Clin Exp Immunol 2000; 120:363±368
Treatment of passively transferred experimental autoimmune
myasthenia gravis using papain
K. POULAS, T. TSOULOUFIS & S. J. TZARTOS Department of Biochemistry, Hellenic Pasteur Institute, Athens, Greece
(Accepted for publication 7 January 2000)
SUMMARY
Antibody-mediated acetylcholine receptor (AChR) loss at the neuromuscular junction, the main cause of
the symptoms of myasthenia gravis, is induced by bivalent or multivalent antibodies. Passive transfer of
experimental autoimmune myasthenia gravis (EAMG) can be induced very efficiently in rats by
administration of intact MoAbs directed against the main immunogenic region (MIR) of the AChR, but
not by their monovalent Fab fragments. We tested whether papain, which has been used therapeutically
in autoimmune and other diseases, is capable of preventing EAMG by in vivo cleavage of the circulating
anti-AChR antibodies into Fab fragments. EAMG was induced in 4-week-old female Lewis rats by i.p.
injection of anti-MIR mAb35. A total of 0´75 mg of papain was given as one or three injections 3±7 h
after MoAb injection. The mAb35 1 papain-treated animals developed mild weakness during the first
30 h and subsequently recovered, while all animals that received only mAb35 developed severe
myasthenic symptoms and died within 24±30 h. Animals treated only with papain showed no apparent
side effects for up to 2 months. Serum anti-AChR levels in mAb35 1 papain-treated rats decreased
within a few hours, whereas in non-papain-treated rats they remained high for at least 30 h. Muscle
AChR in mAb35 1 papain-treated animals was partially protected from antibody-mediated degradation.
These results show that treatment of rats with papain can prevent passively transferred EAMG without
any apparent harm to the animals, and suggest a potential therapeutic use for proteolytic enzymes in
myasthenia gravis.
Keywords acetylcholine receptor autoimmune disease
experimental autoimmune myasthenia gravis myasthenia gravis
INTRODUCTION
Myasthenia gravis (MG) is a well-defined organ-specific autoimmune disease characterized by the functional loss of acetylcholine receptors (AChR) at the neuromuscular junction which is
mediated by autoantibodies directed against the AChR. Although
within a population of MG patients there is not a good correlation
between antibody titre and disease severity, for a given patient
there is a very significant correlation between these two
parameters [1].
Treatment of MG mainly involves the use of acetylcholinesterase inhibitors, immunosuppressive drugs, thymectomy,
plasmapheresis, and the i.v. administration of human immunoglobulins [2,3]. Although such treatment results in significant
improvement of disease, new alternative or supplementary
treatments are needed.
Anti-AChR antibodies exert their effect by inducing AChR
loss or, less efficiently, by blocking the AChR ion channel. AChR
loss is mediated by: (i) antigenic modulation, i.e. acceleration of
Correspondence: Dr S. J. Tzartos, Department of Biochemistry,
Hellenic Pasteur Institute, 127 Vas. Sofias Avenue, Athens 115 21, Greece.
E-mail: [email protected]
q 2000 Blackwell Science
papain treatment
AChR internalization due to antibody cross-linking of AChR
molecules [1], and (ii) complement-mediated post-synaptic
membrane destruction [4]. Both mechanisms depend on the
bivalent nature of the immunoglobulins and/or on their Fc region;
monovalent anti-AChR Fab and Fv fragments are unable to cause
AChR loss in muscle cell cultures [5±7]. Thus, if anti-AChR
antibodies could be cleaved in vivo, their pathogenicity might be
reduced. Since the half-life of Fab fragments is short [5, 8], even
the blocking effect of some Fabs to the ion channel, which causes
a reduction to the mean number of end-plate channels [9], should
be dramatically reduced due to quick removal of circulating Fab
fragments.
