Sponsored document from
Molecular Immunology
Published as: Mol Immunol. 2008 January ; 45(2): 395–405.
Sponsored Document
Prevention of experimental autoimmune myasthenia gravis by rat
Crry-Ig: A model agent for long-term complement inhibition in vivo
Natalie J. Hepburna, Jayne L. Chamberlain-Banouba, Anwen S. Williamsb, B. Paul
Morgana, and Claire L. Harrisa⁎
aDepartment of Medical Biochemistry and Immunology, School of Medicine, Cardiff University, Heath Park,
Cardiff CF14 4XN, United Kingdom.
bDepartment
of Rheumatology, School of Medicine, Cardiff University, Heath Park, Cardiff CF14 4XN,
United Kingdom.
Abstract
Sponsored Document
Despite its vital role in innate immunity, complement is involved in a number of inflammatory
pathologies and has therefore become a therapeutic target. Most agents generated for anticomplement therapy have short half-lives in plasma, or have been of mouse or human origin, thereby
limiting their use either to murine models of disease or to short-term therapy. Here we describe the
generation of a long-acting rat therapeutic agent based on the rat complement inhibitor, Crry.
Characterisation of various soluble forms of Crry demonstrated that the amino-terminal four shortconsensus repeat domains were required for full regulatory and C3b-binding activities. Fusion of
these domains to rat IgG2a Fc generated an effective complement inhibitor (rCrry-Ig) with a
circulating half-life prolonged from 7 min for Crry alone to 53 h for rCrry-Ig. Systemic administration
of rCrry-Ig over 5 weeks generated a weak immune response to the recombinant agent, however this
was predominantly IgM in nature and did not neutralise Crry function or cause clearance of the agent
from plasma. Administration of rCrry-Ig completely abrogated clinical disease in a rat model of
myasthenia gravis whereas soluble Crry lacking the immunoglobulin Fc domain caused a partial
response. rCrry-Ig not only ablated clinical disease, but also prevented C3 and C9 deposition at the
neuromuscular junction and inhibited cellular infiltration at this site. The long half-life and low
immunogenicity of this agent will be useful for therapy in chronic models of inflammatory disease
in the rat.
Sponsored Document
Abbreviations
AChR, acetylcholine receptor; ADEAE, antibody-augmented demyelinating EAE; AP, alternative
pathway; BuTx, Bungarotoxin; C, complement; CHO, Chinese hamster ovary; CP, classical pathway;
CR1, complement receptor 1; CRegs, complement regulators; DAF, decay accelerating factor; EAE,
experimental autoimmune encephalitis; EAMG, experimental autoimmune myasthenia gravis;
FACS, fluorescent activated cell sorting; HRPO, horseradish peroxidase; MAC, membrane attack
complex; NMJ, neuromuscular junction; OPD, 1,2-phenylenediamine dihydrochloride; PBS,
phosphate-buffered saline; sCR1, soluble recombinant CR1; SCR, short-consensus repeat; sCrry,
© 2008 Elsevier Ltd.
⁎Corresponding author. Tel.: +44 2920745254; fax: +44 2920744001. [email protected].
This document was posted here by permission of the publisher. At the time of deposit, it included all changes made during peer review,
copyediting, and publishing. The U.S. National Library of Medicine is responsible for all links within the document and for incorporating
any publisher-supplied amendments or retractions issued subsequently. The published journal article, guaranteed to be such by Elsevier,
is available for free, on ScienceDirect.
Hepburn et al.
Page 2
soluble recombinant form of rat Crry containing the four N-terminal SCRs; SPR, surface plasmon
resonance
Keywords
Sponsored Document
Complement; Crry; EAMG; Myasthenia gravis; Therapy
1 Introduction
Sponsored Document
The complement (C) system forms part of the innate immune system; activation brings about
target cell death or damage, opsonisation of pathogens, induction of inflammatory responses
and clearance of immune complexes. The critical activation step is enzymatic cleavage of C3
or C4 by multi-molecular enzymes termed convertases, and deposition of the active
components (C3b or C4b, respectively) on the target membrane. Nascent C3b and C4b bind
indiscriminately to pathogens and adjacent host cells. To prevent inappropriate damage, selfcells express on their membranes complement regulatory proteins (CReg). These function by
inactivating the convertases formed during C activation or by preventing formation of the
membrane attack complex (MAC). In humans, membrane cofactor protein (MCP; CD46),
decay accelerating factor (DAF; CD55) and complement receptor 1 (CR1; CD35) inactivate
the convertases by accelerating their natural decay or by acting as a cofactor for the factor I
(fI) mediated cleavage of C3b and C4b (Kim and Song, 2006). Rodents have a unique regulator
of the convertases called Crry which both increases decay of the convertases as well as acting
as a cofactor for fI (Wong and Fearon, 1985; Quigg et al., 1993; Takizawa et al., 1994; Li et
al., 1993; Kim et al., 1995). All these CReg are comprised from short-consensus repeats (SCR),
domains of approximately 60 amino acids in which the functional activity of the protein resides.
CR1 consists of 30 SCR domains, DAF and MCP have four whilst rat Crry is comprised from
either 6 or 7 SCRs due to alternative splicing of the gene.
Sponsored Document
In health, CReg are sufficient to protect the host from complement damage. However, in
disease, dysregulated C activation can damage self-cells and exacerbate pathology.
Complement is implicated in ischaemia/reperfusion injury, transplant rejection and a number
of inflammatory diseases, such as systemic lupus erythematosus, autoimmune arthritis,
multiple sclerosis and myasthenia gravis where activation triggers and sustains a ‘vicious cycle’
of inflammation and tissue damage (Morgan and Harris, 2003). In myasthenia gravis, an
antibody-mediated disease of the neuromuscular junction (NMJ), C activation products have
been found at the motor end plate (Lennon et al., 1978; Engel and Arahata, 1987; Sahashi et
al., 1980). Involvement of C in pathology is demonstrated in rodent models by protective effects
of C5 or C6 deficiency (Christadoss, 1988; Chamberlain-Banoub et al., 2006), amelioration of
disease by anti-C therapeutics targeted either to activation pathways or to C5 (Piddlesden et
al., 1996; Hepburn et al., 2007), and enhancement of disease in mice deficient in CReg (Morgan
et al., 2006).
Due to the implication of C in inflammatory diseases a number of different anti-C therapeutics
has been developed. Some of these reagents have reached clinical trials. A soluble, recombinant
form of CR1, sCR1, has been tested in numerous animal models and reached clinical trials for
acute conditions such as adult respiratory distress syndrome and cardiopulmonary bypass
(Zimmerman et al., 2000; Rioux, 2001). Pexelizumab, a single chain Fv (scFv) which binds
human C5 and prevents its cleavage, is in advanced stages of development for use in myocardial
infarction (Whiss, 2002; Fleisig and Verrier, 2005). These reagents have been limited to acute
therapies as they have short circulating half-lives necessitating constant administration in vivo.
Eculizumab, an intact monoclonal antibody that binds C5, has a longer half-life in vivo and
Published as: Mol Immunol. 2008 January ; 45(2): 395–405.
Hepburn et al.
Page 3
has been particularly successful in treatment of paroxysmal nocturnal haemoglobinuria and is
being trialled in other chronic autoimmune indications (Hill et al., 2005; Kaplan, 2002).
