| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Vol. 50, Issue 1, 59-88, March 1998
Department of Cell Biology, PRAECIS Pharmaceuticals, Inc., Cambridge, Massachusetts
I. Introduction
II. The Complement System and Its Regulation
A. The Classical Pathway
B. The Alternative Pathway
C. The Membrane Attack Complex
III. Modified Native Complement Components That Block Complement Activation
A. Soluble Complement Receptor Type 1
B. Soluble Complement-Receptor Type 1 Lacking Long Homologous Repeat-A
C. Soluble Complement Receptor Type 1-Sialyl Lewisx
D. Complement Receptor Type 2
E. Soluble Decay Accelerating Factor
F. Soluble Membrane Cofactor Protein
G. Soluble CD59
H. Decay Accelerating Factor-CD59 Hybrid
I. Membrane Cofactor Protein-Decay Accelerating Factor Hybrid
J. C1 Inhibitor
K. C1q Receptor
IV. Complement-Inhibitory Antibodies
A. Anti-C5 Monoclonal Antibody
B. Anti-C5 Single Chain Fv
V. Synthetic Inhibitors of Complement Activation
A. Peptides and Analogs
B. Organic Molecules
VI. Naturally Occurring Compounds That Block Complement Activation
VII. Complement Inhibition in Xenotransplantation
VIII. Bispecific Antibodies for Immune Complex Removal
IX. Summary
Acknowledgments
References
 |
I. Introduction |
|---|
 Top NextReferences
|
|---|
Activation of the complement system plays a key role in normal
inflammatory response to injury but may cause substantial injury when
activated inappropriately. The cytolytic properties of serum were first
described more than a century ago (Bordet, 1896
), but there is still no
therapeutic compound available on the market for complement inhibition.
This is about to change as several companies and academic investigators
are actively engaged in the development of complement therapeutics
(Morgan, 1995a; Pascual and French, 1995). The molecular cloning and
biochemical dissection of the many components of the complement pathway
during the last 2 decades has led to a detailed understanding of the
mechanisms of complement activation in inflammation. This, in turn, has
allowed for the potential for drug development based on the genetic
engineering of receptors and other components of the complement
pathway. Coupled with the ability to express human transgenes in animal
organs, these developments hold promise for the therapeutic management of complement-mediated injury in certain diseases.
Although complement activation is probably not the primary etiology of
many diseases, the damage to tissues in certain conditions is clearly
complement-mediated. Indeed, the inappropriate activation of complement
is at the core of a long list of disease pathologies (Morgan, 1994)
that affect the immune, renal, cardiovascular, neurological, as well as
other, systems in the body (table 1). Examination of the evidence for the involvement of complement in these
conditions is beyond the scope of this review. References are provided
in table 1 for further reading, and several excellent reviews have
covered clinical complementology (Ross and Densen, 1984; Frank, 1987;
Morgan, 1990, 1994, 1995b; Morgan et al., 1997; Homeister and Lucchesi,
1994; Kalli et al., 1994; Asghar, 1995; Baldwin et al., 1995;
Mossakowska and Smith, 1997). In addition, only a brief overview of the
complement system is provided here as this area has been covered
extensively (Liszewski et al., 1996; Ross, 1986; Rother and Till, 1988;
Fearon and Wong, 1983; Reid, 1986; Müller-Eberhard, 1988;
Dalmasso, 1986; Baldwin et al., 1995; Frank, 1994; Morgan and Meri,
1994). The main objective in this study is to review the published
literature on the use of inhibitors for the therapeutic abrogation of
pathological complement activation. A section is also included on the
use of bispecific antibodies in human disease. This latter approach
does not attempt to inhibit complement, but rather uses components of
the complement system to facilitate the clearance of blood-borne
pathogens from the circulation.
|
| |
II. The Complement System and Its Regulation |
|---|
|
TopPreviousNextReferences
|
|---|
The complement system consists of more than 30 serum and cellular
proteins, including positive and negative regulators, linked in two
biochemical cascades, the classical and alternative pathways (fig.
1). The activation of complement
encompasses a series of initiation, amplification, and lytic steps and
their discrete reactions (Parker, 1992; Liszewski et al., 1996). The
system is regulated at multiple levels temporally as well as spatially. This regulation facilitates recognition of self from foreign tissue (Farries and Atkinson, 1987) and, therefore, allows for control over
the potent tissue-damaging capabilities of complement activation. It
has been recognized that some of the endogenous complement regulatory
proteins might serve as potential therapeutic agents in blocking
inappropriate activation of complement in human disease. Soluble and
membrane-bound variants of complement regulators have been produced and
shown to be effective in blocking complement activation in vitro as
well as in animal models of complement-mediated pathologies (Homeister
and Lucchesi, 1994).
|
A. The Classical Pathway
The classical pathway is usually initiated when a complex of
antigen and IgM or IgG antibody binds to the first component of
complement C1. Activation of this step of complement is regulated by
the C1 inhibitor that binds to C1r and C1s and dissociates them from
C1q (Liszewski et al., 1996). Activated C1 cleaves both C4 and C2 to
generate C4a and C4b, as well as C2a and C2b. The C4b and C2a fragments
combine to form the C3 convertase, which, in turn, cleaves the third
component of complement, C3, to form C3a and C3b. The binding of C3b to
the C3 convertase yields the C5 convertase, which cleaves C5 into C5a
and C5b, the latter becoming part of the membrane attack complex
(MAC)b. It must be noted that activators
other than antibodies are capable of initiating the classical pathway.
For example, in the absence of antibody, 
-amyloid activates
complement in the brain by binding to the collagen-like domain of the
C1q A chain (Rogers et al., 1992; Jiang et al., 1994; Velazquez et al.,
1997; Webster et al., 1997; Cadman and Puttfarcken, 1997). These
observations have therapeutic implications for Alzheimer's disease
(Barnum, 1995; Pasinetti, 1996; Chen et al., 1996).
The three peptides released during these steps, C3a, C4a, and C5a, are
known as anaphylatoxins (Hugli and Müller-Eberhard, 1978), and
they differ in their relative potencies. C5a is the most potent
anaphylatoxin, followed by C3a, which, in turn, is 10- to 100-fold more
active than C4a (Cui et al., 1994; Hugli and Müller-Eberhard,
1978; Liszewski et al., 1996). The anaphylatoxins mediate multiple
reactions in the acute inflammatory response, including smooth muscle
contraction, changes in vascular permeability, histamine release from
mast cells, neutrophil chemotaxis, platelet activation and aggregation
(Morgan, 1986; Hugli, 1989; Gerard and Gerard, 1994), as well as
up-regulation of adhesion molecules that can also play key roles in
neutrophil recruitment (Foreman et al., 1994; Mulligan et al., 1996,
1997; Schmid et al., 1997a). Recently, C3a and C5a have been shown to
be potent chemotactic factors for human mast cells (Hartmann et al.,
1997). The anaphylatoxins are rapidly inactivated by carboxypeptidase
N, which cleaves the carboxyl terminal arginyl residue from each
anaphylatoxin, thus converting them into their des-Arg forms (Bokisch
et al., 1969; Bokisch and Müller-Eberhard, 1970; Chenoweth,
1986). A C5a-inactivating enzyme isolated from human peritoneal fluid
has been described (Ayesh et al., 1995).
