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Enzyme Therapy in Gaucher Disease Type 1: Effect of Neutralizing
Antibodies to Acid b -Glucosidase
By Elvira Ponce, Jay Moskovitz, and Gregory Grabowski
Gaucher disease type 1, a non-neuronopathic lysosomal
storage disease, is caused by mutations at the acid b-glucosidase locus. Periodic infusions of macrophage-targeted acid
b-glucosidase reverse hepatosplenomegaly, hematologic,
and bony findings in many patients. Two patients receiving
enzyme therapy developed neutralizing antibodies to acid
b-glucosidase that were associated with a lack of improvement or progressive disease. After initial improvement, case
1 had no additional response to 2 years of high-dose (50 U/
kg every 2 weeks) enzyme therapy. Similarly, case 2 initially
showed a favorable response to enzyme therapy that plateaued after 1 year of treatment. Both patients developed
minor allergic reactions and antibodies to acid b-glucosidase
within the first 6 months of treatment. Enzyme therapy was
discontinued in case 1, with resultant disease progression
and need for splenectomy. An immunosuppression/tolerization protocol was initiated in case 2 because of disease progression and stable neutralizing antibody titers. The IgG
neutralizing antibodies rapidly and completely inactivated
the wild-type, but not the N370S, acid b-glucosidase in vitro.
Antibodies to human serum albumin and chorionic gonadotropin also developed. The finding of neutralizing antibodies
to acid b-glucosidase during enzyme therapy for Gaucher
disease has significant implications for monitoring the therapeutic responses and for potential alternative future therapies for Gaucher disease.
q 1997 by The American Society of Hematology.
G
human serum albumin (HSA) and 4-methylumbelliferyl-b-D-glucopyranoside (4MU-Glc; Genzyme Corp, Boston, MA); human chorionic gonadotropin (HCG; National Hormone and Pituitary Program,
National Institutes of Health, Bethesda, MD); 4-methylumbelliferone, Triton X-100, bovine serum albumin (BSA), and Lowry reagents (Sigma Chemical Co, St Louis, MO); SF 900 culture media,
fetal calf serum (FBS), and nonimmune goat serum (GIBCO, Grand
Island, NY); immobilon polyvinyl fluoride (PVDF) transfer membranes (Millipore, Bedford, MA); alkaline phosphatase (AP)-conjugated goat-antihuman IgG (Calbiochem, La Jolla, CA); AP-substrate
reagents (BioRad, Hercules, CA).
AUCHER DISEASE type 1 is a common lysosomal
storage disease that results from numerous mutations
at the acid b-glucosidase locus.1,2 The deficiency of acid
b-glucosidase activity is present in all cells but there is a
preferential accumulation of its major natural substrate, glucosylceramide, within macrophages of the liver, spleen, and
bone marrow (BM).1 In addition to causing hepatosplenomegaly and pancytopenia, pathologic involvement of bones
can include osteoporosis, pathologic fractures, and acute
bony pain.1,2 Periodic infusions of macrophage-targeted acid
b-glucosidase lead to the reversal of hepatic, splenic, and
hematologic abnormalities3-7 and has positive effects on bony
disease.8,9
At least 1,200 Gaucher disease type 1 patients have received enzyme therapy with resultant improvement in their
disease signs and symptoms. Of these patients, about 15%
develop antibodies to the administered acid b-glucosidase,4,5
and approximately half of these patients (É7% of total patients) have allergic manifestations related to these antibodies.5 These reactions include hives and pruritus.5,10 In general,
the allergic manifestations are managed with antihistamines,
and only rarely do they necessitate discontinuation of therapy. In addition, substantial negative therapeutic effects have
not been reported in antibody-positive patients.10 These anti–
acid b-glucosidase antibodies develop in patients with
Gaucher disease despite the presence of residual mutant enzyme in all their cells.1 The specificity and origin of these
anti–acid b-glucosidase antibodies have not been evaluated
in detail.
In this communication, we report the development of neutralizing antibody to acid b-glucosidase during the course
of enzyme therapy in two patients with Gaucher disease type
1. One patient showed a lack of response to enzyme therapy,
whereas the second showed progressing Gaucher disease
signs and symptoms concordant with the development of
the neutralizing antibody. The development of neutralizing
antibodies to acid b-glucosidase has significant implications
for ongoing therapeutic regimens for the treatment of
Gaucher disease type 1.