Sponsored Document
A number of different approaches have been used to increase the circulating half-lives of
therapeutic reagents. One approach has been generation of antibody-like molecules where the
therapeutic moiety replaced the Fab arms of antibody. This approach was used to increase the
circulating half-life of the C inhibitor, DAF, from 20 min to 33 h and to create a murine CrryIg with a half-life of 40 h (Harris et al., 2002; Quigg et al., 1998). Such reagents have proved
therapeutically effective in antigen-induced arthritis, glomerulonephritis and intestinal
ischaemia–reperfusion injury (Harris et al., 2002; Quigg et al., 1998; Rehrig et al., 2001).
Reagents generated to date have been based upon human or mouse proteins, limiting their use
in rat models due to their immunogenicity. In order to enable the testing of such reagents in
rats we generated rCrry-Ig, a rat CReg-rat Fc protein consisting of rat Crry and the Fc of rat
IgG2a. We compared rCrry-Ig to soluble Crry lacking an Fc domain both in vitro and in vivo.
Tethering Crry to the Fc created a reagent with a greatly increased in vivo half-life that was
capable of protecting against disease in a model of myasthenia gravis. This result paves the
way for testing such reagents in chronic disease models in the rat.
2 Materials and methods
2.1 Materials
Sponsored Document
Chemicals and reagents were from Fisher Scientific (Loughborough, UK) or Sigma (Poole,
UK) unless otherwise stated. All tissue culture reagents and plastics were from Invitrogen Life
Technologies (Paisley, UK). pDR2ΔEF1α was a gift from Dr. I. Anegon (INSERM U437,
Nantes, France; Charreau et al., 1994). The cell line expressing the monoclonal antibody,
TLD1C11, was from Prof. W. Hickie (Dartmouth, USA). Chinese hamster ovary (CHO) cells
expressing surface-bound rat Crry were from Dr. O.B. Spiller (Cardiff, UK). Sheep
erythrocytes in Alsever's solution were from TCS microbiology (Claydon, UK), guinea pig
erythrocytes and rat serum were from the local animal facility. Rabbit anti-rat Crry polyclonal
antiserum and polyclonal sheep anti-human C9 were prepared in-house using standard
immunisation procedures. The hybridoma expressing mouse anti-human C3b (C3-30) was a
gift from Novartis (Horsham, UK; Kemp et al., 1992). Polyclonal goat anti-rat C3c was from
Nordic Laboratories (Copenhagen, Denmark) and polyclonal sheep anti-human C3c antiserum
was obtained from The Binding Site (Birmingham, UK). The rat hybridoma TIB-175, secreting
the anti-AChR mAB35 was obtained from the American Tissue Culture Collection; antibody
was purified as described previously (Tzartos et al., 1987; Chamberlain-Banoub et al., 2006).
Mouse anti-rat CD68 was obtained from Serotech (Oxford, UK).
Sponsored Document
PBS is 8.1 mM Na2PO4, 1.5 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, pH 7.4. CFD
(Complement fixation diluent; Oxoid) is 2.8 mM barbituric acid, 145.5 mM NaCl, 0.8 mM
MgCl2, 0.3 mM CaCl2, 0.9 mM sodium barbital, pH 7.4. Alternative pathway (AP) buffer is
5 mM sodium barbitone, 150 mM NaCl, 7 mM MgCl2, 10 mM EGTA, pH 7.4. FACS buffer
is PBS containing 0.1% sodium azide, 1% BSA and 10 mM EDTA. Borate-buffered saline is
100 mM boric acid, 25 mM sodium borate, 75 mM NaCl, pH 8.3.
2.2 Generation of cell lines expressing recombinant proteins
DNA encoding the signal peptide and the three (C-terminal residue Leu193) or four (C-terminal
residue Lys256) N-terminal SCR of rat Crry was amplified by PCR from rat oligodendroglioma
cell cDNA (33B; obtained from ECACC), the antisense primer encoded a stop codon. DNA
was ligated into the pDR2ΔEF1α expression vector. To generate the rCrry-Ig protein, DNA
encoding the Fc (hinge, CH2 and CH3 domain) of rat IgG2a was ligated into pDR2ΔEF1α to
generate the pDR2-Fc vector and DNA encoding the signal peptide and four SCR of rat Crry
Published as: Mol Immunol. 2008 January ; 45(2): 395–405.
Hepburn et al.
Page 4
(without stop codon) was cloned into the vector upstream and in frame with the DNA encoding
the Fc. Vent proof reading polymerase (NEB, Hitchin, UK) was used for the PCR and
sequencing confirmed that no errors had been introduced. CHO cells were transfected with the
vectors using LipofectAMINE (Invitrogen Life Technologies) according to the manufacturer's
instructions. Stable lines were established by selection with 400 μg/ml Hygromycin B.
Sponsored Document
2.3 Protein purification
Supernatant was collected from transfected cells and purified by affinity chromatography on
monoclonal anti-Crry. This column was generated by coupling 100 mg TLD1C11 (anti-Crry)
to CNBr-activated Sepharose (GE Healthcare, St. Albans, UK) according to the manufacturer's
instructions. Supernatant was passed over the column, the column washed with PBS and the
recombinant proteins eluted using 0.1 M glycine/HCl pH 2.5. Eluted protein was neutralised
with 1 M Tris, dialysed into PBS and concentrated by ultrafiltration.
2.4 SDS-PAGE and Western blot analysis
Sponsored Document
Purified proteins were subjected to SDS-PAGE and separated proteins were stained with
Coomassie R-250. For Western blot analysis, proteins were transferred to nitrocellulose (VWR,
Lutterworth, UK), blocked with 5% (w/v) non-fat milk in PBS and incubated with the relevant
antibodies. To detect rCrry-Ig the membrane was incubated with donkey anti-rat IgG HRPO
(Jackson ImmunoResearch, Luton, UK) at 1/10,000 in 5% PBS milk. To detect sCrry proteins,
the membrane was incubated with TLD1C11 at 1 μg/ml in 5% PBS milk followed by donkey
anti-mouse IgG HRPO (Horseradish peroxidase; Jackson ImmunoResearch) at 1/10,000 in 5%
PBS milk. To detect C3 fragments the membrane was incubated with polyclonal anti-human
C3c antiserum (cross-reactive with rat C3) at 1/1000 followed by anti-sheep Ig HRPO (Binding
site, Birmingham, UK) at 1/1000. Between primary and secondary antibody incubations and
prior to development membranes were washed three times in PBS/0.1% Tween-20 followed
by three washes in PBS. Bands were visualised using ECL (GE Healthcare) and
autoradiographic film.
2.5 SPR analysis
Sponsored Document
The surface plasmon resonance (SPR) analysis was carried out on a Biacore 3000 (Biacore
International, Uppsala, Sweden) following previously reported methods (Harris et al., 2007).
In brief, a small nidus of human C3b (50RU) was amine-coupled to a chip surface, human
factors B and D were flowed to form a C3 convertase which in turn was used to deposit rat
C3b (1000RU) on the chip surface. Rat C3b was covalently coupled to the surface by
nucleophilic attack on the internal thioester, giving native orientation. Human C3b does not
interact with rat Crry, therefore there is no interference in the Crry-rat C3b interaction (our
unpublished observation). Different concentrations of the sCrry proteins were separately
flowed over the chip surface and the dissociation equilibrium constant (KD) calculated from
steady state kinetics.