The C3 and C5 convertases of the classical pathway (fig. 1) are
controlled by members of the Regulators of Complement Activation (RCA)
family (Rey-Campos et al., 1987; Carroll et al., 1988; Campbell et al.,
1988; Hourcade et al., 1989; Morgan and Meri, 1994) (fig. 2; table
2). This protein family includes the
membrane-bound regulators complement receptor type 1 (CR1; C3b/C4b
receptor; CD35), complement receptor type 2 (CR2; CD21; Epstein-Barr
virus receptor), membrane cofactor protein (MCP; CD46; measles virus receptor), decay-accelerating factor (DAF; CD55), and the serum proteins factor H and C4b-binding protein (C4bp).
|
|
B. The Alternative Pathway
This arm of the complement system is triggered by microbial
surfaces and a variety of complex polysaccharides. C3b, formed by the
spontaneous low-level cleavage of C3, can bind to nucleophilic targets
on cell surfaces and form a complex with factor B that is subsequently
cleaved by factor D (fig. 1). The resulting C3 convertase is stabilized
by the binding of properdin (P) that increases the half-life of this
convertase (Fearon and Austen, 1975). Cleavage of C3 and binding of an
additional C3b to the C3 convertase give rise to the C5 convertase of
the alternative pathway (fig. 1). Subsequent reactions are common to
both pathways and lead to the formation of the MAC. The C3 and C5
convertases of the alternative pathway are controlled by CR1, DAF, MCP,
and by factor H. These regulators differ in their mode of action, i.e.,
their decay-accelerating activity (ability to dissociate convertases)
and ability to serve as required cofactors in the degradation of C3b or
C4b by factor I (tables 2 and 3). In
addition, CR2 may have a minor role in regulating complement activation (Fearon and Carter, 1995).
|
C. The Membrane Attack Complex
The C5 convertases in both the classical and alternative pathways
cleave C5 to produce C5a and C5b. Thereafter, C5b sequentially binds to
C6, C7, and C8 to form C5b-8 that catalyzes the polymerization of C9 to
form the MAC (Tschopp et al., 1982). This structure inserts into target
membranes and causes cell lysis (Hu et al., 1981; Podack et al., 1982).
However, deposition of small amounts of the MAC on cell membranes of
nucleated cells may mediate a range of cellular processes without
causing cell death (Morgan, 1992; Nicholson-Weller and Halperin, 1993;
Benzaquen et al., 1994).
Three different molecules are known to be involved in the control of
the MAC formation. Vitronectin controls fluid-phase MAC by binding to
the C5b-7 complex, preventing its insertion into membranes (Podack et
al., 1977). Similarly, clusterin (SP-40,40; cytolysis inhibitor;
sulfated glycoprotein 2; apolipoprotein J) (Liszewski et al., 1996)
blocks fluid-phase MAC by binding to the C5b-7 complex (Jenne and
Tschopp, 1989; Choi et al., 1989; Murphy et al., 1989). CD59 blocks MAC
formation by binding to C8 and C9, and inhibiting the incorporation and
subsequent polymerization of C9 (Rollins et al., 1991). An additional
protein, homologous restriction factor (Zalman, 1992), may be involved
in MAC regulation, but it has been suggested that the functional
activity reported for homologous restriction factor might possibly be
due to a contamination by CD59 aggregates during purification
(Liszewski et al., 1996).
| |
III. Modified Native Complement Components That Block Complement Activation |
|---|
|
TopPreviousNextReferences
|
|---|
A. Soluble Complement Receptor Type 1
The primary structure of the human CR1 (CD35) has been derived
from its complementary deoxyribonucleic acid (cDNA) sequence (Klickstein et al., 1987, 1988; Hourcade et al., 1988). The mature protein of the most common allotype of CR1 contains 1998 amino acid
residues: an extracellular domain of 1930 residues, a transmembrane region of 25 residues, and a cytoplasmic domain of 43 residues. The
entire extracellular domain is composed of 30 repeating units (fig. 2)
referred to as short consensus repeats (SCRs) or complement control
protein repeats (CCPRs), each consisting of 60 to 70 amino acid
residues. Within each SCR a loop structure is maintained by disulfide
linkages between the conserved cysteines-1 and -3, and -2 and -4 (Ahearn and Fearon, 1989). The SCR motif, first shown in
2-glycoprotein I (Lozier et al., 1984), or a
variation thereof, is found in other complement proteins as well as in
a large number of noncomplement proteins (Reid and Day, 1989). In CR1,
groups of seven SCRs have been organized into four long homologous repeats (LHRs), so that only the SCRs 29 and 30 are not part of a LHR
(fig. 2). CR1 has 25 Asn-X-Ser(Thr) sequence motifs (Klickstein et al.,
1988) that confer potential N-linked glycosylation (Winzler, 1973) (fig. 2). The ligand-binding active sites of CR1 (fig. 2) were
originally identified by Klickstein et al. (1988) who demonstrated a
C4b-binding site within LHR-A and C3b-binding sites within both LHR-B
and LHR-C. These observations were subsequently confirmed by
site-directed mutagenesis studies (Krych et al., 1991, 1994). Optimal
binding affinities equivalent to those of native CR1 were later
demonstrated to reside within SCRs 8-11 and 15-18 for C3b (Kalli et
al., 1991; Makrides et al., 1992) and in SCRs 1-4 for C4b (Reilly et
al., 1994). CR1 has extrinsic activity (Medof et al., 1982), i.e., it
inactivates convertases assembled on external membranes, and it also
exhibits intrinsic activity (Kinoshita et al., 1986; Makrides et al.,
1992), i.e., it inactivates convertases formed on the same membrane on
which it is expressed. Among the members of the RCA family (table 3),
CR1 is the only one that possesses decay-accelerating activity for both
C3 and C5 convertases in both the classical and alternative pathways,
as well as factor I cofactor activity for the degradation of both C3b
and C4b (Fearon, 1991). Recent data indicate that C1q binds
specifically to human CR1 (Klickstein et al., 1997). Thus, CR1
recognizes all three complement opsonins, namely C3b, C4b, and C1q.