The proteolytic enzyme, papain, is routinely used for in vitro
cleavage of immunoglobulins; its site of action is the immunoglobulin hinge region, resulting in the production of two antigenbinding Fab fragments and one Fc fragment, which carries the
complement and Fc receptor binding sites [10]. Thus, if this
enzyme could be used for in vivo cleavage of immunoglobulins,
without causing any deleterious effects to the recipients, it would
be a likely candidate for therapeutic use. Proteolytic enzymes,
including papain, have been used experimentally and in clinical
practice for various diseases and have proved effective, especially
363
364
K. Poulas, T. Tsouloufis & S. J. Tzartos
in autoimmune diseases [11±15], vascular diseases [16±18],
trauma therapy [19], inflammation [20], bacterial and viral
infections [21], and tumour growth and metastasis [22,23]. Their
mode of action seems to be multiple and not well defined.
Although their protein-cleaving activity is not sufficiently specific
(they attack small consensus amino acid sequences present in
many proteins), treatment of patients with various polyenzyme
preparations administered orally [24], intramuscularly [17], or
intravenously [25] has been found to be quite safe, with very few
side effects.
In experimental autoimmune MG (EAMG), the disease is
caused by immunization of laboratory animals with purified
AChR [26], or by injection with anti-AChR antibodies [5,27]. As
in MG, the primary factor causing impairment of neuromuscular
transmission is the loss of muscle AChRs mediated by antigenic
modulation and complement [28,29]. MoAbs directed against the
main immunogenic region (MIR) of the AChR are especially
efficient at inducing EAMG [30±32]. Their bivalent F(ab)2
fragments are less efficient, but can still cause MG symptoms,
whereas their monovalent Fab fragments do not [5]. In fact, Fab
fragments of anti-MIR MoAbs protect the AChR against the
destructive activity of MG antibodies [7,33]. Thus, EAMG
provides an ideal autoimmune model for studying intervention
strategies in MG. In the present study we show that papain,
administered intraperitoneally into rats previously treated with an
anti-MIR MoAb, can cleave anti-AChR antibodies in vivo and
prevent MG symptoms, with no adverse side effects. Thus, these
results suggest a potential therapeutic use for a proteolytic enzyme
in MG.
MATERIALS AND METHODS
Monoclonal antibody preparation
Monoclonal antibody 35 is a rat IgG1 that binds to the MIR of the
AChR from several species, including Torpedo, rat, and human
[34]. The mAb35 preparation consisted of serum-free hybridoma
supernatants, concentrated approximately 100 times by Amicon
ultrafiltration, then dialysed against 20 mm Tris±HCl pH 8´5,
applied to a 10-ml DEAE-Sepharose Fast Flow column (Pharmacia, Uppsala, Sweden), pre-equilibrated with 20 mm Tris±HCl
pH 8´5, and eluted using a salt gradient (0±1 m NaCl) in the same
buffer. The purity of the preparations, evaluated by SDS±PAGE,
was . 90%. The antibody preparations were dialysed against
Ringer's buffer (140 mm NaCl, 5´4 mm KCl, 1 mm CaCl2, 2´4 mm
NaHCO3, pH 7´4), and stored in aliquots of approximately 5 mg/
ml at 2208C. mAb35 was used because it is a reference anti-MIR
MoAb capable of inducing EAMG.
Treatment of animals
Four-week-old female Lewis rats, weighing approximately 65±
80 g, were used for EAMG induction by administration of mAb35
and in protection experiments. All animals were bred under
standard pathogen-free conditions. Each rat received one i.p.
injection of the indicated amounts of mAb35 (usually 0´15 mg) in
0´5 ml Ringer's buffer. A crystallized suspension of mercuripapain (Sigma Chemical Co., St Louis, MO) was dissolved in 0´2 m
cysteine in Ringer's buffer and used for i.p. injections.
Clinical assessment
Disease severity was assessed on the basis of weight loss and
clinical symptoms. The rats were weighed at regular intervals. The
level of weakness was scored in terms of their ability to grasp,
hang and run when provoked. The results were expressed as: 0, no
clinical symptoms; 1, first signs of weaker grasp after a few trials;
2, incomplete paralysis of hind limbs; 3, hind limbs paralysed and
unable to stand; 4, moribund; 5, dead.