2.6 Haemolysis assays
Classical pathway (CP) haemolysis assays were carried out as described previously (Harris et
al., 2002). In brief, antibody-coated sheep erythrocytes were incubated with a concentration
of serum titrated to give 60% lysis and different concentrations of the test protein. Similarly,
in the AP haemolysis assay, 1.5% guinea pig erythrocytes were incubated with a concentration
of rat serum titrated to give 60% lysis and different concentrations of the test protein; the AP
assay was carried out in AP buffer. Lysis was calculated by release of haemoglobin from
erythrocytes.
Published as: Mol Immunol. 2008 January ; 45(2): 395–405.
Hepburn et al.
Page 5
Sponsored Document
The in vivo complement inhibitory activity of the proteins was determined following a
previously reported method (Hepburn et al., 2007). In brief, antibody-coated sheep erythrocytes
were incubated with differing concentrations of pre-treatment serum or with serum from rats
administered the recombinant proteins. For each time point after reagent administration, the
concentration required to give 50% lysis was calculated and expressed relative to the
concentration of pre-treatment serum required to give the same degree of lysis.
2.7 Cofactor assay
The ability of the recombinant forms of Crry to act as a cofactor for the fI-mediated cleavage
of C3b-like C3 (C3MA) was investigated following a previously reported method (Fraser et al.,
2002). C3 was incubated with methylamine in borate-buffered saline pH 8.3 to form C3MA.
C3MA (2.5 μg) was incubated with fI (0.5 μg) and a test Crry protein (2 μg) for 4 h at 37 °C.
Cleavage by fI was detected by resolving the incubations on a reducing SDS-PAGE gel and
Western blotting. Cleavage of the α-chain was detected by probing the blot with polyclonal
anti-human C3c antiserum.
2.8 In vivo half-life
Sponsored Document
The in vivo half-lives of the recombinant Crry proteins were established as described
previously (Harris et al., 2002). Five female Lewis rats (Charles River, average weight 180 g)
were administered 125I labelled rCrry-Ig or sCrry intravenously. Plasma was removed at
specific times following administration, protein was precipitated and protein-bound 125I
calculated. Clearance was calculated according to the method of Fraker and Speck (1978).
2.9 Immune response to therapeutic reagent
Female Wistar rats (five per group, average weight 180 g; Charles River) were injected with
1 mg/kg rCrry-Ig on days 0, 3, 7, 14, 21 and 28 through the tail vein. Blood samples were
collected via the tail vein at specific time points and serum isolated. An ELISA was used to
detect the generation of anti-rCrry-Ig antibodies. The ELISA plate was coated with rCrry-Ig,
incubated with test serum (diluted 1/50) from each time point and rCrry-Ig specific antibodies
were detected using HRPO-conjugated antibodies against rat IgM, IgG1, IgG2b or IgG2c
(Southern Biotech, Birmingham, AL, USA). The ELISA was developed using OPD and the
reaction stopped with 10% sulphuric acid. The presence of rCrry-Ig in the serum was detected
using an ELISA plate coated with TLD1C11 (mouse anti-rat Crry), incubated with serum or
standard concentrations of rCrry-Ig and probed with anti-rat IgG HRPO (Jackson
ImmunoResearch). rCrry-Ig present in serum was quantified from the standard curve.
Sponsored Document
To establish whether the generated antibodies were function-blocking, CHO cells expressing
transmembrane rat Crry (50 μl at 2 × 106 ml−1) were incubated with serum (10%, v/v) from
test animals or rabbit polyclonal anti-Crry antiserum (10%, v/v) for 1 h at 4 °C. Cells were also
incubated with non-immune rabbit or rat serum as negative controls, all sera were heat
inactivated. Following three washes in FACS buffer, the cells were incubated at 37 °C for
30 min in 2% (v/v) rat serum. Cells were washed and stained with mouse anti-human C3b
(C3-30; cross-reactive with rat C3b) for 30 min at 4 °C followed by an incubation with donkey
anti-mouse Ig RPE (Dako) for 1 h at 4 °C. The cells were washed, resuspended and analysed
on a Becton Dickinson FACSCalibur.
2.10 In vivo complement inhibitory function
Female Lewis rats (five per group, average weight 180 g; Charles River) were injected
intravenously with rCrry-Ig at 20 mg/kg or sCrry at 10 mg/kg. Blood was collected from the
tail vein at specific time points, serum was isolated and processed as described in Section 2.6
above.
Published as: Mol Immunol. 2008 January ; 45(2): 395–405.
Hepburn et al.
Page 6
2.11 Experimental autoimmune myasthenia gravis (EAMG)
Sponsored Document
EAMG was induced in female Lewis rats (Charles River; average weight 180 g) as described
previously (Chamberlain-Banoub et al., 2006; Piddlesden et al., 1996). In brief, disease was
induced on day 0 using 1 mg/kg mAb35 in PBS (i.p.). mAb35, produced against Electrophorus
electricus electric organ muscle-type nicotinic AChR, binds the alpha subunit of the AChR
and cross-reacts with rat AChR. At the same time as disease induction, animals were given
PBS (1 ml), rCrry-Ig (20 mg/kg) or sCrry (10 mg/kg; equimolar amount of Crry) through the
tail vein (six animals per group). Weight change and clinical score were monitored; increasing
clinical score was indicative of muscle weakness and paralysis. At 52 h post-induction, all the
PBS group were sacrificed due to disease severity along with one animal from each of the sCrry
and rCrry-Ig groups for comparative histology, the remaining animals were sacrificed at 183 h
post-induction. The soleus muscles were isolated from the sacrificed animals, flash frozen and
cut into 10 μm sections. The sections were processed and stained for C3, C9 and inflammatory
cell infiltrate as described previously (Hepburn et al., 2007).
3 Results
3.1 Generation of soluble Crry proteins
Sponsored Document
To generate the soluble Crry proteins, DNA encoding the three or four N-terminal SCR
domains of rat Crry was ligated into the pDR2ΔEF1α vector. CHO cells were transfected with
the plasmids and stable cell lines were generated. Supernatant was harvested and proteins were
purified using a monoclonal anti-Crry affinity column. Purified proteins were analysed by
SDS-PAGE (Fig. 1). The molecular weight of sCrry was 28 kDa under reduced and nonreduced conditions. The molecular weight of sCrry (3SCRs) was 21 kDa under non-reducing
conditions and 25 kDa under reducing conditions, an increase in weight characteristic of SCRcontaining proteins. The molecular weights were corroborated by mass spectrometry (sCrry
28.248 Da; sCrry (3SCRs) 21.216 Da; data not shown).
3.2 Definition of the minimal functional unit of Crry
Sponsored Document
To identify the minimal functional unit of Crry, the binding of sCrry and sCrry (3SCRs) to rat
C3b was compared using SPR. Rat C3b was coupled to the chip surface and different
concentrations of each protein flowed across the surface. The binding of sCrry (3SCRs) to C3b
was dramatically reduced compared to sCrry (Fig. 2a and b). The affinity of the two proteins
for C3b was calculated using steady state analysis (insets Fig. 2a and b). The dissociation
equilibrium constant (KD) for sCrry protein interaction with C3b was 5.1 × 10−6 M (χ2 = 0.4)
whereas that for the sCrry (3SCRs) protein was too low to calculate accurately; there was
minimal binding even at 33 μM. The sCrry (3SCRs) protein also demonstrated weak inhibitory
activity in AP haemolysis assays (IH50 = 545 nM ± 51; sCrry IH50 = 74.1 nM ± 1.8; data not
shown).