A soluble version of recombinant human CR1 (sCR1) lacking the
transmembrane and cytoplasmic domains was produced and shown to retain
all the known functions of the native CR1 (Weisman et al., 1990a,b).
Initial studies centered on the use of sCR1 in animal models of
ischemia/reperfusion injury. Although thrombolytic agents have been
used effectively in ischemic myocardium to induce reperfusion, blood
reflow into ischemic tissue may induce necrosis because of complement
activation, neutrophil accumulation in the microvasculature, and
consequent damage to the endothelium (Homeister and Lucchesi, 1994).
Administration of sCR1 in a rat model of ischemia/reperfusion injury
reduced myocardial infarct size by 44% assessed at 7 days postdosage
and minimized the accumulation of neutrophils within the infarcted
area, probably because of a decreased generation of the anaphylatoxin
C5a (Weisman et al., 1990a,b). In addition, sCR1 attenuated the
deposition of the C5b-9 MAC. This was the first demonstration that a
recombinant soluble form of a member of the RCA family might provide a
potential therapeutic agent in inflammation. The cardioprotective role
of sCR1 in animal models of ischemia/reperfusion injury has been
confirmed (Shandelya et al., 1993; Smith et al., 1993; Homeister et
al., 1993). Similarly, sCR1 reduced tissue injury in
ischemia/reperfusion of mouse skeletal muscle (Pemberton et al., 1993),
rat intestine (Hill et al., 1992), rat liver (Chávez-Cartaya et
al., 1995), and remote organs after lower torso ischemia in the rat
(Lindsay et al., 1992). In addition to its efficacy in models of
ischemia/reperfusion, sCR1 has been shown to reduce complement-mediated
tissue injury in animal models with a wide range of human acute and
chronic inflammatory diseases. These include dermal vascular reactions
(Yeh et al., 1991; Mulligan et al., 1992b), lung injury (Rabinovici et
al., 1992; Mulligan et al., 1992a,b), trauma (Kaczorowski et al.,
1995), myasthenia gravis (Piddlesden et al., 1996), glomerulonephritis
(Couser et al., 1995), multiple sclerosis (Piddlesden et al., 1994),
allergic reactions (Lima et al., 1997), and asthma (Regal et al.,
1993). In addition, sCR1 protects against vascular injury and cellular infiltration in allografts (Pratt et al., 1996a,b) and attenuates hyperacute rejection in xenografts (Baldwin et al., 1995; Ryan, 1995;
Levin et al., 1996) (see Section VII.).
Pharmacokinetic studies of earlier preparations of sCR1 showed that the
-phase half-life (t1/2) was
approximately 1.7 h in rats, and 8 h in humans (J. Levin,
unpublished data). A longer circulating half-life might permit
bolus-dosage administration, allow lower dosages of a drug to achieve
comparable therapeutic effects and reduce the cost per therapeutic
dosage. One experimental approach used to extend the circulating
half-life of sCR1 utilized the albumin-binding terminus "BA" or
"BABA" of Streptococcal protein G as a fusion partner with sCR1
(Makrides et al., 1996). The resulting sCR1-BA fusion construct
exhibited a significantly longer half-life (297 min) than sCR1 (103 min) in rats (Makrides et al., 1996). Fearon and colleagues chose the
Ig molecule as a fusion partner with SCRs 8-11, the C3b-binding site
of CR1. The (CR1)2-F(ab')2 chimera was as effective as sCR1 in binding to C3b dimers, promoting cleavage of C3b by factor I and inhibiting activation of the
alternative pathway (Kalli et al., 1991). A potential application of
this finding is the fusion of CR1 active units to full-length IgG to create chimeras with a longer half-life than sCR1 because of the long
plasma half-life of the Fc moiety (Capon et al., 1989). An additional
advantage of an Ig fusion partner is the potential for targeting
complement inhibition to specific tissues (Kalli et al., 1994). Thus, a
monoclonal antibody (mAb) specific for antigen localized in the area of
complement activation could be used to construct a CR1/Ig molecule that
might act as a local rather than a systemic complement blocker.
However, none of the above molecular constructions is likely to be
therapeutically useful because of the potential immunogenicity of the
fusion partners. The genetic engineering or "humanization" of
antibodies (Co and Queen, 1991; Rapley, 1995; Morrison and Shin, 1995)
might minimize immunogenic reactions but not completely eliminate
anti-idiotypic effects. Most important, the problems associated with
the short half-life of sCR1 appear to have been solved. Thus, a
subsequent preparation of sCR1 obtained using modified culture
conditions showed a t1/2 of approximately
30 h in humans (Dellinger et al., 1995, 1996). The reason for the longer half-life of sCR1 is unknown but may be related to a potentially altered glycosylation pattern resulting from the culture conditions.
It has been suggested that the effects of complement on the endothelium
are mediated primarily by the MAC because the human vascular
endothelium is apparently devoid of receptors for anaphylatoxins (conference discussion cited in Morgan, 1995a). Although the expression of the C5a receptor (C5aR) was thought to be limited to leukocytes, the
molecular cloning of the human C5aR demonstrated its expression on
nonmyeloid cells, including the vascular endothelium (Haviland et al.,
1995; Wetsel, 1995). Similarly, the human C3a receptor (C3aR) has
recently been cloned by three groups (Ames et al., 1996; Roglic et al.,
1996; Crass et al., 1996). The C3aR, originally thought to be an orphan
receptor (Roglic et al., 1996), was shown to be expressed in
endothelial cells (Roglic et al., 1996). It is now clear that
complement activation products have many diverse effects on endothelial
cells, and, in fact, the endothelium may be a major target of the
complement system (Ward, 1996).
The ability of sCR1 to block activation of both the classical as well
as the alternative pathways has been thought (Evans et al., 1995) to
potentially reduce its therapeutic value because it inhibits generation
of C3b, a C3 opsonic product that is critical for antibacterial
defenses (Ross and Densen, 1984). The possibility that a global
inhibitor of complement activation might compromise antibacterial
defenses was recognized by Becker (1972) who concluded that "this
risk might not be unacceptably high." To date, there is no credible
evidence that sCR1 compromises bacterial defenses in animal models of
inflammation. More importantly, two phase I clinical trials of sCR1 in
patients with myocardial infarct or burn-induced adult respiratory
distress syndrome revealed no safety issues in this regard, including
rates of bacterial infection (J. Levin, personal communication). The
adult respiratory distress syndrome trial is of particular relevance in
this context because people who are severely burned may die of
bacterial sepsis. In this environment, sCR1 had no effect on systemic
bacterial infections (J. Levin, personal communication).