MATERIALS AND METHODS
Materials
The following were from commercial sources: Ceredase (alglucerase for injection; 80 U/mL), Cerezyme (imiglucerase for injection),
Methods
Acid b-glucosidase activity. For consistency with standard medical practice, the amount of enzyme activity administered to the
patients will be expressed in the units (U; mmol/min), as determined
by Genzyme Corp using p-nitrophenyl b-D glucoside. The exact
assay conditions have not been specified. The assays presented here
use 4 methyl-umbelliferyl-b-D-glucoside as the substrate. The activity is expressed as mU.
Acid b-glucosidase neutralizing antibody assay. Serum was obtained by incubation of whole blood for 1 hour (47C), and then
centrifugation for 15 minutes at 4,000 rpm. The serum was removed
and stored (0207C). Before assay, the pH was adjusted to 5.5 by
addition of 1 mol/L HEPES (pH 5.5, 10 mL) to 50 mL of serum.
For titer determination, the serum was buffered with 0.1 mol/L
HEPES, pH 5.5. Ceredase was diluted 1:7,000 with 0.04 mol/L
citrate/phosphate, pH 5.5, containing 1% HSA. For assay, 50 mL of
diluted Ceredase wase preincubated for 30 minutes (377C) with vary-
From the Division of Human Genetics, Children’s Hospital Research Foundation, Cincinnati, OH.
Submitted November 12, 1996; accepted February 27, 1997.
Supported by grants from the National Institutes of Health (DK
36729) and the Lucille P. Markey Charitable Trust, and by the
Children’s Hospital Research Foundation.
Address reprint requests to Gregory Grabowski, MD, Director,
Division of Human Genetics, Children’s Hospital Research Foundation, 3333 Burnet Ave, Cincinnati, OH 45229-3039
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.
q 1997 by The American Society of Hematology.
0006-4971/97/9901-0005$3.00/0
Blood, Vol 90, No 1 (July 1), 1997: pp 43-48
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PONCE, MOSKOVITZ, AND GRABOWSKI
ing aliquots of serum diluted in 0.1 mol/L HEPES, pH 5.5 (final
volume Å 75 mL). Acid b-glucosidase activity was determined after
an additional incubation of the preincubation mixture for 30 minutes
(377C) with the substrate. The final reaction mixture included 4
mmol/L 4MU-Glc, 1% Triton X-100, 0.04 mol/L citrate/phosphate,
pH 5.5, in a total volume of 200 mL. The reaction was stopped with
2.3 mL of 0.1 mol/L ethylenediamine, pH 11.0. Substrate cleavage
was quantified fluorometrically.11 Remaining activity is expressed
as the percent of acid b-glucosidase activity in the absence of serum.
Sera from antibody-positive and -negative Gaucher disease patients,
and normal individuals were used as controls.
Gel electrophoresis and immunoblots. For electrophoresis, samples were mixed 4:1 with 51 sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE) loading buffer (12.5% SDS, 25%
b-mercaptoethanol, 0.05% bromophenol blue, 50 mmol/L Tris HCl
pH 8.0, 5 mmol/L EDTA pH 8.0, and 10% glycerol), and heated
for 3 minutes (957C). Separation of the proteins in 12.5% or 20%
SDS-PAGE gels and electroblotting onto PVDF membranes was
performed in the Phast-System according to the manufacturer’s instructions (Pharmacia Biotech, Inc, Piscataway, NJ). After separation, membranes were briefly washed in Tris-buffered saline (TBS;
50 mmol/L Tris-HCl, pH 7.5, 200 mmol/L NaCl) and dried until
used. The day of the assay, membranes were prewet in methanol
containing solutions and sequentially treated as follows: (1) 30minute incubation with TBS containing 3% BSA and 3% nonimmune goat serum (TBS blocking solution); (2) 1-hour incubation
with patient or control serum in TBS blocking solution (1:1,500
dilution); (3) three (10-minute) washes with T-TBS (0.05% Tween
20 in TBS); (4) 45-minute incubation with goat-antihuman-AP conjugated antibody (1:1,500 dilution); (5) two (10-minute) washes in
T-TBS, one rinse with TBS; and (6) BioRad AP-developer solution
was added according to manufacturer’s instructions, and developed
for 20 minutes. All procedures were done at room temperature.