Measurement of serum levels of mAb35
Rats were bled after mAb35 injection to measure anti-AChR
antibody titres. Sera were assayed for antibody binding to the
AChR using a radioimmunoassay similar to that described by
Lindstrom et al. [35]. In brief, Triton X-100 extracts of Torpedo
electric organ membranes were labelled with 125I-a -bungarotoxin,
then 1 m l of serum was incubated overnight at 48C with 0´3 pmol
of 125I-a -bungarotoxin-labelled Torpedo AChR (total volume
50 m l). Bound antibody±antigen complexes were precipitated by
the addition of a second antibody (rabbit anti-rat immunoglobulins), then the samples were centrifuged (3000 g for 10 min),
washed with 0´5% Triton X-100 in PBS and counted on a gamma
counter. Fab fragments were precipitated equally well using the
same antiserum.
Quantification of muscle AChR
The animals were killed 24 h after antibody administration. The
hind limb muscles of each rat were dissected and weighed, and an
equal weight of muscle from each animal was homogenized in
0´1 m NaCl, 0´01 m Na2HPO4, 0´01 m EDTA, 0´01 m EGTA,
0´001 m PMSF, 0´01 m iodoacetamide, 0´05% NaN3, 5 U/ml of
aprotinin and 0´5 m g/ml of pepstatin, pH 7´5. The homogenates
were washed twice in 10 volumes of PBS pH 7´2, then the pellet
was resuspended in an equal volume of 2% Triton X-100 in PBS
and incubated overnight at 48C. After centrifugation (12 000 g for
30 min) to remove insoluble material, the muscle AChR content
was determined as described by Lindstrom et al. [35]. In brief, 10m l aliquots of each muscle extract were labelled with 0´12 pmol
of 125I-a -bungarotoxin and the complexes precipitated using an
excess of a mixture of MoAbs 192 and 195. Specific precipitation
was calculated by subtracting the values obtained by pretreating
the samples with 10 pmol of non-radioactive a -bungarotoxin prior
to addition of 125I-a -bungarotoxin. The percentage of AChR
remaining in mAb35-treated rats (with or without papain
treatment), was calculated using the equation: remaining AChR
(%) ˆ 100 (AChR(MoAb)/AChR(Ringer's)), where AChR(MoAb)
and AChR(Ringer's) are the AChR concentrations of animals
treated with MoAb (with or without papain) and Ringer's buffer,
respectively.
Statistical analysis
Clinical EAMG scores, weight changes and AChR loss were
evaluated for statistical significance using a two-tailed unpaired
Student's t-test, using the MS Excel 7.0 program.
RESULTS
Determination of mAb35 and papain doses
Initially, we determined the amount of mAb35 required to induce
severe EAMG symptoms in the rats. Four groups of rats were
injected intraperitoneally with 0´05±0´3 mg of mAb35. Figure 1
shows that symptoms appeared as early as 6 h after MoAb
injection. mAb35 (0´15 mg; 4516 pmol determined using Torpedo
AChR and 112´9 pmol determined using rat AChR) was capable of
causing severe clinical weakness resulting in death within 24±30 h;
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Papain treatment of experimental myasthenia
Fig. 1. Clinical course of passive experimental autoimmune myasthenia
gravis (EAMG) in groups of three rats receiving single i.p. injections of
increasing amounts of mAb35. Points represent mean clinical scores. V,
0´05 mg mAb35; B, 0´10 mg mAb35; O, 0´15 mg mAb35; X, 0´30 mg
mAb35. The difference between all groups was found to be statistically
significant (P , 0´05), s.d. were , 0´6.
0´10 mg resulted in mild symptoms (score up to 2) and total
recovery was observed 3 days after antibody administration, while
0´05 mg had no effect. We therefore used the dose of 0´15 mg of
mAb35 in all subsequent experiments.
We then determined the approximate amount of papain
required for reversal of myasthenic symptoms. Table 1 shows
the results from three groups of three animals, which received
0´15 mg of mAb35 followed by three different amounts of papain
3 h later. The dose of papain selected for subsequent experiments
was that resulting in 100% survival of the treated animals, i.e.
0´75 mg/animal.
