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Complement inhibition abrogates nerve terminal injury in Miller Fisher syndrome.

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Complement Inhibition Abrogates Nerve
Terminal Injury in Miller
Fisher Syndrome
Susan K. Halstead, PhD,1 Peter D. Humphreys, MSc,1 John A. Goodfellow, BSc,1 Eric R. Wagner, BSc,1
Richard A. G. Smith, PhD,2 and Hugh J. Willison, FRCP1
A large body of clinical and experimental data indicate that complement activation is an important mechanism for
neuronal and glial injury in Guillain–Barré syndromes. Inhibition of complement activation therefore might be expected
to limit the progression of the disease. Using in vitro and in vivo models of the Guillain–Barré syndrome variant, Miller
Fisher syndrome, we have shown previously that anti-GQ1b ganglioside antibodies target the presynaptic motor nerve
terminal axon and surrounding perisynaptic Schwann cells, thereby mediating destructive injury through deposition of
membrane attack complex. Here, we have used this model to investigate the effects of a novel therapeutic inhibitor of
complement activation, APT070 (Mirococept), both in vitro and in vivo. In these models, APT070 completely prevents
membrane attack complex formation, and thereby has a major neuroprotective effect at the nerve terminal, as assessed by
immunohistology of perisynaptic Schwann cell and axonal integrity. These data provide a rationale for considering
clinical trials of APT070 in Guillain–Barré syndrome, its variant forms, and other complement dependent neuromuscular
Ann Neurol 2005;58:203–210
Serological, immunological, and pathological studies all
implicate a role for complement-mediated damage to
neuronal and Schwann cell membranes in Guillain–
Barré syndromes (GBS).1– 8 Modeling of the GBS variant Miller Fisher syndrome (MFS) in the mouse has
used a range of complement fixing anti-GQ1b antibodies.9,10 In one model, the pore-forming membrane
attack complex (MAC) of complement is critical in
mediating neuronal and perisynaptic Schwann cell
(pSC) injury at the presynaptic neuromuscular junction (NMJ).11
Inhibition of complement might be expected to attenuate the disease process. Complement activation is
negatively regulated by soluble and membrane-bound
molecules including CR1, CD59, and decay accelerating factor (CD55).12 Studies on experimental allergic
neuritis have shown that complement inhibition or depletion with soluble CR1 or cobra venom factor provide protection against disease advancement and tissue
APT070 is a novel type of complement regulator derived from a bacterially expressed region of CR1 combined with a membrane-localizing peptide that confers
unique cell- and tissue-binding properties.15 APT070
protects complement-mediated tissue injury in animal
models of vascular shock,15 rheumatoid arthritis,16 and
renal transplant reperfusion injury.17 It is well tolerated
when given systemically to healthy volunteers and by
local administration to patients with rheumatoid arthritis and patients undergoing renal transplantation (Inflazyme Pharmaceuticals, British Columbia, Canada).
In this study, we investigated the complement inhibitory effects of APT070 in mouse models of MFS to
establish whether this agent might be a suitable candidate for treatment trials of antibody-mediated,
complement-dependent forms of GBS in humans.
From the 1Division of Clinical Neurosciences, Institute of Neurological Sciences, Southern General Hospital, Glasgow, Scotland; and
Inflazyme Pharmaceuticals, Suite 425, 5600 Parkwood Way, Richmond, British Columbia, Canada.
Published online Jul 27, 2005 in Wiley InterScience
( DOI: 10.1002/ana.20546
Received Dec 5, 2004, and in revised form May 16, 2005. Accepted
for publication May 17, 2005.
Material and Methods
APT070 was prepared by posttranslational linkage of an
N-myristoylated 17-mer peptide to a C-terminus cysteine
added to the first three short consensus repeat (CCP) domains of human CR1 expressed in Escherichia coli as described previously.15–17 Bovine serum albumin (BSA) was
obtained from Sigma (Poole, United Kingdom).