3.3 Inhibitory function of rCrry-Ig
Given that the four N-terminal SCRs of rat Crry were identified as the minimal functional unit
of Crry, a rCrry-Ig fusion protein was generated and purified as described for the sCrry reagents
(Fig. 3a). The Fc domain was added to prolong the in vivo half-life as that of 4SCR was
predicted to be short (Harris et al., 2002). The rCrry-Ig fusion protein had a molecular weight
of 140 kDa by non-reducing SDS-PAGE and 70 kDa when the disulphide bonds at the hinge
region were reduced (Fig. 3a; lane 2).
The abilities of rCrry-Ig and sCrry to inhibit C in vitro and the ability of the proteins to act as
cofactors for the fI-mediated cleavage of C3b were compared. Both reagents inhibited the CP
(Fig. 3b). The proteins were compared in terms of Crry molarity as approximately half the
mass of rCrry-Ig is comprised of the Fc. This comparison revealed that rCrry-Ig was more
Published as: Mol Immunol. 2008 January ; 45(2): 395–405.
Hepburn et al.
Page 7
potent than sCrry at inhibiting the CP (IH50 rCrry-Ig = 16.7 ± 0.6 nM; IH50
sCrry = 31.0 ± 0.7 nM). In the AP, sCrry was more effective at inhibiting lysis (Fig. 2c; rCrryIg IH50 = 167.8 ± 17.8 nM; sCrry IH50 = 74.1 ± 1.8 nM, p < 0.05).
Sponsored Document
In the cofactor assay, both proteins supported production of a 43 kDa fragment from rat
C3MA demonstrating that rCrry-Ig and sCrry are effective cofactors for the fI-mediated
cleavage of C3b (Fig. 3d). Incubation with the positive control, sCR1, also produced this
fragment.
3.4 Circulating half-life of rCrry-Ig
To assess the effect the Fc had on the in vivo clearance of Crry, rCrry-Ig and sCrry were
radiolabelled with 125I. A single intravenous dose of either reagent was administered to rats
and samples of blood were removed at intervals and protein bound counts were measured.
sCrry was rapidly cleared from the circulation with a half-life of 7 min. rCrry-Ig had a
significantly prolonged half-life of 53 h (p < 0.001) (Fig. 4).
3.5 Generation of an immune response to rCrry-Ig
Sponsored Document
We investigated whether generation of a Crry/antibody Fc chimeric protein created an
immunogenic neoepitope at the join between the two proteins. Animals were administered
rCrry-Ig intravenously on days 0, 3, 7 and weekly thereafter to 28 days. Blood was collected
at weekly intervals, serum harvested and production of antibodies was assessed by ELISA. A
weak immune response was detected to rCrry-Ig (Fig. 5a). All animals produced an IgM
response in early bleeds (from day 7) with subsequent class switching to IgG1, IgG2b and
IgG2c from day 21. The IgG2a response could not be assessed in this experiment as the ELISA
coat contained the Fc of IgG2a resulting in cross-reactivity. Coating an ELISA with rCrry-Ig,
sCrry and a control fusion protein with the same Fc revealed that the immune response had
been mounted against all domains in the chimeric molecule and not just the join between Crry
and the Fc domain (data not shown).
Sponsored Document
The ability of these antibodies to inhibit the function of rCrry-Ig was investigated using CHO
cells expressing surface-bound Crry. Incubation of CHO cells with test sera resulted in antibody
binding (data not shown), deposition of C3 on the cell surface was assessed following
incubation with rat serum as a source of C. Incubation with polyclonal anti-Crry as a positive
control resulted in enhanced C3 deposition compared to buffer or serum only (Fig. 5b), whereas
pre-incubation with test sera did not enhance C3 deposition demonstrating the non-function
blocking nature of the antibodies (Fig. 5c). In support of this, quantification by ELISA of rCrryIg remaining in the circulation throughout 5 weeks of repeated administration demonstrated
steady state levels of reagent without evidence of enhanced clearance (Table 1).
3.6 C inhibition in vivo
A single dose of the proteins was administered intravenously to normal rats in order to assess
their C inhibitory ability. Serum was harvested at specific time intervals and haemolytic activity
was assessed. Administration of sCrry had no discernable effect on serum haemolytic activity
even at the early time points, likely due to rapid clearance, whereas animals administered rCrryIg had reduced serum haemolytic activity when compared to a pre-bleed, 40–50% for the first
8 h after administration (p < 0.05; Fig. 6). Reduction of serum haemolytic activity was detected
for 24 h post-administration. It should be noted that this assay, because it involves dilution of
test serum, underestimates the true inhibitory effect of the agent, which is highly concentration
dependent.
Published as: Mol Immunol. 2008 January ; 45(2): 395–405.
Hepburn et al.
Page 8
3.7 rCrry-Ig protection against EAMG
Sponsored Document
The therapeutic effect of rCrry-Ig and sCrry was assessed in a rat model of EAMG. Untreated
animals rapidly developed severe weight loss and paralysis and had to be sacrificed by 52 h.
Therapy with rCrry-Ig prevented weight loss (Fig. 7a) and totally ablated clinical disease (Fig.
7b). Animals treated with sCrry had mild disease with weight loss and an increasing clinical
score until 52 h post-induction when they began to recover; clinical disease was always less
severe than in control animals. Immunohistochemical analysis at the end plate demonstrated
significantly reduced C3 and C9 deposition in the rCrry-Ig animals compared to the control
and sCrry animals (Fig. 8; Table 2). Immunohistochemical analysis and H&E staining
demonstrated reduced levels of cellular infiltration in the rCrry-Ig-treated animals (Table 2).
4 Discussion
Sponsored Document
The demonstration of an involvement of C in various inflammatory pathologies has been the
driving force behind the generation of recombinant biologicals designed to inhibit C in disease.
Amongst these reagents are soluble recombinant CReg, including sCR1. This reagent has been
protective against tissue injury in several animal models including EAMG, antibodyaugmented demyelinating experimental allergic encephalomyelitis (ADEAE), antigeninduced arthritis (AIA), glomerulonephritis and ischaemia/reperfusion injuries (Piddlesden et
al., 1994, 1996; Goodfellow et al., 1997; Weisman et al., 1990). However, the use of soluble
recombinant CRegs has been limited by rapid clearance in vivo resulting in the need for
repeated administration, rendering them unsuitable for long-term therapy. A number of
different approaches have been employed to increase the half-lives of such reagents. These
include addition of an albumin binding site to CR1, targeting of CReg to membranes or the
site of C activation and generation of CReg-Ig fusion proteins (Makrides et al., 1996; Smith
and Smith, 2001; Song et al., 2003; Atkinson et al., 2005).
Sponsored Document
Fusion of proteins to Ig domains has been used widely to increase the half-life of many
therapeutic proteins, including CTLA-4 and TNF-α receptor (Linsley et al., 1992; Pugsley,
2001). We have previously described CReg-Ig fusion proteins where the rat CReg, DAF or
CD59, was fused to a human immunoglobulin Fc domain (Harris et al., 2002). DAF-Ig had
prolonged circulating half-life and was effective in a rat model of AIA; we could not test this
reagent in models of chronic disease due to immunogenicity of the human Fc domain. Others
have generated an Fc fusion protein comprising mouse Crry and murine IgG1 Fc, this has also
proven efficacious in many murine models of disease (Quigg et al., 1998; Rehrig et al., 2001;
Leinhase et al., 2006). We describe here the generation of a reagent comprising rat Crry fused
to rat IgG2a Fc; as both domains are derived from rat proteins this agent should have low
immunogenicity and be suitable for therapy in both acute and chronic models of inflammatory
disease in the rat.