Effect of papain on EAMG symptoms
Three groups of six animals were injected intraperitoneally with
0´15 mg of mAb35 per animal, while another group received only
Ringer's buffer (control). The first group of MoAb-treated animals
received no other treatment and developed severe signs of muscle
weakness, as shown in Fig. 2, with the first signs of weakness
(score ˆ 0´5) appearing 5 h after MoAb administration. Three i.p.
injections, each of 0´25 mg of papain, were given to the second
Table 1. Dose response of papain-mediated protection*
Amount of
papain given
intraperitoneally (mg)
0
0´25
0´50
0´75
Symptoms after 24 h
Survival of animals
5
5
3
2
0/3
0/3
1/3
3/3
*Three groups of three rats were injected intraperitoneally with
0´15 mg of mAb35. Three hours later, they were injected intraperitoneally
with the indicated amounts of papain. Their clinical symptoms were tested
24 h after mAb35 injection. They were also followed for survival for a
week. The clinical score was evaluated as 0±5 as described in Materials
and Methods (0 ˆ healthy, 5 ˆ dead).
365
Fig. 2. Clinical course of passive experimental autoimmune myasthenia
gravis (EAMG) in rats receiving mAb35 with or without papain-mediated
protection. Points represent mean clinical scores. Three groups of six rats
were injected intraperitoneally with 0´15 mg mAb35; two of the groups
then received 0´75 mg of papain in one or three i.p. doses. Another two
groups of three rats received only Ringer's buffer or only 0´75 mg papain.
The clinical score was evaluated as 0±5 as described in Materials and
Methods (0 ˆ healthy, 5 ˆ dead). V, mAb35-treated rats; X, mAb35 1
papain-treated rats (0´75 mg as a single injection 5 h after the MoAb); B,
mAb35 1 papain-treated rats (three injections, each of 0´25 mg of papain,
at 3, 5 and 7 h after the MoAb); O, Ringer's buffer only or papain only.
The difference between the two groups that received papain and the one
that received mAb35 only was found to be statistically significant
(P , 0´02), s.d. were , 0´5.
group of animals, 3, 5 and 7 h, respectively, after injection of
mAb35; these animals developed mild weakness for the first 30 h,
then recovered. Similar results were obtained with the third group
of animals that received 0´75 mg papain as a single injection
given 5 h after mAb35 administration (Fig. 2). Administration of
papain (0´75 mg) to a fifth group of three rats that did not receive
MoAb had no apparent effect on the animals. A better protective
effect (score , 2) was obtained when the single bolus of enzyme
was administered even earlier (2 h after mAb35 administration)
before any symptoms of EAMG were evident (data not shown).
Figure 3 shows the variations in weight of the animals during
the experiment. Twelve hours after administration of MoAb, the
animals receiving only MoAb showed signs of weight loss
(approx. 5%), which increased with time to 20% just before death,
24±30 h after mAb35 administration. All the mAb35 1 papaintreated animals exhibited a much less pronounced weight loss than
the mAb35-treated animals. The weight of the mAb35 1 papaintreated animals stopped decreasing 48 h after mAb35 administration, then the animals started to regain weight (data not shown).
In order to determine whether the protective activity of papain
was due to cleavage of anti-AChR antibodies or to some other
mechanism, 0´75 mg of papain was administered to healthy
animals 2 h and 3 h before they were injected with 0´15 mg of
mAb35. All the animals developed severe signs of myasthenia
(clinical score ˆ 3, 16 h after mAb35 injection) and no protection
was seen in either case. This suggests that papain is only active in
the animals for a short space of time (, 2 h).
In order to test the toxicity of papain, another two groups of
three untreated rats were given a single injection of 2´25 mg or
4´5 mg of papain per animal, i.e. doses 3 and 6 times greater than
that used in the protection experiments. The first group remained
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K. Poulas, T. Tsouloufis & S. J. Tzartos
Fig. 3. Change in weight following injection of anti-acetylcholine
receptor (AChR) mAb35 with or without papain. Points represent mean
percentage change in weight. The groups of animals used were those
described in Fig. 2. V, mAb35-treated rats; X, mAb35 1 papain-treated
rats (0´75 mg in one injection 5 h after the MoAb); B, mAb35 1 papaintreated rats (three injections, each of 0´25 mg of papain, at 3, 5 and 7 h
after the MoAb); O, Ringer's buffer only. The difference between the
mAb35 1 papain-treated animals and the other groups was found to be
statistically significant (P , 0´02), s.d. were , 1´6.
healthy for at least 2 months, while the animals in the second
group died within 3 h.