Address correspondence to Dr Willison, University of Glasgow, Department of Neurology, Ground Floor Neurology Block, Southern
General Hospital, Glasgow, G51 4TF, Scotland. E-mail:
© 2005 American Neurological Association
Published by Wiley-Liss, Inc., through Wiley Subscription Services
Mice, Antibodies, and Sera
Male Balb/c mice were obtained from Harlan (Bicester,
Oxor, United Kingdom) and used at 4 to 6 weeks old.
The IgM anti-ganglioside monoclonal antibody CGM3
was derived from mice inoculated with a GT1a-bearing
Campylobacter jejuni lipooligosaccharide. CGM3 reacts with
gangliosides GQ1b, GD3, and GT1a.18,19 This antibody
has similar specificity and pathogenic effects to human MFS
sera.10,20 CGM3 concentration was measured using quantitative enzyme-linked immunosorbent assay (Bethyl Laboratories, Montgomery, TX). An MFS serum positive for antiGQ1b IgG antibodies (titre ⬎1/12,500) was studied with
institutional ethical approval according to local guidelines.
Normal human serum (NHS) from a single donor stock was
fresh frozen and stored in aliquots at ⫺70°C to preserve
complement activity for use as a source of heterologous complement. CGM3, MFS serum, and NHS were dialyzed
against Ringers’ solution (116mM NaCl, 4.5mM KCl, 1mM
MgCl2, 2mM CaCl2, 1mM NaH2PO4, 23mM NaHCO3,
11mM glucose, pH 7.4) for 24 hours at 4°C.
Unfixed hemidiaphragm cryostat sections (8 –20␮m) were
air-dried, then stored at ⫺20°C. To localize NMJs, we used
Texas Red and dipyrromethene boron difluoride (BODIPY)labeled ␣-bungarotoxin (␣-BTx; diluted 1/750 to 1.3␮g/ml;
Molecular Probes, Eugene, OR) that binds to postsynaptic
nicotine acetylcholine receptors (nAChr). Complement component C4 was detected with rabbit anti-C4 (1:200; Sigma),
followed by fluorescein isothiocyanate (FITC)–conjugated
goat anti–rabbit IgG (1:300; Southern Biotechnology Associates, Birmingham, AL). C3c was detected with FITClabeled rabbit anti-C3c (1/300; Dako, Ely, United Kingdom). MAC was detected using mouse anti–human C5b-9
(1/50; Dako), followed by FITC-conjugated goat anti–
mouse IgG (1/300). Mouse IgM deposits were identified
with FITC-labeled anti–mouse IgM antibodies (1/300;
Southern Biotechnology Associates). All antibody incubations were for 1 hour at 4°C.
S100 staining was used to label pSCs in whole-mount triangularis sterni muscle preparations (see later). Tissue was
fixed with 4% formaldehyde in phosphate-buffered saline
(PBS) for 20 minutes, then rinsed with PBS for 10 minutes.
Muscle samples then were incubated with 0.1M glycine for
10 minutes, rinsed in PBS for 10 minutes, and incubated
with rabbit anti-S100 (1/200; Dako) in blocking solution
(1% goat serum, 0.4M L-lysine, 0.5% Triton X-100 [Sigma
Labs, St. Louis, MO] in PBS) overnight at 4°C. Samples
were rinsed three times in blocking solution for 5 minutes,
incubated in goat anti–rabbit IgG–conjugated CY5 (1:200;
Southern Biotechnology Associates) for 3 hours at 4°C, followed by three 5-minute rinses in blocking solution.
For neurofilament (NF) staining, sections of unfixed tissue
were preincubated for 1 hour at room temperature (RT)
with Texas Red–conjugated ␣-BTx, rinsed, immersed in ethanol at ⫺20°C for 20 minutes, then incubated overnight at
RT with the rabbit polyclonal serum 1211 (1/750; reactive
with phosphorylated NF; Affiniti Research Products, Exeter,
United Kingdom), followed by goat anti–rabbit IgG (1/300;
Southern Biotechnology Associates).
