Frontiers in Cardiovascular Research
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Theme Issue Article
Complement depletion with humanised cobra venom factor:
Efficacy in preclinical models of vascular diseases
Carl-Wilhelm Vogel1,2; David C. Fritzinger1; W. Brian Gorsuch3; Gregory L. Stahl3
1University
of Hawaii Cancer Center, University of Hawaii at Manoa, Honolulu, Hawaii, USA; 2Department of Pathology, John A. Burns School of Medicine, University of Hawaii at
Manoa, Honolulu, Hawaii, USA; 3Center for Experimental Therapeutics and Reperfusion Injury, Department of Anesthesia, Perioperative and Pain Medicine, Brigham and Women’s
Hospital, Harvard Institutes of Medicine, Boston, Massachusetts, USA
Summary
The complement system is an intrinsic part of the immune system and
has important functions in both innate and adaptive immunity. On the
other hand, inadvertent or misdirected complement activation is also
involved in the pathogenesis of many diseases, contributing solely or
significantly to tissue injury and disease development. Multiple approaches to develop pharmacological agents to inhibit complement
are currently being pursued. We have developed a conceptually different approach of not inhibiting but depleting complement, based on
the complement-depleting activities of cobra venom factor (CVF), a
non-toxic cobra venom component with structural and functional
Correspondence to:
Carl-Wilhelm Vogel, MD, PhD
University of Hawaii Cancer Center
701 Ilalo Street
Honolulu, HI 96813, USA
Tel.: +1 808 564 5979, Fax: +1 808 586 3077
E-mail: [email protected]
homology to complement component C3. We developed a humanised
version of CVF by creating human complement component C3 derivatives with complement-depleting activities of CVF (humanised CVF) as
a promising therapeutic agent for diseases with complement pathogenesis. Here we review the beneficial therapeutic effect of humanised CVF in several murine models of vascular diseases such as
reperfusion injury.
Keywords
Cobra venom factor, humanised cobra venom factor, CVF, complement
depletion, reperfusion injury
Received: April 2, 2014
Accepted after minor revision: May 7, 2014
Epub ahead of print: July 17, 2014
http://dx.doi.org/10.1160/TH14-04-0300
Thromb Haemost 2015; 113: 548–552
Introduction
The complement system is an intrinsic part of the immune system
and has important functions in both innate and adaptive immunity. On the other hand, inadvertent or misdirected complement activation is also involved in the pathogenesis of many diseases, and
in some cases, represents the major pathogenetic mechanism ([1]
and references therein). A few of these diseases include: rheumatoid arthritis (2), lupus erythematosus (3), myasthenia gravis (4),
macular degeneration (5), and paroxysmal nocturnal haemoglobinuria (PNH) (6). Several diseases with complement pathogenesis
involve the vascular and coagulation systems, with reperfusion injury being a prominent example (7–10).
Cobra venom factor (CVF) is the complement-activating protein in the venom of the Indian cobra (Naja naja) and other cobra
species (1, 11). CVF is a structural and functional analogue of
complement component C3 (1, 12, 13). CVF activates complement
analogously to C3b, the activated form of C3 (14, 15). Both CVF
and C3b bind to complement component Factor B. Once in complex with CVF or C3b, Factor B is cleaved by Factor D, resulting in
the formation of the bimolecular complex CVF,Bb or C3b,Bb, respectively (1, 16–18). Both complexes are enzymes, called C3/C5
convertase (EC 3.4.21.47), activating both C3 and C5 (15, 19, 20).
Whereas C3b-dependent complement activation occurs on the
surface of a target cell, and all regulatory mechanisms of the complement system restrict activation to the target site, CVF-depend-
ent complement activation occurs in the fluid phase, and both
CVF and CVF,Bb are completely resistant to inactivation (21, 22).
Accordingly, CVF will continuously activate C3 and C5, leading to
the depletion of serum complement activity.
Ever since it was shown, more than 40 years ago, that CVF can
be safely administered to laboratory animals, leading to in vivo
complement depletion (23–25), CVF has been used as a tool to
study both the biological functions of complement as well as its
role in the pathogenesis of diseases by comparing normal (complement-sufficient) animals with complement-depleted animals (1,
11). This approach has led to the delineation of multiple physiological activities of complement as well as the identification of the
complement system in the pathogeneses of many diseases.