Serum levels of mAb35
Fab fragments have a much shorter in vivo half life than intact
antibodies [5, 9], thus cleavage of MoAb by papain should result
in a decrease in total (intact plus fragments) anti-AChR antibody
levels. We therefore measured the total anti-AChR activity in the
sera of rats treated with either mAb35 or mAb35 1 papain (the
second antibody used for precipitation was equally effective with
whole MoAb and the Fab fragment). Three hours after mAb35
administration the anti-Torpedo AChR titre was found to be
204´6 nm and 5´1 nm for rat AChR, which remained relatively
stable for at least 24 h. Figure 4 shows that antibody levels in
the mAb35 1 papain-treated animals fell soon after papain
Fig. 4. Serum anti-acetylcholine receptor (AChR) antibody concentrations in the groups of rats of Fig. 2. The animals used were those in Fig. 2.
V, mAb35-treated rats; X, mAb35 1 papain-treated rats (0´75 mg in one
injection 5 h after the MoAb); B, mAb35 1 papain-treated rats (0´25 mg
papain in three injections 3, 5 and 7 h after the MoAb). The difference
between the two groups of mAb35 1 papain-treated animals and the one
mAb35-treated was found to be statistically significant (P , 0´05).
Fig. 5. Muscle acetylcholine receptor (AChR) content of treated rats.
Three groups of six rats were used. Group 1 received only Ringer's buffer,
group 2 were injected with 0´15 mg of mAb35 followed by 0´75 mg
papain 5 h later, and group 3 were injected only with 0´15 mg of mAb35.
Their symptoms were observed at 24 h, then the animals were killed
immediately. AChR-containing muscle extracts were prepared from the
hind limbs and their AChR content measured. The results are expressed as
the muscle AChR content per unit weight of muscle in rats treated with
MoAb or MoAb 1 papain as a percentage of that in rats receiving
Ringer's buffer only. Insert: clinical symptoms at 24 h. The difference
between the two groups of rats, mAb35-treated and mAb35 1 papaintreated, was found to be statistically significant (P , 0´05).
administration, the reduction 11 h after mAb35 injection being
about 50% when compared with antibody levels in rats treated
only with mAb35. The decrease in antibody levels was greater in
the animals that received the treatment in three injections
compared with one. The remaining antibodies in the blood of
the papain-treated animals probably represent intact antibodies
which escaped proteolysis, since Fab fragments should have been
eliminated from the circulation within a few hours.
Muscle AChR levels
In order to measure the effect of papain on muscle AChR levels, a
new set of three groups of six animals was used. The animals in
the first group received Ringer's buffer, those in the second group
received mAb35 followed by a single i.p. injection of 0´75 mg
papain 5 h later, while those in the third group received only
mAb35. All the animals were killed 24 h after mAb35 administration. At this time, as shown in the inset of Fig. 5, the MoAbtreated animals showed signs of severe weakness (score ˆ 4),
while the MoAb 1 papain-treated animals had only mild
myasthenic symptoms (score ˆ 2). Figure 5 also shows that
papain administration significantly, though only partially, protected muscle AChR from antibody-mediated degradation, the
muscle AChR content in MoAb 1 papain-treated animals being
reduced by 40%, while that in MoAb-treated rats was reduced by
62%.
DISCUSSION
Current treatment of MG is only partially effective and is
currently limited mainly to the use of acetylcholinesterase
inhibitors, immunosuppressives, plasmapheresis, intravenous
immunoglobulin, and thymectomy [1,2]. The goal of several of
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Papain treatment of experimental myasthenia
Q1
these treatments is to decrease levels of circulating autoantibodies
against the AChR. Various procedures have been developed to
achieve this. Using plasmapheresis, a large fraction of the
patient's plasma, including the pathogenic anti-AChR antibodies,
is eliminated [3]. An improvement on this approach is to pass the
plasma through a protein-A column, which binds and removes
most immunoglobulins, including most anti-AChR autoantibodies
[36]. Plasmapheresis is an expensive and laborious procedure.