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pSC viability was assessed using ethidium homodimer 1
(EthD-1; 2␮M, a membrane impermeant dye that labels
with red fluorescence the nucleic acids of membranepermeable cells; Molecular Probes) as described previously.11
NMJs were identified in 15␮m cryostat sections by staining
with bodipy-conjugated ␣-BTx, and the percentage of NMJs
with EthD-1–positive nuclei at end-plates was calculated.
In Vitro Nerve–Muscle Preparations
Mice were killed by CO2 inhalation, subject to UK Home
Office guidelines. Hemidiaphragms were dissected and prepared for in vitro studies in pregassed (95% O2/5% CO2)
Ringers’ solution at RT (20 –22°C).21 The APT070 dose–
response curve was established using four mouse hemidiaphragms on three separate occasions. Untreated control sections of diaphragm were removed from each muscle
preparation for baseline immunohistological analysis of NF,
activated complement proteins, and IgM levels. Muscles were
preincubated with 48, 24, 12, or 0␮g/ml APT070 in Ringers’ solution (1 hour at 32°C), rinsed in Ringers’ solution,
followed by CGM3 (50␮g/ml) plus APT070 (48, 24,12, or
0␮g/ml), normalized to a protein concentration of 48␮g/ml
with BSA for 2 hours at 32°C, then 30 minutes at 4°C, then
equilibrated for 10 minutes at RT. Muscle preparations then
were rinsed in Ringers’ solution and exposed to 40% NHS
plus APT070 (48, 24, 12, or 0␮g/ml) for 1 hour at RT.
In subsequent bioassays, hemidiaphragm preparations
were incubated with APT070 or BSA as a placebo control
(48␮g/ml) in Ringers’ solution for 1 hour at 32°C. Hemidiaphragms were rinsed in Ringers’ solution, incubated as described earlier with CGM3 (50␮g/ml) or MFS serum (diluted 1:1 in Ringers’ solution) plus APT070 or BSA (48␮g/
ml), then exposed to 40% NHS plus APT070/BSA (48␮g/
ml) for 1 hour at RT. Tissue was assessed for levels of mouse
IgM, C4 or C3c, MAC, NF, and pSC viability using
EthD-1. For illustrations of nerve terminals containing
whole NMJs with innervating nerve fibers, we removed and
treated triangularis sterni muscles with CGM3, APT070 (or
BSA as placebo), and NHS as described earlier. Wholemount preparations then were stained for MAC, NF, S100,
and EthD-1.
Passive Immunization
To demonstrate APT070 regulatory effects in vivo, we conducted passive immunization of live mice as described previously.11 Mice were injected intraperitoneally (IP) with 1.5mg
CGM3, followed 15 hours later by an intravenous (IV) injection of APT070 (580␮g), or an equivalent dose of BSA.
Two hours later, mice were given 0.5ml NHS by IP injection. In a further series of experiments, a comparison was
made between prophylactic treatment with APT070 (580␮g,
followed 1 hour later by NHS) and simultaneous administration of APT070 (580␮g) with NHS (ie, treatment at onset of neurological injury). Animals were observed for 3
hours after dosing with NHS; then they were killed by CO2
asphyxiation before tissue removal for immunohistology. Diaphragms were assessed for levels of IgM, C3c, MAC, NF,
and EthD-1 as described earlier.
Image Acquisition, Quantitation,
and Statistical Analysis
Digital images were captured using both Zeiss Pascal (Carl
Zeiss, Welayn Garden, UK) and Bio-Rad MRC1024 (BioRad, Hemel Hempstead, UK) confocal laser-scanning microscopes. Image-analysis measurements were made using Scion
Image (Scion Corporation, Frederick, MD) analysis software.