Given the involvement of complement in the pathogenesis of
many diseases, for the last two decades many investigators have
devised experimental approaches to inhibit complement with the
aim to develop pharmacological agents for therapy of complement-mediated diseases (26–29). Whereas a detailed description
of complement inhibitory therapeutics is beyond the scope of this
article, complement inhibitors are either directed to prevent the
activation of a given complement component or to inhibit the action of an activated complement component. In contrast, we have
developed a conceptually different approach to treat complementmediated diseases. Our approach is not based on inhibition of
complement but on depletion of complement, mimicking the
complement-depleting activity of CVF (1, 30–32). Whereas CVF
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itself represents a potential therapeutic agent for complement-depletion, and has served as the gold standard in experimental models of disease for evaluating the efficacy of complement inhibitors
(33), both the limited availability of its natural source, cobra
venom, and its immunogenicity severely limit its clinical usefulness.
To overcome these two limitations, we expressed active recombinant CVF in insect cells (34, 35). We also identified that the
C-terminal region of the CVF β-chain, corresponding to the C-terminal region of the C3 α-chain, harbours the crucial structures in
CVF allowing its uncontrolled activation of the complement system (36, 37). In a third step, we generated human C3 derivatives
with functional features of CVF by replacing the C-terminal portion of the C3 α-chain with the homologous sequence from the
CVF β-chain (31, 32, 38, 39). These human C3 derivatives are collectively referred to as “humanised CVF” (hCVF) and represent
experimental therapeutic agents for complement depletion in diseases with complement pathogenesis. hCVF protein HC3–1496 is
human C3 in which the C-terminal 168 amino acid residues in its
α-chain have been replaced with the corresponding CVF sequence
(1, 32) (▶ Figure 1). Even within this stretch of 168 amino acids,
human C3 and CVF sequences are 44% identical (64% similar);
and the overall sequence identity between HC3–1496 and human
C3 is 94% (96% similarity) (13, 40). We have shown the efficacy of
HC3–1496 as an experimental therapeutic agent in multiple preclinical models of disease including PNH (41), collagen-induced
arthritis (30), monoclonal antibody therapy of lymphoma (42),
and macular degeneration (43). Here we will review and report on
the efficacy of complement-depletion with HC3–1496 in two different preclinical models of reperfusion injury as well as ventilatorinduced lung damage.
Myocardial ischaemia/reperfusion injury
To assess the therapeutic potential of complement depletion with
hCVF, we used a murine model of experimental myocardial ischaemia/reperfusion injury (MI/RI) (44, 45). Mice were intubated,
ventilated, and anesthesia was maintained with isoflurane. The
Figure 1: Schematic representation of the chain structures of human
pro-C3, pro-CVF, and humanised CVF protein HC3–1496. Human
pro-C3 is a single-chain protein of 1641 amino acid residues. It is processed
by the removal of four arginine residues into the mature C3 protein consisting of the α- and β-chain. Pro-CVF is a single-chain protein of 1620 amino
acid residues. It is processed in the venom gland into the mature three-chain
protein involving the removal of the four arginine residues, the C3a domain,
and the C3dg-like domain. Humanised CVF protein HC3–1496, like human
chest was opened and a suture was placed on the left anterior descending coronary artery and tightened. After 30 minutes (min) of
ischaemia, the ligation was loosened and the myocardium was
reperfused for 4 hours (h). Decomplementation with hCVF protein HC3–1496 at 250 μg/kg was performed by i.p. injection 2 h
prior to the induction of anesthesia (46).
Complement-depletion with hCVF protein HC3–1496 was an
effective therapeutic approach to reduce reperfusion injury as
demonstrated functionally (better ejection fraction), morphologically (smaller infarct size), and immunohistochemically (less deposition of C3b) (▶ Figure 2) (46). Better left ventricular function
in animals depleted with hCVF was also demonstrated by an improved fractional shortening as measured by echocardiography
(46).
Gastrointestinal ischaemia/reperfusion injury
We used a murine model of gastrointestinal ischaemia/reperfusion
injury (GI/IR) to assess the effect of complement-depletion. In this
model, mice were anesthetised with isoflurane, and intestinal ischaemia was produced for 20 min by occlusion of the superior
mesenteric artery (47–49). Subsequently, 3 h of reperfusion was
achieved by removal of the surgical clips. The intestinal permeability was assessed by luminal enteral administration of FITC-conjugated dextran with subsequent determination of FITC-dextran
concentrations in serum by fluorescence spectrophotometry (47).
