be that serum binding is not relevant. Antibodies may, in fact, enhance
the uptake of the enzyme via Fc receptors. Very few patients had their
clinical response deleteriously effected by the development of
antibodies to GCR and clearly developed neutralizing antibodies. One of
these patients stopped therapy, while the other three
patients22,23 were treated with immunosuppressive regimens,
but continued receiving enzyme therapy due to deterioration of their
condition. This very low incidence of clinically relevant
neutralization stands noticeably different from the experience with
Factor VIII.
An interesting trend described in our previous study was the
development of humoral tolerance with continued treatment. Our current
study confirmed this finding, that with time, the antibody response to
GCR is not sustained. The serologic response became abrogated despite
the fact these patients continue to be infused with enzyme at the same
dose and frequency. These findings suggest that patients developed a
state of B-cell unresponsiveness toward the therapeutic molecule. This
observation has also been reported with other therapies. Immunologic
tolerization was reported for interferon alpha treatment in hairy cell
leukemia.30 After a median of 14.5 months, nonneutralizing
antibody patients became seronegative. Within 10 months of the
development of neutralizing antibodies, 50% of those patients became
nonneutralizing and another 30% became seronegative.30
With alglucerase treatment, 80% of the patients developed humoral
tolerance to the molecule within 2 years of treatment and 93% within
30 months. In addition, most of the antibodies detected were not
neutralizing, differentiating the reactivity to GCR from that reported
generally for other protein therapies.
In conclusion, this evaluation demonstrated that alglucerase therapy
does not cause major immunological consequences for Gaucher patients.
The molecule is not immunogenic in the majority of the patients. In a
small percentage (12.8%) of the treated population, patients
seroconvert, but became tolerized with time. In general, the presence
of antibodies does not affect the efficacy of the therapy. Of the 74 patients experiencing putative immune-related adverse events, only 34 were found to have developed IgG specific to GCR, and only one patient
has developed IgE antibodies to GCR. This incidence was considerably
lower than what has been reported for many other protein therapies. The
mechanism by which nonseroconverted patients manifest immune-mediated
symptoms is unclear, but could be related to release of other factors
such as cytokines. This study supports the rationale that continued
treatment of patients who have developed antibodies is warranted.
| |
ACKNOWLEDGMENT |
The authors wish to thank N. Moore and M. Hesselton for their excellent
technical assistance and J. Angell for statistical analysis. We are
also grateful to the participating ICGG registry patients and
physicians as well as the staff for patient information required to
analyze this retrospective study and to J. McPherson and R. Moscicki
for critically reviewing this manuscript.
| |
FOOTNOTES |
Submitted December 31, 1997; accepted November 9, 1998.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Mireille Rosenberg, PhD, Genzyme Corp, 1 Mountain Rd, Framingham MA 01701-9322.
| |
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ABSTRACT
INTRODUCTION