For quantitative analysis of IgM, C3c, MAC, and NF, three
staining runs of each marker were performed on tissue from
at least three individual hemidiaphragm preparations or passively immunized mice, and they were quantified as previously described.21 All studies were observer blinded. For immunohistological analysis of nonparametric data, statistical
comparisons were made using the Mann–Whitney U test at
a 5% level of significance. For comparison of EthD-1–positive pSCs at the NMJ, the ␹2 test was used at a 1% level of
Protective Effects of APT070 In Vitro
To assess the dose range of APT070 that might be required for neuronal and pSC protection in the neuropathy model in vitro, we first conducted a dose finding
study. APT070 was assessed across the concentration
range of 48, 24, 12, and 0␮g/ml and administered in
all incubation steps to maximize the possibility of finding a beneficial therapeutic effect.
Across this APT070 dose range, there was a gradual
reduction in the deposition of the intermediate complement protein, C3c, at the NMJ (Fig 1). MAC deposits were dramatically reduced to undetectable levels
at all three concentrations of APT070 (see Figs 2A, B).
Thus, APT070 had a more pronounced inhibitory effect on MAC deposition than C3c deposition across
this concentration range.
In the absence of APT070, NF immunoreactivity
was abolished over nerve terminals (see Figs 1, 2C, and
2D), indicating that a severe level of MAC-mediated
neuronal injury has occurred.11 In the presence of all
concentrations of APT070, NF levels were protected,
with a minor dose-dependent effect. Thus, tissue was
protected (to levels equal to untreated control tissue)
when incubated with 48␮g/ml APT070 ( p ⫽ 0.488).
NF signal was also significantly different to untreated
controls in the samples incubated with 12 ( p ⫽ 0.017)
and 24␮g/ml ( p ⫽ 0.001). pSC death was monitored
Fig 1. APT070 dose–response curve. Mouse hemidiaphragm preparations incubated with APT070 (0, 12, 24, and 48␮g/ml) at all
stages of the in vitro preparation compared with untreated control tissue from the same mouse. An increasing concentration of
APT070 results in dose-dependent reduction of the intermediate complement protein C3c. Membrane attack complex (MAC) deposits were greatly reduced at all concentrations of APT070 examined, indicating that APT070 has a stronger inhibitory effect on the
C5 convertase compared with the C3 convertase. Neurofilament (NF) signal (a measure of axonal integrity) demonstrates NF preservation to control levels (p ⫽ 0.488) at 48␮g/ml APT070. nAChr ⫽ nicotinic acetylcholine receptor. Mann–Whitney U test:
asterisk denotes significantly different from control, and number sign indicates significantly different from 0␮g/ml APT070 (maximal lesion).
Halstead et al: Complement Inhibition in MFS
Fig 2. Illustrative images of whole-mount muscles demonstrating neuronal and perisynaptic Schwann cell (pSC) protection
at the neuromuscular junctions (NMJs) treated with APT070.
Mouse triangularis sterni in vitro preparations incubated with
APT070 or bovine serum albumin (BSA) at 48␮g/ml in the
presence of CGM3 and normal human serum (NHS). (A)
Membrane attack complex (MAC; green) deposits overlying
two BSA-treated NMJs (red). Severe MAC-mediated injury to
the pSCs has resulted in loss of S100 labeling (blue) directly
over the NMJ, although S100 labeling is still present in myelinating Schwann cells. (B) No MAC (green) is present at
NMJs (red) treated with the complement inhibitor APT070,
and pSC integrity is indicated by the presence of S100 labeling (blue) over the NMJ. (C, D) Neurofilament signal (green)
at the NMJs (red). BSA-treated NMJs (C) show loss of neurofilament (NF) signal over the terminal suggesting axonal injury, compared with APT070-protected NMJs (D). (E, F)
Ethidium homodimer (EthD-1; green) was used to assess pSC
membrane integrity. (E) BSA-treated NMJs have EthD-1–
positive pSC nuclei over the NMJ (red), which are protected
in the presence of APT070 (F). Asterisks (E, F) indicate pSC
nuclei. Scale bar ⫽ 20␮m.
by EthD-1 uptake and was found to be extensive, as
expected, in tissue exposed to CGM3 plus complement
in the absence of APT070.11 Thus, in the APT070untreated control sample, 1,480 of 2,313 (64%) NMJs
had 1 or more EthD-1–positive nuclei compared with
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16% (n ⫽ 2,281; ␹2 test, p ⬍ 0.01) at 12␮g/ml
APT070, 3% (n ⫽ 1,879; ␹2 test, p ⬍ 0.01) at
24␮g/ml APT070, and 1% (n ⫽ 2,824; ␹2 test, p ⬍
0.01) at 48␮g/ml APT070 (see Figs 2E, F). The concentration of CGM3 added to the organ bath was constant in all experiments, and abundant IgM deposits
over nerve terminals were observed under all conditions studied (data not shown).