Crry contains both decay accelerating and cofactor activities and is a powerful regulator of C
(Li et al., 1993; Kim et al., 1995). Therapeutic molecules combining both activities in one
molecule have proven to be more effective than those comprising one activity alone
(Christiansen et al., 1996; Higgins et al., 1997). We first defined the domains of rat Crry
necessary for regulatory function; our previous findings have shown that the 4 N-terminal SCRs
were capable of regulating the CP and act as a cofactor for the fI mediated cleavage of C3b
(Fraser et al., 2002). Recombinant Crry constructs were generated that comprised either the
first three or four N-terminal SCR of Crry (Fig. 1), these were compared for their ability to
bind the ligand, rat C3b. The affinity of sCrry for C3b was 5.1 μM, deletion of the fourth SCR
markedly decreased ability of the reagent to bind C3b, revealing that the fourth N-terminal
SCR contains an important C3b binding site (Fig. 2). We therefore generated a construct
comprising the N-terminal 4SCR fused to rat IgG2a Fc and tested its ability to inhibit C in vitro
(Fig. 3). Both rCrry-Ig and sCrry inhibited in a CP haemolysis assay, rCrry-Ig showed improved
Published as: Mol Immunol. 2008 January ; 45(2): 395–405.
Hepburn et al.
Page 9
Sponsored Document
function presumably due to the ability of the two Crry moieties in the protein to act
synergistically. A similar effect has been noted previously with CR1 where multiple binding/
functional sites enhanced inhibition of the C3 and C5 convertases (Krych-Goldberg et al., 1999,
2005). In the case of CR1, increased binding avidity may explain enhanced regulation of the
C3 convertase, whereas ability to bind to both C3b/C4b subunits within the enzyme may
explain enhanced regulation of the C5 convertase where exact spacing between the active sites
is crucial for efficient decay. Either mechanism could be responsible for enhanced function of
Crry-Ig in the CP that we describe here. Inhibition of C by rCrry-Ig in an AP haemolysis assay
was slightly less efficient when compared to the same reagent lacking an Fc domain. We have
demonstrated that SCR4 of Crry is required to bind C3b (Fig. 2), location of this domain close
to the antibody hinge and Fc may therefore restrict access to the ligand. This effect has been
noted previously with DAF-Ig where steric hindrance imparted by tethering DAF to the Fc
reduced function two-fold (Harris et al., 2002).
Sponsored Document
Tethering the four N-terminal SCRs of rat Crry to an Fc dramatically increased the circulating
half-life as expected (Fig. 4), an effect of increasing size of the protein (limiting filtration by
the kidney) and interaction with Fc receptors which serve to actively retain the protein in the
circulation (Ghetie and Ward, 1997; Ghetie and Ward, 2000). Long-term administration of
rCrry-Ig resulted in a weak immune response, although the antibodies generated were
predominantly IgM and did not result in clearance of the reagent or inhibit its function (Fig.
5). Similar immune responses have been noted to other therapeutics, but this has not limited
their use in the clinic. The immune response may have been caused by different posttranslational modifications carried out by the CHO cells used to express the reagent in vitro.
Others have shown that high concentration of ammonium ions, produced by cellular
metabolism, alters glycosylation in CHO cells (Chen and Harcum, 2005). Recombinant follicle
stimulating hormone (FSH) expressed in CHO cells differed from native FSH in terms of
sialyation of carbohydrates (Recombinant Human FSH Product Development Group, 1998).
Selection of an alternative cell line for expression of the recombinant therapeutic may ablate
the observed immunogenicity.
Sponsored Document
At the doses administered in our experiments, animals treated with rCrry-Ig had reduced serum
C activity (50% when compared to pre-bleed; Fig. 6), this was sufficient to prevent any clinical
score in rat EAMG (Fig. 7). Treatment with rCrry-Ig was so effective that no C3 or C9
deposition was evident at the NMJ (Fig. 8). Despite its short half-life, sCrry reduced clinical
score, although this was not as dramatic as therapy with rCrry-Ig. As EAMG is an acute disease,
the presence of sCrry during the initial C activation may have reduced C amplification and
therefore lessened the degree of injury. We have previously shown that an acute phase response
early in the course of this disease increases plasma complement activity (Hepburn et al.,
2007), the presence of sCrry at initial stages may dampen the acute response with therapeutic
benefit. Both C3b and C9 deposition were detectable at the NMJ in the sCrry-treated group,
showing that the reagent did not completely halt C activation suggesting it had a role in
regulating the initial injury during disease establishment. The C3-staining evident in Fig. 8
may represent iC3b generated through the cofactor activity of sCrry, again accounting for some
therapeutic benefit in the acute phase of the disease. The degree of cellular infiltration differed
between the two groups, rCrry-Ig-treated animals had virtually no cellular infiltration, unlike
the sCrry-treated animals. This reduction in cellular infiltration again confirms the efficacy of
rCrry-Ig in regulation of C and reduction in levels of the anaphylatoxins C3a and C5a as well
as MAC, all of which recruit inflammatory cells.
In conclusion, we have generated and characterised both in vitro and in vivo a long-lived and
powerful inhibitor of rat C, rCrry-Ig. This reagent totally ablated clinical score in a rat model
of acute inflammatory disease, EAMG, and inhibited C activation at the site of tissue damage
(NMJ). rCrry-Ig had low immunogenicity when given long-term, thereby facilitating its use in
Published as: Mol Immunol. 2008 January ; 45(2): 395–405.
Hepburn et al.
Page 10
rodent models of chronic inflammation, such as collagen-induced arthritis, and enabling the
efficacy and side-effects of prolonged therapy in treatment of chronic inflammatory diseases
to be assessed.
Acknowledgements
Sponsored Document
We thank Dr. Jim Neale for scoring the cellular infiltration and Nicholas Amos for invaluable advice on half-life
analysis. This work was funded by the Welsh Office of Research and Development (WORD; DTA01/1/014) and the
Wellcome Trust (068823).
References
Sponsored Document
Sponsored Document
Atkinson et al, 2005. Atkinson C. Song H. Lu B. Qiao F. Burns T.A. Holers V.M. Tsokos G.C. Tomlinson
S. Targeted complement inhibition by C3d recognition ameliorates tissue injury without apparent
increase in susceptibility to infection. J. Clin. Invest. 2005;115:2444–2453. [PubMed: 16127466]
(epub August 25, 2005)
Chamberlain-Banoub et al, 2006. Chamberlain-Banoub J. Neal J.W. Mizuno M. Harris C.L. Morgan B.P.
Complement membrane attack is required for endplate damage and clinical disease in passive
experimental myasthenia gravis in Lewis rats. Clin. Exp. Immunol. 2006;146:278–286. [PubMed:
17034580]
Charreau et al, 1994. Charreau B. Cassard A. Tesson L. Le Mauff B. Navenot J.M. Blanchard D. Lublin
D. Soulillou J.P. Anegon I. Protection of rat endothelial cells from primate complement-mediated
lysis by expression of human CD59 and/or decay-accelerating factor. Transplantation
1994;58:1222–1229. [PubMed: 7527603]
Chen and Harcum, 2005. Chen P. Harcum S.W. Effects of elevated ammonium on glycosylation gene
expression in CHO cells. Metab. Eng. 2005;24:24.