Lymphocyte acid b-glucosidase. Mutant N370S and wild-type
acid b-glucosidase were from immortalized or freshly isolated lymphocytes from Gaucher disease patients or normal individuals, respectively. The patients were shown to have one or more N370S
alleles12; case 1 was homozygous for the N370S allele and case 2
had an N370S/ ‘‘unknown’’ genotype. The soluble lymphoid extracts
were obtained by incubation of lymphocyte pellets with 5% NP40
on ice for 10 minutes, and centrifugation at 12,000g for 15 minutes
(47C). Analyses were done by SDS-PAGE.
Recombinant wild-type and mutant acid b-glucosidase. Normal
and N370S mutant acid b-glucosidase cDNAs were subcloned into
the EcoRI(5*)/Xba I(3*) polylinker sites of the plasmid pAC610 and
were used to produce high-titer recombinant baculovirus in Sf9 insect
cells by homologous recombination with wild-type baculovirus.11
Heterologous expression of human recombinant acid b-glucosidase
in protein-free media (Sf900) was obtained after infection of 2 1
107 Sf9 cells with enough virus inoculum to give 1 to 5 multiplicity
of infection (MOI). For the analysis, cells and media containing
the recombinant protein were obtained 3 days postinfection, and
separated by centrifugation for 10 minutes at 800 rpm. After three
washes (800 rpm; 10 minutes) with saline, the pelleted cells were
stored (0207C) until used. Both the wild-type and N370S recombinant proteins have similar biochemical and immunological properties
to their natural counterparts.11
Case Reports
Case 1 is a 22-year-old Jewish man who was diagnosed with
Gaucher disease due to hepatosplenomegaly at 10 years of age. The
diagnosis was confirmed by demonstration of decreased acid bglucosidase activity in lymphocytes and the identification of homozygosity for the N370S allele. His younger brother, age 20, also has
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Table 1. Effect of Enzyme Therapy in Case 2
Enzyme
Therapy
(mo)
Liver Volume
Splenic Volume
(mL/fold normal)
30 U/kg every 2 wk
0
1,749/1.21
7
1,655/1.01
13
1,623/1.01
21
1,758/1.21
Hemoglobin
(g%)
Platelets
(mL)
1,620/141
1,056/81
1,004/71
1,230/91
11.3
12.6
13.6
12.7
75,000
92,000
99,000
75,000
1,372/101
905/6.51
12.2
12.7
79,000
103,000
Stop enzyme therapy
25-29
60 U/kg every wk
29
1,590/1.01
32
1,766/1.21
Gaucher disease type 1. Case 1 and his brother have Hashimoto’s
thyroiditis.
At 18.5 years of age, case 1 began enzyme therapy (50 U/kg
every 2 weeks), because of massive splenomegaly (É13 fold normal)
and thrombocytopenia (21,000/mL). During the first four infusions
of Ceredase, he experienced sweatiness and chills during and several
hours after alglucerase infusions. These symptoms subsided spontaneously. Anti–acid b-glucosidase antibody (IgG) was detected
within the first 7 months. He had experienced no improvement of
his Gaucher disease over 26 months of enzyme therapy. At that time
he came under our care and his serum was found to inhibit acid bglucosidase activity. Because of the lack of effectiveness of enzyme
therapy and the finding of a neutralizing antibody, this treatment
was discontinued and the patient was followed up. Over the next
24 months, progressive thrombocytopenia to 17,000/mL necessitated
splenectomy. To date, his affected brother, treated with enzyme
therapy for 4 years, remains antibody negative.