Decomplementation with hCVF protein HC3–1496 or natural
CVF at 250 μg/kg was performed by i.p. injection two hours prior
to the induction of anesthesia. Injection of phosphate-buffered saline (PBS) served as control.
▶ Figure 3 demonstrates that complement-depletion with
hCVF protein HC3–1496 results in a significantly decreased
serum concentration of FITC-dextran, indicating significantly reduced reperfusion injury compared to PBS-treated animals. Complement-depletion with natural CVF, which served as positive
control, resulted in an identical reduction of reperfusion injury
(▶ Figure 3).
C3, has 1641 amino acid residues. Recombinant production of hCVF in
Drosophila S2 cells results in a mixture of a C3-like and a C3b-like two-chain
protein (1, 31, 34). The C-terminal 146 amino acid residues of both C3 and
CVF constitute the C345C domain which has been shown by x-ray crystallography to bind Bb in the convertase (16, 18). Not surprisingly, this region in
CVF harbors the important structures responsible for forming a stable
convertase.
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549
Frontiers in Cardiovascular Research
Vogel et al. Complement depletion with humanised cobra venom factor
Vogel et al. Complement depletion with humanised cobra venom factor
Frontiers in Cardiovascular Research
550
Figure 3: Effect of complement depletion with humanised CVF protein HC3–1496 in gastrointestinal infarction reperfusion injury. Mice
were anaesthetised with isoflurane and, after a mid-line laparotomy, intestinal ischaemia was produced for 20 min by occlusion of the superior mesenteric artery with surgical clips (47–49). Subsequently, 3 h of reperfusion was
achieved by removal of the clips. The intestinal permeability was assessed by
luminal enteral administration of FITC-conjugated dextran 4000 (47). The
animals were gavaged 5 min prior to ischaemia with FITC-dextran at 200
μg/g of body weight. Blood was obtained by cardiac puncture at the time of
euthanasia. Blood samples were allowed to clot at 4°C and centrifuged.
Serum FITC-dextran concentrations were determined by fluorescence spectrophotometry (47). Decomplementation with hCVF protein HC3–1496 or
natural CVF at 250 μg/kg was performed by i.p. injection 2 h prior to the induction of anesthesia. Injection of PBS served as control. Shown is the uptake
of FITC-labelled dextran into the blood stream from the intestine. PBS and
natural CVF served as negative and positive controls, respectively.
Ventilator-induced lung injury
Figure 2: Effect of complement depletion with humanised CVF protein HC3–1496 in myocardial infarction reperfusion injury. Mice were
intubated and maintained under anesthesia with isoflurane. The chest was
opened by a diagonal incision, and a suture was placed on the left anterior
descending coronary artery and tightened. After 30 minutes of ischaemia, the
suture was removed, the chest wall was surgically closed, the intubation was
discontinued, and myocardial reperfusion was allowed for 4 h prior to sacrificing the animals (44–46). Mice in the treatment group were decomplemented with hCVF protein HC3–1496 by i.p. injection at 250 μg/kg 2 h prior
to the induction of anesthesia. Injection of PBS served as control (46). Heart
sections were prepared and stained with goat anti-mouse C3 and donkey
anti-goat IRDye800, the infarct size was determined using antegrade perfusion with 1% (w/v) Evans Blue and stereoscopic zoom microscope imaging,
and transthoracic echocardiography was performed to determine the ejection fraction as described (44–46). The upper panel shows C3 deposition in
the heart muscle. The centre panel shows the infarcted area as percent of the
left ventricle. The lower panel shows the ejection fraction. (Modified from [1,
46]).
Ventilator-induced lung injury (VILI) is a major course of morbidity and mortality in critically ill patients (50). A murine model of
VILI was used to assess the effect of complement depletion with
hCVF (51). Mice were anaesthetised with i.p. injection of avertin,
and a tracheotomy was performed, followed by intubation with a
catheter. Mice were ventilated for 2 h on a warm plate using a
small animal mechanical ventilator. Complement-depletion was
performed by s.c. injection of hCVF protein HC3–1496 at 250
μg/kg 1 h prior to mechanical ventilation.