Christadoss, 1988. Christadoss P. C5 gene influences the development of murine myasthenia gravis. J.
Immunol. 1988;140:2589–2592. [PubMed: 3356901]
Christiansen et al, 1996. Christiansen D. Milland J. Thorley B.R. McKenzie I.F. Mottram P.L. Purcell
L.J. Loveland B.E. Engineering of recombinant soluble CD46: an inhibitor of complement
activation. Immunology 1996;87:348–354. [PubMed: 8778018]
Engel and Arahata, 1987. Engel A.G. Arahata K. The membrane attack complex of complement at the
endplate in myasthenia gravis. Ann. N.Y. Acad. Sci. 1987;505:326–332. [PubMed: 3318619]
Fleisig and Verrier, 2005. Fleisig A.J. Verrier E.D. Pexelizumab—a C5 complement inhibitor for use in
both acute myocardial infarction and cardiac surgery with cardiopulmonary bypass. Expert Opin.
Biol. Ther. 2005;5:833–839. [PubMed: 15952913]
Fraker and Speck, 1978. Fraker P.J. Speck J.C. Protein and cell membrane iodinations with a sparingly
soluble chloroamide, 1,3,4,6-tetrachloro-3a,6a-diphrenylglycoluril. Biochem. Biophys. Res.
Commun. 1978;80:849–857. [PubMed: 637870]
Fraser et al, 2002. Fraser D.A. Harris C.L. Smith R.A. Morgan B.P. Bacterial expression and membrane
targeting of the rat complement regulator Crry: a new model anticomplement therapeutic. Protein
Sci. 2002;11:2512–2521. [PubMed: 12237472]
Ghetie and Ward, 1997. Ghetie V. Ward E.S. FcRn: the MHC class I-related receptor that is more than
an IgG transporter. Immunol. Today 1997;18:592–598. [PubMed: 9425738]
Ghetie and Ward, 2000. Ghetie V. Ward E.S. Multiple roles for the major histocompatibility complex
class I-related receptor FcRn. Annu. Rev. Immunol. 2000;18:739–766. [PubMed: 10837074]
Goodfellow et al, 1997. Goodfellow R.M. Williams A.S. Levin J.L. Williams B.D. Morgan B.P. Local
therapy with soluble complement receptor 1 (sCR1) suppresses inflammation in rat mono-articular
arthritis. Clin. Exp. Immunol. 1997;110:45–52. [PubMed: 9353148]
Harris et al, 2007. Harris C.L. Pettigrew D.M. Lea S.M. Morgan B.P. Decay-accelerating factor must
bind both components of the complement alternative pathway C3 convertase to mediate efficient
decay. J. Immunol. 2007;178:352–359. [PubMed: 17182573]
Harris et al, 2002. Harris C.L. Williams A.S. Linton S.M. Morgan B.P. Coupling complement regulators
to immunoglobulin domains generates effective anti-complement reagents with extended half-life
in vivo. Clin. Exp. Immunol. 2002;129:198–207. [PubMed: 12165074]
Published as: Mol Immunol. 2008 January ; 45(2): 395–405.
Hepburn et al.
Page 11
Sponsored Document
Sponsored Document
Sponsored Document
Hepburn et al, 2007. Hepburn N.J. Williams A.S. Nunn M.A. Chamberlain-Banoub J.C. Hamer J. Morgan
B.P. Harris C.L. In vivo characterization and therapeutic efficacy of a C5-specific inhibitor from
the soft tick Ornithodoros moubata. J. Biol. Chem. 2007;282:8292–8299. [PubMed: 17215252]
Higgins et al, 1997. Higgins P.J. Ko J.L. Lobell R. Sardonini C. Alessi M.K. Yeh C.G. A soluble chimeric
complement inhibitory protein that possesses both decay-accelerating and factor I cofactor
activities. J. Immunol. 1997;158:2872–2881. [PubMed: 9058824]
Hill et al, 2005. Hill A. Hillmen P. Richards S.J. Elebute D. Marsh J.C. Chan J. Mojcik C.F. Rother R.P.
Sustained response and long-term safety of eculizumab in paroxysmal nocturnal hemoglobinuria.
Blood 2005;106:2559–2565. [PubMed: 15985537]
Kaplan, 2002. Kaplan M. Eculizumab (alexion). Curr. Opin. Investig. Drugs 2002;3:1017–1023.
Kemp et al, 1992. Kemp P.A. Spragg J.H. Brown J.C. Morgan B.P. Gunn C.A. Taylor P.W.
Immunohistochemical determination of complement activation in joint tissues of patients with
rheumatoid arthritis and osteoarthritis using neoantigen-specific monoclonal antibodies. J. Clin.
Lab. Immunol. 1992;37:147–162. [PubMed: 1285067]
Kim and Song, 2006. Kim D.D. Song W.C. Membrane complement regulatory proteins. Clin. Immunol.
2006;118:127–136. [PubMed: 16338172]
Kim et al, 1995. Kim Y.U. Kinoshita T. Molina H. Hourcade D. Seya T. Wagner L.M. Holers V.M.
Mouse complement regulatory protein Crry/p65 uses the specific mechanisms of both human decayaccelerating factor and membrane cofactor protein. J. Exp. Med. 1995;181:151–159. [PubMed:
7528766]
Krych-Goldberg et al, 2005. Krych-Goldberg M. Hauhart R.E. Porzukowiak T. Atkinson J.P. Synergy
between two active sites of human complement receptor type 1 (CD35) in complement regulation:
implications for the structure of the classical pathway C3 convertase and generation of more potent
inhibitors. J. Immunol. 2005;175:4528–4535. [PubMed: 16177096]
Krych-Goldberg et al, 1999. Krych-Goldberg M. Hauhart R.E. Subramanian V.B. Yurcisin B.M.
Crimmins D.L. Hourcade D.E. Atkinson J.P. Decay accelerating activity of complement receptor
type 1 (CD35). Two active sites are required for dissociating C5 convertases. J. Biol. Chem.
1999;274:31160–31168. [PubMed: 10531307]
Leinhase et al, 2006. Leinhase I. Schmidt O.I. Thurman J.M. Hossini A.M. Rozanski M. Taha M.E.
Scheffler A. John T. Smith W.R. Holers V.M. Stahel P.F. Pharmacological complement inhibition
at the C3 convertase level promotes neuronal survival, neuroprotective intracerebral gene
expression, and neurological outcome after traumatic brain injury. Exp. Neurol. 2006;199:454–464.
[PubMed: 16545803]
Lennon et al, 1978. Lennon V.A. Seybold M.E. Lindstrom J.M. Cochrane C. Ulevitch R. The
immunopathology of myasthenia gravis. Role of complement in the pathogenesis of experimental
autoimmune myasthenia gravis. Hum. Pathol. 1978;9:541–551. [PubMed: 309428]
Li et al, 1993. Li B. Sallee C. Dehoff M. Foley S. Molina H. Holers V.M. Mouse Crry/p65.