B. Soluble Complement-Receptor Type 1 Lacking Long Homologous Repeat-A
A mutant version of sCR1 lacking LHR-A sCR1[desLHR-A] was
constructed with the objective of generating a selective inhibitor of
the alternative pathway (Scesney et al., 1996). The rationale for this
is based on the fact that C4b is a component of the classical pathway
exclusively, whereas C3b functions in both classical and alternative
pathways (fig. 1). Thus, removal of the C4b-binding LHR-A from sCR1
would be expected to abrogate the ability of sCR1[desLHR-A] to
accelerate the decay of the C3 and C5 convertases in the classical pathway (fig. 1). Indeed, sCR1[desLHR-A] was shown to be
quantitatively equivalent to sCR1 in its ability to inhibit the
alternative pathway in vitro (Scesney et al., 1996). On the other hand,
sCR1[desLHR-A] was less effective than sCR1 in blocking activation of
the classical pathway in vitro. Both sCR1[desLHR-A] and sCR1
exhibited equal capacities to serve as cofactor in the degradation of
fluid-phase C3b by factor I (Scesney et al., 1996). These results are
consistent with the observations of Kalli et al. (1991) who constructed
a chimera between SCR 8-11 of CR1 and an antibody
F(ab')2 fragment, thus allowing the bivalent
presentation of SCR 8-11 (C3b-binding site). In that case, the chimera
(CR1)2-F(ab')2 and sCR1
were shown to be equivalent in their capacity to inhibit the
alternative pathway of complement activation, although the chimera,
which lacks the C4b-binding site found in LHR-A, was considerably less effective at inhibiting the classical pathway (Kalli et al., 1991).
The availability of sCR1[desLHR-A] facilitated examination of the
relative contributions of the classical and alternative pathways in a
model of discordant xenotransplantation in which an isolated perfused
heart from a rabbit is exposed to human plasma that serves as a
complement source (Homeister et al., 1992). The interaction of rabbit
heart tissue with plasma activates complement, leading to the
production of anaphylatoxins and the generation of C5b-9 membrane
attack complex. Both sCR1 and sCR1[desLHR-A] had a cardioprotective
effect in the rabbit heart perfused with human plasma (Gralinski et
al., 1996). Complement activation was also shown to attenuate
endothelium-dependent relaxation in rabbit tissue (Lennon et al.,
1996). This attenuation was dependent on the formation of C5b-9 via the
classical and alternative pathways, as demonstrated through the use of
human serum depleted in factor B, C2, or C8. The use of sCR1 and
sCR1[desLHR-A] decreased the loss of endothelium-dependent relaxation
in rabbit thoracic aortic rings (Lennon et al., 1996). Murohara et al.
(Murohara et al., 1995a) examined the relative contribution of the
classical and alternative pathways in a rat model of ischemia and
reperfusion injury using either C1 esterase inhibitor (see Section
III.J), a classical pathway inhibitor, or sCR1[desLHR-A]. These
authors concluded that both the classical and alternative pathways
contribute to reperfusion injury in myocardial ischemia by a
neutrophil-dependent mechanism. Selective inhibition of the classical
pathway appeared to be slightly more effective in limiting tissue
injury than the selective inhibition of the alternative pathway in this
model (Murohara et al., 1995a).
C. Soluble Complement Receptor Type 1-Sialyl Lewisx
This compound is designed to simultaneously inhibit both
complement activation and neutrophil recruitment at sites of
inflammation (C. Rittershaus, personal communication). The rationale
behind the development of this complement inhibitor is based on the
current understanding of the interaction between complement and
selectins in inflammation (Lefer et al., 1994a; Lefer, 1995; Mulligan
et al., 1996) and the demonstration that C5a up-regulates P-selectin (Foreman et al., 1994; Mulligan et al., 1997). The migration of leukocytes to sites of inflammation is a complex and highly regulated process that is orchestrated by chemoattractants and a large number of
adhesion molecules that are involved in cell-cell and cell-matrix interactions. These adhesion molecules are members of the four families
of receptors, the selectins, the integrins, the Ig superfamily, and the
cadherins (Zimmerman et al., 1992; Pardi et al., 1992; Mackay and
Imhof, 1993; Albelda et al., 1994; Springer, 1994; Malik and Lo, 1996;
Butcher and Picker, 1996). The selectins, L-, P-, and
E-selectins, participate in the initial "rolling" adhesions,
bringing the circulating leukocytes into close proximity with
chemoattractants released from endothelial cells of the vessel wall.
Chemoattractants bind to G protein-coupled receptors on leukocytes,
signaling the activation of integrins that, together with members of
the Ig superfamily effect the arrest and subsequent migration of
leukocytes into the tissue (Springer, 1994). Although this model of
neutrophil extravasation suggests that rolling is a necessary precursor
to subsequent adhesive events, experimental evidence indicates the
possibility of simultaneous, rather than sequential activity of the
various adhesion molecules of the inflammatory cascade (Doerschuk et
al., 1993; Hogg and Doerschuk, 1995; Ward, 1995; Lowe and Ward, 1997).
Selectin function, unlike that of most other adhesion molecules,
appears to be restricted to interactions between leukocytes and the
vascular endothelium (Tedder et al., 1995). The selectins bind
carbohydrate ligands containing fucose, including
SLex (Neu5Ac
2-3Gal1-4(Fuc1-3)GlcNAc-)
(Phillips et al., 1990; Polley et al., 1991; Foxall et al., 1992; Rosen
and Bertozzi, 1994; Bertozzi, 1995; McEver et al., 1995). Other
proteins, including PSGL-1, CD34, and GlyCAM-1 have been identified as
high-affinity ligands for selectins (Lasky, 1995; Kansas, 1996). There
is a diversity of opinions as to the identities of the physiologically
relevant ligands for selectins (Varki, 1994, 1997; Kansas, 1996).
Nevertheless, the observation that SLex can
inhibit neutrophil adhesion mediated by both E- and P-selectins (Phillips et al., 1990; Lasky, 1992) led to vigorous efforts to develop
compounds for the therapeutic disruption of the
selectin-SLex interaction in inflammation. Such
antagonists include SLex and its analogs
(Mulligan et al., 1993a; Rao et al., 1994; Bertozzi et al., 1995; Flynn
et al., 1996; Lefer et al., 1994b; Buerke et al., 1994; Murohara et
al., 1995c; Maaheimo et al., 1995; Lin et al., 1996; Tojo et al., 1996;
Zhang et al., 1996), antibodies against SLex
(Dinh et al., 1996; Seko et al., 1996) or against P-selectin (Lefer et
al., 1996; Doerschuk et al., 1996), peptides (Briggs et al., 1995; Geng
et al., 1992; Heavner et al., 1993; Briggs et al., 1996; Martens et
al., 1995; Norman et al., 1996), oligonucleotides (Murohara et al.,
1996; Hicke et al., 1996; O'Connell et al., 1996), fucoidin (Kubes et
al., 1995), inositol polyanions (Cecconi et al., 1994), sulfatides
(Mulligan et al., 1995), heparin-derived oligosaccharides (Nelson et
al., 1993), sulfated neoglycopolymers (Manning et al., 1997), a
hydroxamic acid-based peptide inhibitor of matrix metalloproteases
(Walcheck et al., 1996), and chimeric proteins (Mulligan et al., 1993b;
Fujise et al., 1997). Recently, the 3'-sulfated
Lewisa pentasaccharide was demonstrated to
prevent ischemia-reperfusion lung injury in a rat model (Reignier et
al., 1997). The 3'-sulfated Lewisa has been shown
to be a more potent ligand for E- and L-selectins as
compared with SLex (Green et al., 1995; Yuen et
al., 1994). The biological effects of many of these compounds in
selectin-dependent animal models of inflammation have been critically
reviewed (Lowe and Ward, 1997).