Case 2 is a 13-year-old Hispanic/German girl who was diagnosed
at age 11 years with Gaucher disease due to asymptomatic splenomegaly. This diagnosis was confirmed by the deficient activity of
acid b-glucosidase in lymphocytes and the presence of one N370S
allele. The second allele was not identified by screening for the three
other common mutations (84GG, L444P, IVS2/).13-15 She began
enzyme therapy (30 U/kg every 2 weeks) due to severe fatigue,
progressive thrombocytopenia, increasing bone pain (particularly in
the knees), and the progressive hepatosplenomegaly. During the first
6 months of enzyme therapy she showed substantial improvement,
particularly in her energy level, diminution in bone pain and partial
regression of splenomegaly (Table 1). The pretreatment splenic volume was 1,620 mL (É14 1 predicted normal) and the liver volume
was 1,749 mL (1.25 1 predicted normal). From 6 to 12 months of
enzyme therapy, the splenic volume decreased by 35% to 38%,
respectively, from initial and the liver volume remained essentially
normal. The improvement in energy level was quite dramatic with
decreasing sleep requirements from 12 hours to 8 hours per day and
participation in extracurricular school activities for the first time in
several years.
From 6 to 12 months after initiation of enzyme therapy, increasing
fatigue, knee and ankle pain, and fullness in the left upper quadrant
of the abdomen was noted by the patient. In addition, she complained
of face flushing and sweatiness during the alglucerase infusions.
Slowing the infusion rate provided nearly complete relief of these
symptoms. Her mother also reported substantially decreased energy
levels. In the next 12 months, splenomegaly increased from 1,004
mL (É8 1 normal) to 1,230 mL (É9 1 normal), or about a 17%
increase. Concomitant with these changes was a return to pretreatment levels of fatigue, falling asleep during the day, and knee pain.
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ANTIBODIES TO ACID b-GLUCOSIDASE
45
Fig 1. Inhibition of acid b-glucosidase activity by sera from cases
1 (Ì) and 2 (●). Controls sera were from normal individuals ("), and
antibody positive (j) and antibody negative (m) Gaucher disease
patients. Acid b-glucosidase, as alglucerase, was incubated with
varying amounts of serum at pH 5.5 in the absence of detergents.
The decrease in enzyme activity at greater than 15 mL of nonimmune
serum are due to denaturation.
A serum sample obtained before initiation of therapy was negative
for acid b-glucosidase antibodies. Anti–acid b-glucosidase seroconversion and neutralizing antibodies were detected in a serum
sample obtained at 3 months of enzyme therapy.
After 2 years, enzyme therapy was discontinued for 4 months
with no major increase in hepatosplenomegaly. During that period,
the fatigue continued worsening. Because of the continuing presence
of inhibitory antibody, progressively increasing fatigue, and bony
complaints, an immunosuppression/acid b-glucosidase tolerization
schedule was instituted. This schedule included Ceredase (60 U/kg)
on day 1 followed by 15 mg Cytoxan/kg body weight intravenously
on days 2 and 3. Cytoxan (50 mg, orally) was administered once
per day from days 4 to 16. Because of severe neutropenia (absolute
neutrophil count [ANC] õ 500), oral Cytoxan was discontinued on
day 16. The neutropenia resolved over the next month. Transient
alopecia developed over the next 2 months and began to resolve
after 4 months. Ceredase (60 U/kg) was administered once per week
throughout this period.
While receiving Ceredase (60 U/kg) once per week, no subjective
improvement was observed after 3 months, and the fatigue and bone
pain continued. Some symptoms could have been related to the
emotional response to the alopecia and rejection by some of her
classmates. However, the spleen decreased in volume by 34% and
her platelets increased by 30%. By 6 months of enzyme therapy (60
U/kg every week), she had less fatigue and was participating is
school activities.
During the first 3 months of Ceredase (60 U/kg every week)
enzyme therapy, she began experiencing severe headaches, flushing,
chills, chest pain, and occasional shortness of breath associated with
the enzyme infusions. She was switched to the recombinant enzyme
preparation, Cerezyme (60 U/kg every week), 5 months later with
resolution of these symptoms and signs.
RESULTS
Cases 1 and 2 had high-titer antibodies that developed
during enzyme therapy and inhibited acid b-glucosidase ac-
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tivity in vitro. To ensure that the neutralizing activity of the
antibody was being tested, several controls were included:
(1) sera from unaffected normal individuals, (2) pre-enzyme
therapy sera from cases 1 and 2, and (3) sera from other
antibody positive Gaucher patients. Since denaturation of
the enzyme occurs at alkaline pH, stability of the added acid
b-glucosidase in Ceredase was optimized by adjusting the
serum pH to 5.5 at a final 1% Triton X-100 concentration.