Characterization of monoclonal antibodies and the tissue distribution of a functional homologue of
human MCP and DAF. J. Immunol. 1993;151:4295–4305. [PubMed: 7691944]
Linsley et al, 1992. Linsley P.S. Wallace P.M. Johnson J. Gibson M.G. Greene J.L. Ledbetter J.A. Singh
C. Tepper M.A. Immunosuppression in vivo by a soluble form of the CTLA-4 T cell activation
molecule. Science 1992;257:792–795. [PubMed: 1496399]
Makrides et al, 1996. Makrides S.C. Nygren P.A. Andrews B. Ford P.J. Evans K.S. Hayman E.G. Adari
H. Uhlen M. Toth C.A. Extended in vivo half-life of human soluble complement receptor type 1
fused to a serum albumin-binding receptor. J. Pharmacol. Exp. Ther. 1996;277:534–542. [PubMed:
8613964]
Morgan et al, 2006. Morgan B.P. Chamberlain-Banoub J. Neal J.W. Song W. Mizuno M. Harris C.L.
The membrane attack pathway of complement drives pathology in passively induced experimental
autoimmune myasthenia gravis in mice. Clin. Exp. Immunol. 2006;146:294–302. [PubMed:
17034582]
Morgan and Harris, 2003. Morgan B.P. Harris C.L. Complement therapeutics; history and current
progress. Mol. Immunol. 2003;40:159–170. [PubMed: 12914822]
Published as: Mol Immunol. 2008 January ; 45(2): 395–405.
Hepburn et al.
Page 12
Sponsored Document
Sponsored Document
Sponsored Document
Piddlesden et al, 1996. Piddlesden S.J. Jiang S. Levin J.L. Vincent A. Morgan B.P. Soluble complement
receptor 1 (sCR1) protects against experimental autoimmune myasthenia gravis. J. Neuroimmunol.
1996;71:173–177. [PubMed: 8982117]
Piddlesden et al, 1994. Piddlesden S.J. Storch M.K. Hibbs M. Freeman A.M. Lassmann H. Morgan B.P.
Soluble recombinant complement receptor 1 inhibits inflammation and demyelination in antibodymediated demyelinating experimental allergic encephalomyelitis. J. Immunol. 1994;152:5477–
5484. [PubMed: 8189065]
Pugsley, 2001. Pugsley M.K. Etanercept immunex. Curr. Opin. Investig. Drugs 2001;2:1725–1731.
Quigg et al, 1993. Quigg R.J. Galishoff M.L. Sneed A.E. Kim D. Isolation and characterisation of
complement receptor type 1 from rat glomerular epithelial cells. Kidney Int. 1993;43:730–736.
[PubMed: 8455373]
Quigg et al, 1998. Quigg R.J. Kozono Y. Berthiaume D. Lim A. Salant D.J. Weinfeld A. Griffin P.
Kremmer E. Holers V.M. Blockade of antibody-induced glomerulonephritis with Crry-Ig, a soluble
murine complement inhibitor. J. Immunol. 1998;160:4553–4560. [PubMed: 9574562]
Recombinant Human FSH Product Development Group, 1998. Recombinant Human FSH Product
Development Group. Recombinant follicle stimulating hormone: development of the first
biotechnology product for the treatment of infertility. Hum. Reprod. Update 1998;4:862–881.
[PubMed: 10098477]
Rehrig et al, 2001. Rehrig S. Fleming S.D. Anderson J. Guthridge J.M. Rakstang J. McQueen C.E. Holers
V.M. Tsokos G.C. Shea-Donohue T. Complement inhibitor, complement receptor 1-related gene/
protein y-Ig attenuates intestinal damage after the onset of mesenteric ischemia/reperfusion injury
in mice. J. Immunol. 2001;167:5921–5927. [PubMed: 11698469]
Rioux, 2001. Rioux P. TP-10 (AVANT immunotherapeutics). Curr. Opin. Investig. Drugs 2001;2:364–
371.
Sahashi et al, 1980. Sahashi K. Engel A.G. Lambert E.H. Howard F.M. Ultrastructural localization of
the terminal and lytic ninth complement component (C9) at the motor end-plate in myasthenia
gravis. J. Neuropathol. Exp. Neurol. 1980;39:160–172. [PubMed: 7373347]
Smith and Smith, 2001. Smith G.P. Smith R.A. Membrane-targeted complement inhibitors. Mol.
Immunol. 2001;38:249–255. [PubMed: 11532286]
Song et al, 2003. Song H. He C. Knaak C. Guthridge J.M. Holers V.M. Tomlinson S. Complement
receptor 2-mediated targeting of complement inhibitors to sites of complement activation. J. Clin.
Invest. 2003;111:1875–1885. [PubMed: 12813023]
Takizawa et al, 1994. Takizawa H. Okada N. Okada H. Complement inhibitor of rat cell membrane
resembling mouse Crry/p65. J. Immunol. 1994;152:3032–3038. [PubMed: 8144902]
Tzartos et al, 1987. Tzartos S. Hochschwender S. Vasquez P. Lindstrom J. Passive transfer of
experimental autoimmune myasthenia gravis by monoclonal antibodies to the main immunogenic
region of the acetylcholine receptor. J. Neuroimmunol. 1987;15:185–194. [PubMed: 3495549]
Waller, 1994. Waller, D. Balliere Tindall; London: 1994. Principles of Medical Pharmacology.
Weisman et al, 1990. Weisman H.F. Bartow T. Leppo M.K. Boyle M.P. Marsh H.C. Carson G.R. Roux
K.H. Weisfeldt M.L. Fearon D.T. Recombinant soluble CR1 suppressed complement activation,
inflammation, and necrosis associated with reperfusion of ischemic myocardium. Trans. Assoc.
Am. Phys. 1990;103:64–72. [PubMed: 2132543]
Whiss, 2002. Whiss P.A. Pexelizumab alexion. Curr. Opin. Investig. Drugs 2002;3:870–877.
Wong and Fearon, 1985. Wong W.W. Fearon D.T. p65; a C3b-binding protein on murine cells that shares
antigenic determinants with the human C3b receptor (CR1) and is distant from murine C3b receptor.
J. Immunol. 1985;134:4048–4056. [PubMed: 2580903]
Zimmerman et al, 2000. Zimmerman J.L. Dellinger R.P. Straube R.C. Levin J.L. Phase I trial of the
recombinant soluble complement receptor 1 in acute lung injury and acute respiratory distress
syndrome. Crit. Care Med. 2000;28:3149–3154. [PubMed: 11008973]
Published as: Mol Immunol. 2008 January ; 45(2): 395–405.
Hepburn et al.
Page 13
Sponsored Document
Fig. 1.
Analysis of purified sCrry reagents by SDS-PAGE. The proteins were purified from cell culture
supernatant by anti-Crry affinity chromatography and subjected to SDS-PAGE (11% gel).
Lanes 1 and 3, sCrry non-reduced and reduced, respectively. Lanes 2 and 4, sCrry (3SCRs)
non-reduced and reduced, respectively.
Sponsored Document
Sponsored Document
Published as: Mol Immunol. 2008 January ; 45(2): 395–405.
Hepburn et al.
Page 14
Sponsored Document
Sponsored Document
Fig. 2.
Sponsored Document
SPR analysis of the interaction of the sCrry reagents with rat C3b. Rat C3b was coupled
covalently to the chip surface using the internal thioester bond and the interaction with sCrry
(a) and sCrry (3SCRs) (b) was analysed. The affinity of the interaction was analysed by steady
state kinetics (see inset).