Immunosuppressives are also used to decrease circulating
autoantibody levels, but have serious side effects [1]. While all
these treatments are partially effective, alternative treatments are
needed.
We considered that papain, by directly digesting the circulating autoantibodies, might be efficacious in MG, and could be used
either as an alternative therapeutic approach or as complementary
to established treatments. In the present study we showed that
systemic administration of papain reduced the levels of circulating
anti-AChR antibodies in an experimental model of MG and
dramatically improved clinical symptoms. EAMG animals
showed significant improvement when 0´75 mg of papain was
administered intraperitoneally. Interestingly, all the mAb35 1
papain-treated animals recovered from myasthenic symptoms,
while all the mAb35-treated animals died.
Papain is the enzyme of choice for cleavage of immunoglobulins into Fab fragments. The in vivo cleavage of the anti-AChR
antibodies into Fab fragments could have three beneficial effects:
(i) Fab fragments usually are not expected to cause AChR loss; (ii)
they are eliminated very rapidly from the body; and (iii) those that
do bind to the AChR may protect it against the binding of intact
pathogenic antibodies. Although the papain-protected animals
were not free of MG symptoms, these were much milder than
those in non-papain-treated rats and were short-lived. These
effects may have resulted from the fact that (i) some AChR
molecules were already destroyed before papain administration,
and (ii) a proportion of the circulating antibodies may have
escaped papain cleavage (Fig. 4) due to the short half-life of
papain in vivo (, 2 h, as suggested by the lack of protection
when papain was administered 2 h before mAb35) and to a
possible sequestration, followed by slow release, of a fraction of
the MoAb in body compartments. Although injection of papain
2 h after MoAb injection resulted in greater protection than
injections at 3±7 h after MoAb administration, and despite the
assumption that co-injection of MoAb and enzyme may elicit
higher protection, we preferred to use papain after symptoms of
EAMG were apparent, a condition mimicking MG more closely.
Despite the notion that in vivo administration of proteases
might be deleterious to the recipients (e.g. causing renal damage),
as they also cleave various other proteins, the available data on
their in vivo experimental and clinical use suggest that they are
safer than expected. It has been repeatedly shown that the
administration of proteases to animals [11,13] and humans [12,
21] has no significant side effects (only cases of allergic reaction
have been observed and a sensation of fullness and flatulence). An
enteric-coated orally administered drug (WOBE-MUGOS; Mucos
Pharma, Munich, Germany), the main ingredient of which is
papain, is officially approved for use in several western countries.
The Federal Drug Administration (FDA) classifies systemic
enzyme therapy as generally recognized as safe (GRAS) [37]. In
the present study, we found that administration of papain to
healthy animals at a dose three times higher than that used for
EAMG protection did not cause any significant effects for a
367
period of at least 2 months. Similarly, Gesualdo et al. [11] and
Nakazawa et al. [13,14] observed that the use of . 1´0 mg of
papain/100 g body weight (i.p.) did not result in any signs of
toxicity.
The investigation of the possible therapeutic effect of papain
in MG must await further studies. The next important step is to
test its therapeutic efficacy in active EAMG induced by
immunization with AChR, which is a better model of the human
disease than the passive transfer model. The effect of orally
administered enteric-coated preparations of papain should also be
tested in animal models.
The possibility of restricting the targets of the proteolytic
enzyme can also be envisaged. Proteases specific for IgA or IgG
immunoglobulins are known [38]; further specificity may be
achieved by phage-display combinatorial approaches. Alternatively, hybrid molecules composed of a protease and an antibody
fragment specific for a constituent of the neuromuscular junction
could direct the enzyme to the target site, thus dramatically
reducing the effective dose of the enzyme.
ACKNOWLEDGMENTS
We are grateful to A. Kokla for excellent technical assistance and to Dr L.
Jacobson for valuable suggestions. Supported by the Hellenic Pasteur
Institute.
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