As shown in the upper panel of ▶ Figure 4, complement depletion with hCVF protein HC3–1496 resulted in significantly reduced deposition of C3 in lung tissue as measured by immunohistochemistry (51). As shown in the lower panel of ▶ Figure 4, complement-depleted mice showed a larger number of single cells in
the bronchoalveolar lavage (BAL) fluid compared to wild-type
mice, where the ventilator-induced complement activation leads to
activation of thrombin and formation of fibrin clots embedding
cells into aggregates (51).
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Vogel et al. Complement depletion with humanised cobra venom factor
As demonstrated in the preclinical models of disease described
here, temporary ischaemia or mechanical injury both cause vascular tissue damage that leads to subsequent activation of complement. In turn, complement activation causes significant tissue
damage well above and beyond the initial tissue damage by ischaemia or mechanical injury. The structural changes induced by ischaemia or mechanical injury that lead to activation of complement are not known. In reperfusion injury, both the lectin pathway and the alternative pathway have been shown to be activated
whereas the classical pathway is not, although IgM binding to neoepitopes leads to MBL binding and subsequent complement activation (8, 52). Regardless of the mechanism of complement activation, the complement-dependent tissue damage is demonstrated
by morphological and, importantly, functional impairment of the
affected organs. In all cases, depletion of complement with humanised CVF prevents the complement-mediated tissue damage.
It is noteworthy to add that no adverse side effects of complement depletion with humanised CVF were observed, neither in
the three preclinical disease models reported here nor in any other
preclinical disease model (1, 30, 42, 43, 45, 51, 53, 54). Moreover,
the only known side effect of massive fluid-phase complement activation by natural CVF is a consequence of the released anaphylatoxins C3a and C5a. Both anaphylatoxins are readily inactivated
by carboxypeptidase N to C3a-des-Arg and C5a-des-Arg, respectively. However, C5a-des-Arg retains its ability to activate neutrophils, which have been shown to be sequestered in the lungs, causing acute but fleeting inflammatory lung injury (55–57). Fortuitously, humanised CVF lacks C5-cleaving activity and does not
generate C5a (1, 31). This is consistent with a complete lack of lung
injury in cynomolgus monkeys after intra-arterial hCVF injection
into the pulmonary artery (1, 58). Another potential complication
is immunogenicity of hCVF. However, recombinantly produced
hCVF lacks the unusual oligosaccharides of native CVF (59), and
exhibits 94% sequence identity with human C3, along with an
identical domain structure (1, 16, 30), suggesting a significantly reduced or potentially absent immunogenicity. Indeed, hCVF displayed no functionally relevant immunogenicity compared to CVF
in a murine model of haemophilia A (60).
Accordingly, complement depletion with humanised CVF represents a promising therapeutic strategy to prevent tissue damage
and functional impairment in reperfusion injury and ventilatorinduced lung damage, as well as other diseases with complement
pathology (1).
Conflicts of interest
CWV, DCF, and GLS were former recipients of research grants
from Incode Biopharmaceutics, Inc. and previously had a financial
interest in the company.
Frontiers in Cardiovascular Research
Conclusion
551
Figure 4: Effect of complement depletion with humanised CVF protein HC3–1496 on ventilator-induced lung injury. Mice were anaesthetised with i.p. injection of avertin at 250 mg/kg. Tracheotomy was performed,
followed by intubation with a catheter which was connected to a small animal mechanical ventilator. Mice were ventilated for 2 h on a warm plate
(37°C), and injected with additional avertin after 1 h at 125 mg/kg. Ventilation settings were 35 ml/kg of tidal volume, with a respiratory rate of
50/min using room air, and 2 cmH2O positive end-expiratory pressure (51, 61,
62). Mice in the treatment group were decomplemented with hCVF protein
HC3–1496 by s.c. injection at 250 μg/kg 1 h prior to mechanical ventilation
(51). After 2 h, mice were sacrificed. Brochoalveolar lavage (BAL) was performed with subsequent counting of single BAL cells, and lungs were sectioned and stained with FITC-conjugated goat anti-mouse C3 as described
(51). The upper panel shows C3 deposition in lung sections. The lower panel
shows the total number of single BAL cells in the BAL lavage fluid. (Modified
from [51]).
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