The initial inhibitory potencies of the serum were similar in
cases 1 and 2 (Fig 1). Under optimal conditions, about 0.5
mL of antisera from either case produced a 50% inhibition
of 100 mU acid b-glucosidase activity. For case 2, this titer
remained unchanged throughout the time of this study (see
below). However, case 1’s serum showed a progressively
diminishing inhibitory potency with time after discontinuing
enzyme therapy. After 1 year off enzyme therapy, about 12.5
mL of serum was required for 50% inhibition of 100 mU of
acid b-glucosidase (Fig 2), ie, about a 25-fold decrease in
inhibitory antibody titer. An 18% to 25% decrease in activity
was obtained at higher amounts of serum from other Gaucher
disease patients, either anti–acid b-glucosidase antibody
negative or positive, or normal individuals (Fig 1). To ensure
that the Igs related to Hashimoto’s thyroiditis did not have
a specific effect on the enzyme, serum from case 1’s affected
brother (anti–acid b-glucosidase antibody negative) was
shown not to inhibit acid b-glucosidase (Fig 2).
Because both patients have N370S alleles, we assessed
the inhibitory effects of their sera on the N370S enzyme
obtained by heterologous expression in the baculovirus system. These results showed that the N370S enzyme was
poorly inhibited (maximum of 30%) by the sera from case
2 (Fig 3). The losses of activity were similar to those ob-
Fig 2. Decreasing inhibitory titres in serum from case 1 after stopping enzyme therapy. Normal control (l), case 1’s brother with
Gaucher disease and Hashimoto’s thyroiditis (j), and case 1 while
on enzyme therapy (●) and after stopping: 1 month (Ì), 8 months
("), and 12 mos (.).
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46
PONCE, MOSKOVITZ, AND GRABOWSKI
Fig 3. Inhibition of acid b-glucosidase activity of recombinant normal (Ì) and N370S (") enzymes expressed in the baculovirus system
and alglucerase (●). Similar inhibitory profiles were obtained with
the recombinant and placental acid b-glucosidase when incubated
with case 2 serum. The recombinant N370S acid b-glucosidase had
a similar activity profile to the normal acid b-glucosidase with nonimmune sera (Fig 1).
tained with control sera and were probably caused by denaturation or other nonspecific effects. In comparison, Western
blots (Fig 4) using inhibitory sera from both patients could
detect the wild-type and N370S human enzymes expressed
in the baculovirus system (Fig 4A). Also, mutant acid bglucosidase was detected in lymphocytes from case 2
(N370S/?) or other N370S Gaucher disease patients and heterozygotes (Fig 4B).
Inhibitory potency of the sera from case 2 was determined
every 3 months during the immunosuppression/acid b-glucosidase tolerization protocol (months 29 to 35, Fig 5). No
change in in vitro inhibitory potency of the sera was detectible during this period. In comparison, simultaneous evaluation of the clinical symptoms and objective measures showed
improvement (Table 1). However, the adverse effects (headaches, chills, etc) increased when on 60 U/kg every week.
Some of these adverse effects may relate to the presence of
anti-HSA antibodies present in case 2’s serum (Fig 6). This
is supported by the cessation of these adverse effects when
Cerezyme was used; this recombinant enzyme that does not
contain HSA.
Fig 4. Western blot analyses of wild-type and Gaucher disease
acid b-glucosidase from recombinant (A) and natural (B) sources,
using case 2 sera. (A) Immunoreactivity of recombinant wild-type
and N370S enzymes expressed in the baculovirus system. Similar
forms of both recombinant enzymes were detected with serum from
case 2. Acid b-glucosidase (lane 1), wild-type enzyme (lanes 2 and
4), N370S enzyme (lanes 3 and 5). (B) Wild-type enzyme from an
unaffected individual with normal levels of enzyme activity (lane 1),
a Gaucher disease patient who was hemizygous for the N370S allele
and a gene deletion (lane 2), the father of the patient in lane 2 who
is hemizygous for the normal allele (lane 3), and case 2 who was
heterozygous for N370S (lane 4). The three bands present in lanes 1,
3, and 4 correspond to different posttranslational glycosylation
stages of acid b-glucosidase.