Published as: Mol Immunol. 2008 January ; 45(2): 395–405.
Hepburn et al.
Page 15
Sponsored Document
Sponsored Document
Sponsored Document
Fig. 3.
Inhibition of C by rCrry-Ig and sCrry. (a) rCrry-Ig was purified from cell culture supernatant
by anti-Crry affinity chromatography and analysed by SDS-PAGE under non-reducing (7.5%
gel; lane 1) or reducing conditions (10% gel; lane 2). (b) Antibody-sensitised erythrocytes were
attacked with rat C and the ability of rCrry-Ig (■) and sCrry (▴) to inhibit CP-mediated
haemolysis was compared with non-regulatory rat Ig (○). (c) Guinea pig erythrocytes were
incubated with rat serum and the ability of rCrry-Ig (■), sCrry (▴) and rat Ig (○) to prevent APmediated haemolysis was determined. Each data point represents the mean value ±1S.D. of
three determinations. (d) Methylamine inactivated C3 (C3MA) was incubated with fI in the
Published as: Mol Immunol. 2008 January ; 45(2): 395–405.
Hepburn et al.
Page 16
presence of a cofactor: sCR1 (lane 3), rCrry-Ig (lane 4) or sCrry (lane 5). Ability of the reagent
to act as cofactor was determined by SDS-PAGE on a 10% reduced gel followed by Western
blotting. The blot was probed with anti-human C3c (cross-reactive with rat C3) followed by
HRPO-conjugated anti-sheep Ig. Controls included C3MA only (lane 1) and C3MA with factor
I (lane 2).
Sponsored Document
Sponsored Document
Sponsored Document
Published as: Mol Immunol. 2008 January ; 45(2): 395–405.
Hepburn et al.
Page 17
Sponsored Document
Fig. 4.
In vivo half-life of rCrry-Ig and sCrry. Radiolabelled rCrry-Ig (■) and the sCrry reagent (▴)
were administered to rats intravenously, blood was removed at specific time points and protein
bound counts determined. This was expressed as a percent of protein bound counts present at
5 min post-administration and the beta phase half-life determined (Waller, 1994). Each data
point represents the mean of five animals ±1S.D.
Sponsored Document
Sponsored Document
Published as: Mol Immunol. 2008 January ; 45(2): 395–405.
Hepburn et al.
Page 18
Sponsored Document
Sponsored Document
Fig. 5.
Immunogenicity of rCrry-Ig. Rats were administered rCrry-Ig at days 0, 3, 7 and weekly
thereafter, and blood was collected at certain time points. The presence of different antibodies
against rCrry-Ig was detected by ELISA in the day 35 serum (a). Each bar represents a different
animal. (b) Rabbit anti-Crry antiserum (shaded; positive control; mean fluorescence 399.6),
buffer (solid line; mean fluorescence 51.3) or normal rabbit serum (dashed line; mean
fluorescence 38.5) was incubated with CHO cells expressing rat Crry prior to incubation with
active rat C. Deposition of C3b on the CHO cells was detected by flow cytometry. (c) Serum
from an immune animal (shaded; mean fluorescence 41.72 ± 7.09), buffer (solid line; mean
fluorescence 30.67) or normal rat serum (dashed line; mean fluorescence 38.5) was incubated
with the CHO-Crry cells. The cells were attacked with C and C3b deposition detected as above.
No increase in C3b deposition was observed following incubation with the immune sera. This
histogram is representative of results from five different animals.
Sponsored Document
Published as: Mol Immunol. 2008 January ; 45(2): 395–405.
Hepburn et al.
Page 19
Sponsored Document
Fig. 6.
Effect of rCrry-Ig on rat C activity in vivo. Rats were administered rCrry-Ig (■) or sCrry (◊)
and blood was collected at specific time intervals. The serum was isolated and used as a source
of C to lyse antibody-coated erythrocytes. The lytic ability of the serum was compared to that
of a pre-bleed. Each data point represents the mean of five animals ±1S.D.
Sponsored Document
Sponsored Document
Published as: Mol Immunol. 2008 January ; 45(2): 395–405.
Hepburn et al.
Page 20
Sponsored Document
Sponsored Document
Fig. 7.
Clinical assessment of rats with EAMG. EAMG was induced in rats and weight change (a) and
clinical score (b) were monitored. Control animals (●) rapidly developed a clinical score and
lost weight, these animals were sacrificed at 52 h and thereafter their score was scored 4. rCrryIg-treated animals (▴) were protected from disease whilst sCrry-treated animals (■) developed
slight clinical disease and then recovered.
Sponsored Document
Published as: Mol Immunol. 2008 January ; 45(2): 395–405.
Hepburn et al.
Page 21
Sponsored Document
Sponsored Document
Sponsored Document
Fig. 8.
C deposition at the NMJ. (a) The soleus muscle isolated from the control group, sCrry- and
rCrry-Ig-treated animals at 52 h was sectioned and stained for AChR using BuTx (left-hand
column) and C3 (right-hand column). (b) Similarly, the isolated soleus muscle was sectioned
and stained for AChR using BuTx (left-hand column) and C9 (right-hand column). Bar
represents 400 μm, representative images are shown.
Published as: Mol Immunol. 2008 January ; 45(2): 395–405.
Hepburn et al.
Page 22
Table 1
Serum concentrations of rCrry-Ig during the immune response study
Sponsored Document
Day
Concentration (μg/ml)
7
10.4
14
7.3
21
4.3
28
4.6
35
6.8
The concentration of rCrry-Ig, assessed by ELISA, in the serum isolated from one representative animal on certain days during the immune response
study.
Sponsored Document
Sponsored Document
Published as: Mol Immunol. 2008 January ; 45(2): 395–405.
Hepburn et al.
Page 23
Table 2
Histological analysis of the NMJ in soleus muscle from EAMG animals
Sponsored Document
Sponsored Document
Group
C3 deposition
C9 deposition
Infiltration (H&E)
Infiltration (ED1 staining)
Control (52 h)
1
2
3
4
5
++
++
++
+
+
+
−
+
+
+
2
2
3
3
3
2–3
2–3
3
2
2–3
sCrry-treated (183 h)
1
2
3
4
5
52 h animal
++
±
++
+
++
++
++
+
++
++
+
++
1
2
3
3
3
1
2–3
2–3
2
3
3
1–2
rCrry-Ig-treated (183 h)
1
2
3
4
5
52 h animal
−
±
+
−
±
±
−
−
−
−
−
−
1
1
1
1
1
0
1
0
0
0
0
0–1
Sections were double-stained for AChR (rhodamine-conjugated α-bungarotoxin) and either activated C3 or C9. Deposition of C3 and C9 were scored as
follows: −, negative; ±, trace; +, weak; ++, strong. Infiltration was assessed following H&E staining and infiltrating macrophages were detected by
immunofluorescence using ED1 (anti-CD68). Infiltration was scored as follows: 0, no infiltration; 1, up to 20% of the whole muscle infiltrated; 2, up to
40% of whole muscle infiltrated; 3, up to 60% of whole muscle infiltrated; 4, over 60% of muscle infiltrated. PBS control group were sacrificed at 52 h
due to disease severity along with one animal from the two treatment groups (52 h animal). All other animals were sacrificed at 183 h.
Sponsored Document
Published as: Mol Immunol. 2008 January ; 45(2): 395–405.