The protective effects of SLex synthetic
analogues have been demonstrated in several models of inflammation,
including feline (Buerke et al., 1994) and canine (Lefer et al., 1994b;
Flynn et al., 1996) models of myocardial ischemia/reperfusion, as well as in a rat model of lung injury (Mulligan et al., 1993a). However, the
use of the SLex analogue CY-1503 did not reduce
myocardial infarct or neutrophil accumulation in dogs subjected to
ischemia/reperfusion injury (Gill et al., 1996). These conflicting
results may in part be explained by the dosing regimes employed by the
different investigators and the relatively short half-life of the
SLex analogue. Of key importance is the high
IC50 (0.5 to 1.0 mM) of the
monovalent SLex tetrasaccharide in inhibiting E-
and P-selectin-dependent adhesion of leukocytes, as determined in
static adhesion assays (Jacob et al., 1995). SLex
multivalency appears to enhance its binding to L-selectin
(Maaheimo et al., 1995). Thus, a synthetic SLex
analog was tested as an inhibitor of L-selectin-mediated
lymphocyte-endothelium interactions in rejecting rat kidney transplant.
Although the nonfucosylated O-glycosidic oligosaccharide did
not possess any inhibitory activity, the monovalent
SLex molecule prevented the binding
significantly, and the divalent SLex saccharide
was the most potent inhibitor (Maaheimo et al., 1995).
Complement activation and, in particular, generation of C5a attract and
stimulate neutrophils, causing their sequestration within capillaries.
Activated neutrophils produce toxic oxygen metabolites that damage
endothelial cells (Mulligan et al., 1996, 1997). C5a is necessary for
up-regulation of vascular P-selectin after systemic activation of
complement (Foreman et al., 1994; Mulligan et al., 1997; Ward, 1996).
To control the damaging effects of both complement and neutrophil
activation during inflammation, sCR1 was produced in a mammalian cell
line capable of SLex glycosylation (Picard et
al., 1996; Sen et al., 1966; Bertino et al., 1996). It was shown that
sCR1 purified from conditioned media possessed
SLex moieties on the N-linked
oligosaccharides. sCR1 potentially has 25 N-glycosylation
sites (Klickstein et al., 1988) and, although not every Asn-X-Ser(Thr)
sequon is an efficient oligosaccharide acceptor (Kasturi et al., 1997),
it is expected that sCR1-SLex would be
extensively decorated with SLex moieties. Thus,
in addition to blocking complement activation, the potential
multivalent interactions between sCR1-SLex and
its selectin counterligands might render this molecule particularly effective at inhibiting neutrophil activation and recruitment to sites
of inflammation on the endothelial surface. It is important to
determine the half-life of sCR1-SLex and,
especially, whether it localizes to sites of inflammation. Notably, the
basic SCR structure of CR1 occurs in selectins, and this might allow
relatively easy structural modifications and a "cassette" approach
to the molecular construction of hybrid molecules. For example,
constructs possessing the ability to home to areas of inflamed
endothelium might be readily combined with elements effecting
multivalent complement inhibition. Spacing of the active segments along
the construct could be varied for optimal interaction with complement
elements while still retaining the affinity of the selectins for
targeted endothelium.
D. Complement Receptor Type 2
The molecular cloning of the human CR2 (Moore et al., 1987; Weis
et al., 1988) facilitated its structural and functional
characterization (Ahearn and Fearon, 1989; Fearon and Carter, 1995;
Carroll and Fischer, 1997). CR2 (CD21; Epstein-Barr virus receptor) is
present on follicular dendritic cells, mature B cells, and a
subpopulation of T cells, and it binds the C3 breakdown fragments, C3dg
and C3d. CR2 has relatively weak cofactor activity for the factor I-mediated breakdown of iC3b to C3dg and C3c (Mitomo et al., 1987), and
it probably plays a minor role in complement regulation. CR2 has B
cell-stimulating functions, as it associates with CD19, a B cell
surface molecule that activates B cells, and participates in T
cell-dependent B cell responses (Fearon and Carter, 1995; Carroll and
Fischer, 1997). Fearon and colleagues provided direct evidence that
attachment of C3d to antigen significantly enhances humoral responses,
a process that is mediated by CR2 (Dempsey et al., 1996). The
immune-augmenting function of C3d was demonstrated by the fusion of
murine C3d to hen egg lysozyme (HEL). Thus, HEL bearing three copies of
C3d was ten thousand-fold more immunogenic than HEL alone, suggesting
that such manipulations might allow for development of effective
strategies for vaccination without the need for adjuvant (Dempsey et
al., 1996).
E. Soluble Decay Accelerating Factor
Decay accelerating factor (DAF) (CD55) is composed of four SCRs
plus a serine/threonine-enriched domain that is capable of extensive
O-linked glycosylation (fig. 2) (Nicholson-Weller and Wang,
1994). DAF is attached to cell membranes by a glycosyl phosphatidyl inositol (GPI) anchor (Davitz et al., 1986; Medof et al., 1986) and,
through its ability to bind C4b and C3b, it acts by dissociating the C3
and C5 convertases in both the classical and alternative pathways (fig.
1). Unlike CR1, which possesses both extrinsic (Medof et al., 1982) and
intrinsic activity (Kinoshita et al., 1986; Makrides et al., 1992), DAF
functions only intrinsically by inactivating convertases assembled on
the same cell membrane on which it is expressed and not those
convertases formed on external membranes (Medof et al., 1984). Soluble
versions of DAF (sDAF) have been shown to inhibit complement activation
in vitro (Christiansen et al., 1996; Moran et al., 1992) as well as in
the reversed passive Arthus reaction in guinea pigs (Moran et al.,
1992) (table 4).
|
The clinical usefulness of a complement blocker depends on several
requirements (Kalli et al., 1994). These include the ability to inhibit
the C5 convertases of both classical and alternative pathways, a high
affinity for the C3b and C4b components of the convertases, the
irreversible inactivation of the convertases, and the ability to
recycle in order to block multiple convertases (Kalli et al., 1994).