The exploratory dose finding study indicated that
APT070 administered at 48␮g/ml appeared amply sufficient to preserve both neuronal and pSC integrity in
vitro. To validate these neuroprotective effects using an
irrelevant placebo control protein (BSA) and to test for
C4 inhibition (as an indicator of classic pathway activation/inhibition), we conducted in vitro hemidiaphragm preparations at 48␮g/ml APT070 (or BSA as
placebo) in three independent experiments. In this set
of experiments, levels of C4-derived products were reduced ( p ⬍ 0.01) compared with BSA placebo, as was
observed for the C3 convertase product, C3c (Fig 3).
This indicates that APT070 is having an effect on C4
cleavage, in addition to an effect on C3 convertase.
MAC deposition also was found to be significantly reduced ( p ⬍ 0.01) compared with the BSA-treated placebo and to a greater extent than C3c or C4. This
indicates that under these conditions APT070 is inhibiting C5 convertase to a greater extent than C3 convertase. APT070 is known to be capable of inhibiting
both C3 and C5 convertases. As expected, immunoreactive NF levels also were significantly greater in the
APT070 incubated preparation compared with the
BSA-treated placebo control ( p ⬍ 0.01), indicating a
level of neuronal protection with APT070. pSC death
was also significantly reduced in the APT070-treated
tissue (5% of NMJs with one or more EthD-1–positive
nuclei; n ⫽ 1,096) compared with the BSA placebo
control sample (45% of NMJs with one or more
EthD-1–positive nuclei; n ⫽ 1,237; ␹2 test, p ⬍ 0.01).
To ensure that APT070 was also protective when
the source of anti-GQ1b antibody was human MFS
serum, we conducted a further study by preincubation
of hemidiaphragm tissue (in the presence of APT070)
with MFS serum in vitro, then NHS as described earlier. In this study, APT070 was protective, as expected
(Fig 4), comprising a reduction in C3c deposits, complete inhibition of MAC formation, and preservation
of NF levels at control values.
Protective Effects of APT070 In Vivo after Passive
To demonstrate that any neuroprotective effects observed in vitro could also occur in vivo, we performed
passive immunization studies (Fig 5). Mice were injected IP with CGM3, followed by either APT070
(580␮g IV, n ⫽ 5) or BSA (580␮g IV, n ⫽ 4), before
NHS administration IP as a source of complement. As-
Fig 3. Mouse hemidiaphragm in vitro preparations incubated with APT070 or bovine serum albumin (BSA) at 48␮g/ml. C4 deposits are reduced at the neuromuscular junction (NMJ) in APT070-treated tissue compared with BSA (p ⬍ 0.01), indicating that
APT070 retains the similar characteristics as CR1. Membrane attack complex (MAC) deposition is greatly reduced in APT070treated tissue compared with BSA placebo control (p ⬍ 0.01). The neurofilament (NF) signal was preserved in APT070-incubated
tissue compared with BSA tissue (p ⬍ 0.01), in which the signal over the NMJ was abolished. nAChR ⫽ nicotinic acetylcholine
receptor. Mann–Whitney U test: asterisk indicates significantly different from BSA.
Fig 4. Mouse hemidiaphragm in vitro preparations were incubated with an anti-GQ1b antibody positive human Miller–Fisher
syndrome (MFS) serum plus normal human serum (NHS) in the presence of APT070 or bovine serum albumin (BSA) as placebo.