DISCUSSION
To date, the factors that influence the variability in response to enzyme therapy of patients with Gaucher disease
type 1 have been poorly characterized. Although antibodies
are present in 15% of patients receiving either natural or
recombinant enzyme products, none, except those reported
here, have had a substantial impact on the effectiveness of
enzyme therapy. In both of our patients the development
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Fig 5. Inhibitory titres of case 2 sera over 24 months. Pretreatment
(●) and seven sera samples (other symbols) from a period of 24
months with inhibitory capacity during enzyme therapy and after
discontinuation (m). No differences in titer were appreciated.
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ANTIBODIES TO ACID b-GLUCOSIDASE
47
Fig 6. Western blot analysis of proteins present in Ceredase (lanes
1 and 2) and Cerezyme (lanes 3 and 4) using case 2 antiserum (1:1,500
dilution). Acid b-glucosidase in Ceredase [0.4 mg (lane 1) and 0.8 mg
(lane 2)] and in Cerezyme [5 mg (lane 3) and 10 mg (lane 4). HSA in
lanes 5 (1 mg) and 6 (2 mg).
of neutralizing antibodies blunted the expected therapeutic
progress by enzyme therapy even though both patients received substantial initial doses of enzyme (50 or 30 U/kg
every 2 weeks). The presence of antibody positivity correlated with the development of allergic-like reactions that
seem likely to be antibody mediated. However, at least in
case 2, these allergic-like reactions were potentially more
related to antibodies to HSA in Ceredase than to those
against acid b-glucosidase. This conclusion is based on the
absence of these symptoms when the recombinant enzyme,
Cerezyme, was used, since this preparation does not contain
HSA. However, the serum from case 2 did react with and
inhibit the acid b-glucosidase, imiglucerase, in Cerezyme.
Case 1 had decreasing inhibitory antibody titers during the
first 8 months after discontinuation of enzyme therapy. This
titer diminished an additional 50% by 12 months off therapy.
This decrease in antibody titer was accompanied by increasing thrombocytopenia and by increasing splenomegaly by
physical examination. Thus, his disease progressed despite
the loss of neutralizing antibody. This clinical finding suggests that enzyme therapy had been controlling the rate of
his disease progression, even in the presence of neutralizing
antibodies.
In comparison, case 2 initially had positive responses to
enzyme therapy and then her disease progressively worsened
concordant with the development of neutralizing antibodies.
Her improvement while on 60 U/kg every week likely relates
to a large amount of free enzyme being present after all
antibody in her serum was bound to enzyme. Although antiidiotypic antibodies16,17 could have been induced by the increased enzyme dosage, these were not detected as a decreasing antibody titer by our in vitro inhibitory assay.
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The development of neutralizing antibodies in these two
patients highlights the need for continuing close monitoring
of patients during the initial phases of therapy since most
patients become antibody positive during their first 6 to 9
months.5,10 Although these two patients had antibodies that
inhibited acid b-glucosidase activity, these antibodies differed somewhat in their response to enzyme therapy withdrawal.
Although the sera from cases 1 and 2 could completely
neutralize acid b-glucosidase activity, other antibodies, with
partial or low-level inhibitory capacity, may exist in other
Gaucher disease patients. These could contribute, in part, to
the less than expected responses to enzyme therapy. In our
group of seven antibody-positive patients, none, other than
cases 1 and 2, had inhibitory antiserum. Clearly, screening
for inhibitory antibodies should be a routine component of
management. The assays for effects of antibodies on acid
b-glucosidase activity are not straightforward, particularly
because these are done in the presence of detergents and
added proteins, ie, serum and HSA, that have significant
effects on activity.18,19 Based on the present cases we would
recommend that all antibody-positive patients should have
their antibodies tested for potential inhibitory effects. This
test is available on a regular basis (Susan Richards, personal
commmunication, February 1997, Genzyme Corp). Particular attention should be directed to low-level and/or partially
neutralizing antibodies. Finding of such antibodies may influence dosage reductions and/or schedules due to the serum’s ability to bind and remove or inhibit enzyme in vivo.
The latter was suggested by the good therapeutic response
of case 2 to a fourfold increase in enzyme dose that was
unaccompanied by decreasing titers of antibody. It is also
clear from the Western blot and neutralizing studies that
several different populations of antibodies are present in the
sera from cases 1 and 2. Some of these inhibit the wild type,
but do not have this effect on the N370S enzymes, whereas
others give immunoreactivity on Western blots.