The modest inhibitory activity of sDAF (Christiansen et al., 1996;
Moran et al., 1992; Kalli et al., 1994) and its lack of factor I
cofactor activity limit its therapeutic potential as a complement
blocker.
F. Soluble Membrane Cofactor Protein
Membrane cofactor protein (CD46; measles virus receptor) (fig. 2)
has factor I cofactor activity but no decay-accelerating activity. It
acts jointly with DAF, which has decay-accelerating activity but no
cofactor activity (table 3) to block C3b/C4b deposition on cell
membranes (Liszewski et al., 1991). MCP is an intrinsic regulator
of complement activation, i.e., it protects cells on which it is
expressed, but it does not protect neighboring cells (Oglesby et al.,
1992). It is expressed primarily as four isoforms, termed BC1, BC2, C1,
and C2, that are formed by alternative splicing of a single gene and
that differ in the domains for O-glycosylation and
cytoplasmic regions (reviewed in Liszewski et al., 1996). The BC
isoforms have been shown to cleave cell-bound C4b more efficiently than
the C isoforms and to provide enhanced cytoprotection against the
classical pathway (Liszewski and Atkinson, 1996). A recombinant sMCP
was shown to inhibit immune complex-mediated inflammation in the
reverse passive Arthus reaction model in rats (Christiansen et al.,
1996). As in the case of sDAF, the single activity of sMCP limits its
potential as an effective therapeutic reagent. However, sMCP may prove
to be a valuable reagent in combination with other complement
inhibitors (see Section III.I.).
G. Soluble CD59
CD59, also known by several other names (Liszewski et al., 1996),
is a single-chain glycoprotein that is GPI-anchored to cell membranes
(Holguin et al., 1989; Davies et al., 1989; Davies and Lachmann, 1993).
The carbohydrate moiety at the single N-glycosylation site
is not required for complement inhibition (Suzuki et al., 1996;
Rushmere et al., 1997). CD59 functions as an inhibitor of the formation
of the MAC on cells by binding to C8 and C9, thereby blocking the
addition of polymerized C9 molecules (Meri et al., 1990; Rollins et
al., 1991). sCD59 has been shown to possess complement inhibitory
activity in vitro (Sugita et al., 1994). However, the potential
usefulness of sCD59 as a therapeutic complement blocker is limited by
its lack of certain functional properties (as discussed in Section
III.E.) (Kalli et al., 1994). Although the inhibition of MAC assembly
would be of benefit in inflammation, the late stage in the complement
cascade at which CD59 acts (fig. 1), leaves the generation of
anaphylatoxins and their pathological sequelae unaffected.
H. Decay Accelerating Factor-CD59 Hybrid
The molecular fusion of different complement regulatory proteins
has been used to create chimeric molecules endowed with novel functions. Fodor and colleagues (Fodor et al., 1995) constructed two
such chimeric complement inhibitors for cell surface expression using a
GPI anchor: CD (NH2-CD59-DAF-GPI) and DC (NH2-DAF-CD59-GPI). The
rationale behind this work was to create a single protein that blocks
C3 and C5 convertase activity as well as the assembly of the MAC. Of
the two molecules, CD retained DAF function, but did not inhibit C5b-9
assembly. The DC chimera, however, exhibited both DAF and CD59
activity. The reason for the differential function of the two molecules
was thought to be the different orientation of the protein domains.
Thus, in the CD molecule, the CD59 moiety occupies a membrane-distal
position where it cannot interact with the C5b-8 and C5b-9 complex,
although in the DC molecule the membrane-proximal position of the CD59
domain facilitates the interaction between CD59 and the MAC (Fodor et
al., 1995). The DC chimera may have utility in the production of
transgenic organs (see also Section VII.) for the inhibition of
hyperacute rejection in xenotransplantation (Kennedy et al., 1994;
Fodor et al., 1994; McCurry et al., 1995a,b; Miyagawa et al., 1995;
Heckl-Östreicher et al., 1996; Kroshus et al., 1996b; Diamond et
al., 1996; Byrne et al., 1997).
I. Membrane Cofactor Protein-Decay Accelerating Factor Hybrid
The molecular fusion of membrane cofactor protein (MCP) and decay
acclerating factor (DAF) brings together the complementary activities
of these two regulatory molecules to create a single protein that has
both factor I cofactor activity and decay-accelerating activity. A
membrane-bound chimeric MCP-DAF was expressed in CHO cells, and its
activity was compared with that of transfectants expressing MCP or DAF
or MCP plus DAF (Iwata et al., 1994). The proteins differed in their
ability to block C3 deposition on sensitized CHO cells through
activation of the classical pathway, in the order of MCP + DAF > DAF > MCP-DAF > MCP. C3 deposition via the alternative
pathway was blocked in the order MCP-DAF > MCP + DAF > DAF > MCP (Iwata et al., 1994). Thus, in this in vitro system, the hybrid surface-bound protein appeared to have greater potency at
blocking alternative rather than classical pathway activation. Similar
studies were performed in vitro in stably transfected swine endothelial
cells exposed to human complement (Miyagawa et al., 1994). In this
model of xenograft hyperacute rejection, mediated mainly by the
classical pathway, the surface-expressed MCP-DAF hybrid inhibited cell
lysis more effectively than MCP alone, and apparently as effectively as
DAF. Differences in lysis, however, were rather small, and the
quantitative differences in the levels of surface expression of the
molecules make it difficult to draw firm conclusions regarding their
relative effectiveness (Iwata et al., 1994; Miyagawa et al., 1994).
Nevertheless, these studies demonstrate the dual functionality and
complement inhibitory activity of the MCP-DAF hybrid.
A soluble version of chimeric MCP-DAF, referred to as complement
activation blocker-2 (CAB-2), possessed factor I cofactor activity and
decay-accelerating activity, and inactivated both classical and
alternative C3 and C5 convertases in vitro as measured by assays of
inhibition of cytotoxicity and anaphylatoxin generation (Higgins et
al., 1997). CAB-2 had inhibitory activity against cell-bound
convertases that was greater than that of either sMCP or sDAF or both
factors combined. This hybrid was shown to inhibit complement
activation in vivo, in the reversed passive Arthus reaction and in the
direct passive Arthus reaction, as well as in the Forssman shock model
in guinea pigs. The t1/2 of CAB-2 in rats was
8 h (Higgins et al., 1997), which is suitable for human therapy.
It is possible that the half-life of CAB-2 may be longer in humans than
in rats, as has been the case for sCR1 (see Section III.A.). One
potential limitation of CAB-2 as a therapeutic is its potential
immunogenicity. The molecular fusion of two otherwise natural proteins
is likely to create novel epitopes, which might trigger an immune
response. In this case, CAB-2 might be useful in acute indications,
depending on the severity of the anti-CAB-2 response.