Complement C3c and membrane attack complex (MAC) deposits were significantly reduced over the neuromuscular junctions
(NMJs) in preparations treated with APT070 compared with BSA. The neurofilament (NF) signal was similarly preserved in the
presence of APT070. nAChR ⫽ nicotinic acetylcholine receptor. Mann–Whitney U test: asterisk indicates significantly different
from control, and number sign indicates significantly different from BSA.
Halstead et al: Complement Inhibition in MFS
Fig 5. Analysis of diaphragm tissue from mice passively immunized with CGM3 and normal human serum (NHS), treated with a
single intravenous injection of APT070 or bovine serum albumin (BSA; 580␮g, after CGM3 and before NHS administration).
C3c and membrane attack complex (MAC) are reduced at the neuromuscular junctions (NMJs) of mice treated with APT070 compared with BSA as placebo (p ⬍ 0.05). Neurofilament (NF) signal over the NMJ was significantly greater in APT070-treated
tissue compared with BSA (p ⬍ 0.05). nAChR ⫽ nicotinic acetylcholine receptor. Asterisk indicates significantly different from
suming a completely even volume of distribution
(20ml) of APT070 throughout the mouse (20gm) with
no metabolism or excretion, this equates to an in vitro
dose of 29␮g/ml. C3c deposits at NMJs were abundant in both samples, although significantly lower in
APT070-injected mice ( p ⬍ 0.01). MAC deposits were
massively reduced in APT070-treated mice compared
with BSA placebo control animals ( p ⬍ 0.01). By corollary, the NF signal was also significantly greater in
APT070-treated animals compared with BSA-treated
animals ( p ⬍ 0.01), indicating a neuroprotective effect.
pSC death was significantly reduced in the APT070treated tissue (4% of NMJs with one or more EthD1–positive nuclei; n ⫽ 1,104) compared with BSA placebo control samples (36% of NMJs with one or more
EthD-1–positive nuclei; n ⫽ 819; ␹2 test, p ⬍ 0.01),
thus demonstrating that APT070 also has a glial protective effect in vivo.
The above experiments demonstrated a prophylactic
protective effect of APT070 in vivo. To demonstrate
that APT070 could be an immediately effective therapy
from the point of administration onward, we conducted a further experiment in which APT070 was coadministered with NHS. This was compared with an
APT070 preadministration period of 1 hour and with
BSA as a placebo control. Immunohistological parameters are shown in Figure 6. Under both APT070
preadministration and coadministration conditions,
C3c and MAC levels were significantly reduced compared with placebo, with APT070 preadministration
Annals of Neurology
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being more effective in attenuating MAC deposition
than APT070 coadministration. Similarly, NF levels
were significantly protected by both preadministration
and coadministration of APT070, but more so in the
former condition.
We have demonstrated previously that functional failure and pathological injury to terminal motor axons
and pSCs is entirely dependent on the deposition of
MAC, directed to the NMJ by anti-GQ1b antibody
binding to nerve terminal gangliosides.11 Here, we
have used APT070 to inhibit the progression of the
complement cascade and preserved NMJ components.
APT070 combines the three N-terminal short consensus repeats of human CR1 with the membranetargeting peptide derivative “2-addressin.”22 CR1 has
both classical and alternative C3 and C5 convertase decay accelerating activity and is also a cofactor for Factor
I cleavage of C4b and C3b. The N-terminal region of
CR1 shares these activities, although not in the same
ratio as found in intact CR1.23
Initial studies were undertaken to assess the effectiveness of APT070 in vitro, and they demonstrated a
dose-dependent effect on complement inhibition. The
C5 convertase regulatory activity of APT070 was more
striking at the concentration range investigated than
C3 convertase or C4 cleavage activity as manifested by
the dramatic reduction in MAC deposits relative to
C3c/C4 deposits. In these in vitro studies, administra-
Fig 6. Analysis of diaphragm tissue from mice passively immunized with CGM3 (1.5mg total dose for 16 hours) followed by a single intravenous bolus of APT070 (580␮g total dose) either 1 hour before or concomitantly with NHS (0.5ml intraperitoneally).