The reactivity of the antisera from cases 1 and 2 toward
the N370S mutant protein raises additional issues. The antibodies to acid b-glucosidase in cases 1 and 2 sera probably
contain numerous specificities to various epitopes on the
enzyme. The lack of inhibition of N370S enzyme activity
by the antiserum suggests an epitope specific antibody for
the region around residue N370. However, the reactivity of
the antiserum from case 2 with the normal and N370S enzymes on Western blots indicates the broader interaction of
the antisera than just the N370 epitope. Of potential concern
is an acceleration of the disease that might follow termination of therapy due to the persistence of the reactive neutralizing antibodies within the serum. For this to occur, free
antibody would need to gain access to intracellular, endogenous acid b-glucosidase. Theoretically, such interaction
could worsen their disease. We did not observe this in either
case. Of equal importance is the potential need to terminate
therapy in some of these patients because of its lack of effect
and/or its great expense, ie, for a 70-kg individual, a 60-U/
kg every week treatment would represent an expense of
É$850,000 per year. This is similar to the cost of factor VIII
used to treat seroconverted hemophiliacs, ie, for a 70-kg
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48
PONCE, MOSKOVITZ, AND GRABOWSKI
patient with neutralizing antibodies to factor VIII, the cost
would range from $210,000 to $1,540,000 per year, depending on the treatment/desensitization protocol. With time
off therapy neutralizing antibodies could disappear, as in
case 1, until exposure to the enzyme again elicits an anamnestic reaction. Other patients’ antibodies will develop and
these could be reactive to their respective variants of acid
b-glucosidase. In all of these patients such antibodies also
could potentially interfere with future therapeutic strategies
by a BM transplantation and/or gene therapy for the treatment of their disease.
The lack of substantial decreases in titers of neutralizing
antibody in case 2 during immunosuppression and high-dose
tolerization indicate continuing and expensive difficulties for
treatment of this patient. Thus, the inability to eliminate the
reactive antibody and reinstitute effectiveness of enzyme
therapy indicates the need to develop improved supportive
therapy for such patients and/or the selective intermittent use
of enzyme therapy for certain types of progressive involvement. Although the development of neutralizing antibodies
is rare, it has had a substantial impact on the effectiveness
of therapy in these two patients with Gaucher disease. It is
anticipated that other patients with other lysosomal storage
diseases, particularly those that are cross-reacting immunologic material negative, can also develop neutralizing antibodies or other types of adverse effects that impact the effectiveness of therapy for their diseases.
REFERENCES
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Gaucher disease, in Scriver CR, Beaudet AL, Sly WS, Valle D (eds):
The Metabolic and Molecular Bases of Inherited Disease. New York,
NY, McGraw-Hill, 1995, p 2641
2. Grabowski GA, Saal H, Wenstrup RJ, Barton NW: Gaucher
disease: A prototype for molecular medicine. Crit Rev Hematol Oncol 23:25, 1996
3. Barton NW, Brady RO, Dambrosia JM, Di Bisceglie AM,
Doppelt SH, Hill SC, Mankin HJ, Murray GJ, Parker RI, Argoff
CE: Replacement therapy for inherited enzyme deficiency—Macrophage-targeted glucocerebrosidase for Gaucher’s disease. N Engl J
Med 324:1464, 1991
4. Grabowski GA, Barton NW, Pastores G, Banerjee TK, McKee
A, Parker C, Schiffmann R, Dambrosia JM, Hill SC, Brady RO:
Enzyme therapy in Gaucher disease type 1: Comparative efficacy
of mannose-terminated glucocerebrosidase from natural and recombinant sources. Ann Intern Med 122:33, 1995
5. Pastores G, Sibille A, Grabowski GA: Enzyme therapy in
Gaucher disease type 1: Dosage efficacy and adverse effects in thirtythree patients treated for six to twenty-four months. Blood 82:408,
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blda
WBS: Blood
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
1997 90: 43-48
Enzyme Therapy in Gaucher Disease Type 1: Effect of Neutralizing
Antibodies to Acid β-Glucosidase
Elvira Ponce, Jay Moskovitz and Gregory Grabowski
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