J. C1 Inhibitor
C1 inhibitor, a member of the "serpin" family of serine
protease inhibitors, is a heavily glycosylated plasma protein that prevents fluid-phase C1 activation (reviewed in Davis, 1988; Davis et
al., 1993). C1 inhibitor regulates the classical pathway of complement
activation (fig. 1) by blocking the active site of C1r and C1s and
dissociating them from C1q (Ziccardi and Cooper, 1979). Studies of the
role of complement activation in myocardial ischemia and reperfusion
injury (reviewed in Homeister and Lucchesi, 1994; Makrides and Ryan,
1997) have used C1 inhibitor in feline (Buerke et al., 1995), rat
(Murohara et al., 1995a), and pig (Horstick et al., 1997) models.
All these studies have demonstrated that blocking the classical pathway
of complement activation by C1 inhibitor is an effective means of
protecting ischemic myocardial tissue from reperfusion injury.
K. C1q Receptor
Several types of human C1q receptors (C1qR) have been described.
These include the ubiquitously distributed 60- to 67-kDa receptor,
referred to as cC1qR because it binds the collagen-like domain of C1q
(Peerschke et al., 1993; Malhotra et al., 1993). This C1qR
variant was shown to be calreticulin (Malhotra et al., 1993; Stuart et
al., 1996); a 126-kDa receptor that modulates monocyte phagocytosis,
designated C1qRp (Guan et al., 1991, 1994; Nepomuceno et al., 1997); and a 28- to 33-kDa protein isolated and
cloned from Raji cells, termed gC1qR because it interacts preferentially with the globular domains of C1q (Ghebrehiwet et al.,
1994; Peerschke et al., 1996). A recent study showed that CR1 also
acts as a receptor for C1q (Klickstein et al., 1997). Experimental
evidence supports the hypothesis that gC1qR is not a membrane-bound
molecule, but rather a secreted soluble protein with affinity for the
globular regions of C1q (van den Berg et al., 1997). Thus, it may act
as a fluid-phase regulator of complement activation. van den Berg et
al. (1997) did not detect surface expression of gC1qR but were able to
demonstrate strong intracellular staining for this protein, as well as
its presence in human and rat sera and in supernatants of cultured
HUVEC. Furthermore, other data are consistent with the molecular
properties of gC1qR. Thus, the cDNA sequence (Ghebrehiwet et al., 1994)
encodes a protein that lacks a membrane-spanning domain (Fasman and
Gilbert, 1990) or a consensus sequence for GPI-anchoring (Medof et al.,
1996). It is possible, however, that under certain conditions gC1qR may be surface-expressed at low levels, or it may bind to cell membranes as
a complex with other fluid-phase molecules (van den Berg et al., 1997).
The ability of C1qR (66 kDa) to inhibit the classical pathway of
complement has been demonstrated in vitro. Membrane-associated C1qR as
well as detergent-solubilized C1qR, purified from polymorphonuclear leukocytes and endothelial cells, blocked complement-mediated lysis of
C1q-sensitized erythrocytes (van den Berg et al., 1995).
The mechanisms by which the different types of C1qR regulate complement
activation in vivo and the physiological significance of the putative
fluid-phase C1qR (van den Berg et al., 1995, 1997) remain unclear.
However, the studies cited here, and the demonstration that C1q is
required for immune complexes to stimulate endothelial cells to express
adhesion molecules (Lozada et al., 1995), suggest a potential
therapeutic use in preventing vascular injury.
| |
IV. Complement-Inhibitory Antibodies |
|---|
|
TopPreviousNextReferences
|
|---|
A. Anti-C5 Monoclonal Antibody
Inhibition of C5 activation using high-affinity
(Kd < 100 pM) anti-C5 monoclonal
antibodies (mAbs) represents another therapeutic approach for blocking
complement activation (Matis and Rollins, 1995; Rinder et al., 1995).
This strategy is aimed at inhibiting the formation of C5a and C5b-9 via
both the classical and alternative pathways (fig. 1), without affecting
the generation of C3b, a C3 opsonic product that is critical for
antibacterial defenses (Ross and Densen, 1984). This is scientifically
sound, although, as discussed above (section III.A.), the on-going
clinical trials using sCR1 have produced no evidence to date that
blockade of the C3 and C5 convertases in both the classical and
alternative pathways compromises bacterial defenses. Another suggested
advantage (Wang et al., 1995) of using monoclonal antibodies to block
C5 activation is the prevention of the direct cleavage and activation of C5 by oxygen radicals (Vogt et al., 1989) or by enzymes released from injured tissue (Wetsel and Kolb, 1982, 1983) during inflammation.
The efficacy of a mAb specific for murine C5 was demonstrated in the
treatment of collagen-induced arthritis, an animal model for human
rheumatoid arthritis. It was shown that the systemic administration of
the anti-C5 mAb in mice blocked complement activation, prevented the
onset of arthritis in immunized animals, and ameliorated established
disease (Wang et al., 1995). The same anti-C5 mAb was tested in mice
that develop an autoimmune disorder similar to human systemic lupus
erythematosus. Continuous treatment with the antibody resulted in
significant reduction in glomerulonephritis and in increased survival
(Wang et al., 1996).
The anti-human C5 mAb N19/8 (Würzner et al., 1991) that does not
inhibit formation of C3a was tested in an in vitro model of
extracorporeal blood flow that activates complement, platelets, and
neutrophils (Rinder et al., 1995). This mAb inhibited the generation of
C5a and soluble C5b-9 and blocked serum complement hemolytic activity,
without affecting the production of C3a. In addition, the anti-C5 mAb
inhibited neutrophil CD11b up-regulation, abolished the increase in P
selectin-positive platelets, and reduced formation of
leukocyte-platelet aggregates (Rinder et al., 1995). Thus, it appears
that C5a and C5b-9, but not C3a contribute to platelet and neutrophil
activation during extracorporeal procedures. Although the N19/8 mAb
could be used in human therapy, it is recognized that chronic
application of monoclonal antibodies would elicit human anti-mouse
antibody responses (Waldmann, 1991; Khazaeli et al., 1994). The
"humanization" of antibodies (Co and Queen, 1991; Rapley, 1995;
Morrison and Shin, 1995) should minimize immunogenic reactions,
although it might be difficult to completely eliminate anti-idiotypic
effects. Recent advances in transgenic animal technology now make it
possible to produce completely human monoclonal antibodies that are
devoid of mouse or other nonhuman sequences (Fishwild et al., 1996;
Brüggemann and Neuberger, 1996; Brüggemann and Taussig,
1997; Jakobovitz, 1995; Sherman-Gold, 1997).