Complement C3c and membrane attack complex (MAC) were significantly reduced when APT070 was either preadministered or
coadministered, but they were significantly more so in the former. Similarly, neurofilament (NF) immunoreactivity was significantly
better preserved with preadministered APT070 compared with coadministered APT070, with both conditions being significantly
superior to placebo. nAChR ⫽ nicotinic acetylcholine receptor. Mann–Whitney U test: asterisk indicates significantly different from
BSA, and number sign indicates significantly different from APT070 preadministration.
tion of APT070 at 48␮g/ml protected both axons and
pSCs at the NMJ, and a similar dose was used in subsequent in vivo studies.
A passive immunization model was used to investigate
APT070 in vivo. In this acute model, complementmediated damage occurs rapidly after NHS administration (ie, within 1 hour). In this model, although small
amounts of mouse complement products, including
MAC, may be deposited at nerve terminals, the majority
of MAC has its origin from the heterologous NHS that
is required for disease induction in the mouse. Thus, a
particular value of this model is that APT070 effects are
being largely assessed on human complement activation
and are therefore of greater relevance to its possible therapeutic effects in human disease.
In initial in vivo studies, APT070 was given prophylactically, 2 hours before dosing with NHS, to ensure a
wide distribution throughout the mouse and to create
optimal conditions for a maximal protective effect.
Subsequently, APT070 was given at the same time as
NHS as the source of complement to create a more
realistic clinical situation. In these passively immunized
mice, the effect of APT070 was that MAC deposition
at nerve terminals was greatly reduced, both when
preadministered and coadministered with NHS, and
both neuronal and pSC integrity was significantly preserved. Importantly, no treatment could be expected to
acutely reverse the severe membrane injury mediated
by MAC pores, but that would ideally be able to halt
the progression of any ongoing MAC pore formation,
thereby allowing initiation of intrinsic repair mechanisms.
Clinical relevance was also assessed by using MFS
serum as a source of complement-fixing anti-GQ1b antibody, and APT070 was found to be similarly protective. Comparisons with other available treatments, including IV immunoglobulin,9,24,25 were not assessed in
this study but would form important considerations
when designing clinical trials.
APT070 contains a membrane-localizing motif,
which is believed to target the complement regulatory
elements of the compound to cell membranes, especially those containing acidic phospholipids.22 Previous
studies on APT070 have shown that it can be incorporated into renal membranes.17 In this study, we have
not directly demonstrated APT070 incorporation into
mouse motor nerve terminal membranes. However, by
inference from the therapeutic benefit, it is likely that
APT070 has been incorporated at this site. APT070
may also have some complement regulatory activity in
a soluble phase that would be occurring in the extracellular fluid environment surrounding the motor
nerve terminal. Unlike molecules such as cobra venom
factor, APT070 does not deplete the serum or tissues
of complement components, but rather simply inhibits
the progression of the activation pathway, and therefore is capable of acting locally at sites of complement
activation.16 Regardless of the precise location of complement inhibition, APT070 clearly has a dramatically
beneficial effect in this model system.
Current treatment of GBS uses the multimechanistic
approaches of plasma exchange and IV immunoglobulin therapy.26 By contrast, these data demonstrated a
clearly protective role through complement inhibition
for APT070 at the NMJ that may augment other approaches such as inhibition of antibody binding or se-
Halstead et al: Complement Inhibition in MFS
lective plasma exchange.19 APT070 may be a valuable
drug for the treatment of GBS, as well as other acute
and chronic complement-mediated neuropathies and
nerve terminal disorders in which active complement
deposition is a pathophysiological feature.
Richard A. Smith has a commercial interest in Inflazyme Pharmaceuticals Ltd that freely provided the
APT070 to the Willison Laboratory where these experiments were conducted.
This work was supported by grants from the Wellcome Trust
(060349, S.K.H., P.D.H., E.R.W., H.J.W.) and Guillain–Barré
Syndrome Support Group UK (J.A.G., H.J.W.).
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