B. Anti-C5 Single Chain Fv
A recombinant single chain (scFv) antibody, constructed from the
variable region of the N19/8 mAb, was shown to inhibit human C5b-9-mediated hemolysis of chicken erythrocytes and to partially inhibit C5a generation (Evans et al., 1995). The ability of this scFv
to protect against complement-mediated myocardial injury was
demonstrated in isolated mouse hearts perfused with 6% human plasma.
Pharmacokinetic analysis in rhesus monkeys revealed a t1/2 of 28 minutes and a
t1/2 of 17 h (Evans et al., 1995). Humanized anti-C5 antibody and scFv have been produced (Thomas et al.,
1996).
| |
V. Synthetic Inhibitors of Complement Activation |
|---|
|
TopPreviousNextReferences
|
|---|
Compared with conventional drugs, recombinant proteins for therapy
remain attractive to date, for reasons having to do with both the
biological properties of proteins and the economics of drug development
(Buckel, 1996). The time required to develop protein drugs is shorter
than that for conventional drugs and, although a therapeutic protein
has a 40% probability of becoming a marketable drug, this figure is
approximately 10% for a new chemical entity, partly because of the
lower toxicity of proteins compared with chemical compounds (Buckel,
1996). However, the high cost of therapeutic proteins is increasingly
becoming a problem (Grindley and Ogden, 1995). The emergence of
structure-based drug design for the development of small synthetic
molecules for therapy holds promise, in spite of formidable technical
challenges (Verlinde and Hol, 1994; Hruby, 1997).
The existing plethora of synthetic blockers of complement prompted
Becker in 1972 to note that "a comprehensive review of all compounds
found to inhibit complement would turn into a catalogue of a chemical
supply house." Twenty five years later, this task becomes even more
daunting. Several excellent reviews on the use of synthetic complement
inhibitors (table 5) for therapeutic, as
well as for other uses, have been published (Becker, 1972; Patrick and
Johnson, 1980; Asghar, 1984; Fujii and Aoyama, 1984; Hagmann and
Sindelar, 1992). The objective here is to present a brief and
selective summary of the findings using synthetic molecules for the
therapeutic inhibition of complement.
|
A. Peptides and Analogs
The anaphylatoxins exert their multiple biological functions
(Gerard and Gerard, 1994; Mulligan et al., 1997; Hartmann et al., 1997)
by binding to their respective receptors (Wetsel, 1995). C5a, the most
potent anaphylatoxin, is a 74-amino acid polypeptide, the sequence of
which (Fernandez and Hugli, 1978) has been used to synthesize peptide
analogs to downregulate the transducing functions of the C5aR, a member
of the G protein-coupled receptor superfamily (Gerard and Gerard, 1991;
Boulay et al., 1991). A series of hexapeptide analogs of the form
NMePhe-Lys-Pro-dCha-X-dArg has been synthesized (Mollison et al., 1992;
Konteatis et al., 1994) and tested for C5aR antagonism. The peptide
C089 (IC50 70 nM) containing Trp at
position X lacked agonist properties and inhibited C5a-induced
degranulation and GTPase activity, a measure of G protein activation
(Konteatis et al., 1994). The in vivo functionality of this peptide has
not been reported. Other C5a peptide derivatives having no
anaphylatoxin or agonist activity have been described (van Oostrum et
al., 1996) and shown to be active in reducing inflammation in animal
models. The elucidation of the tertiary structure of a peptide
antagonist of C5aR (Zhang et al., 1997) should provide information for
the future structure-based design of C5aR antagonists.
A different experimental approach to the design of peptide antagonists
of C5aR is based on the molecular recognition theory proposed by
Blalock (reviewed in Blalock, 1990; Trospha et al., 1992; Blalock,
1995). This theory is based on the concept of "complementary" or
"antisense" peptide and proposes that peptides encoded in the same
reading frame on opposite strands of deoxyribonucleic acid (DNA) can
bind to each other on the basis of their complementary hydropathy
(Blalock, 1995). Furthermore, the theory suggests that receptors and
their cognate ligands may have evolved from complementary regions of
the same nucleotide sequence (see discussion in Baranyi et al., 1996).
Amphiphilic peptides consisting of 8-15 residues and their
corresponding antisense peptides have been identified within proteins
and termed antisense homology boxes (AHB) (Baranyi et al., 1995). These
regions may represent important structural elements that somehow
influence the function of their respective proteins. A peptide derived
from an AHB of the human endothelin A receptor inhibited endothelin in
a smooth muscle relaxation assay and blocked endotoxin-induced shock in
rats (Baranyi et al., 1995). Similarly, computer analysis of human C5a
and the C5aR revealed several AHBs, and peptides derived from the AHBs acted as agonists or antagonists of C5aR function, depending on their
concentration (Baranyi et al., 1996). It is possible that the ability
to locate AHBs in proteins may provide an efficient means to identify
peptides with biological activity. Other peptides that inhibit specific
components of the complement system are summarized in table 5.
B. Organic Molecules
The crystal structure of factor D has been elucidated in a series
of studies designed to produce an inhibitor for the therapeutic modulation of the alternative pathway (Narayana et al., 1994; Kim et
al., 1995; Cole et al., 1997). This strategy is based on the rationale
that factor D is the limiting enzyme in the alternative pathway and is
positioned early in the biochemical cascade. The ability of diisopropyl
fluorophosphate to completely inactivate factor D (Fearon et al., 1974)
has been exploited in crystallographic studies to compare the active
sites between factor D and the diisopropyl fluorophosphate-inhibited
factor D (Cole et al., 1997) with the objective of designing small
molecule inhibitors. This work resulted in the synthesis of a factor D
inhibitor (BCX-1470, IC50 96 nM, see
table 5).
The fungal metabolite K76 (see table 6)
has been modified to yield complement inhibitors of modest
IC50 values (Kaufman et al., 1995a,b). TKIXc, a
K76 derivative, inhibited both the classical and the alternative
pathways (see table 5). Other synthetic inhibitors of complement
activation are listed in table 5.
|
| |
VI. Naturally Occurring Compounds That Block Complement Activation |
|---|
|
TopPreviousNextReferences
|
|---|
There is voluminous literature on naturally-occurring complement
inhibitors isolated from animal and plant tissues (table 6). Some of
these compounds may serve as leads to new chemical structures, although
others have not yet been purified to homogeneity. Heparin and its
related glycosaminoglycan compounds and derivatives have been actively
pursued as complement inhibitors. Heparin is a sulfated copolymer of
uronic acid and glucosamine (Jaques, 1979a,b). Its protein core is
removed during commercial processing to yield glycosaminoglycan
heparin. The anticomplement activity of heparin was first demonstrated
in 1929 (Ecker and Gross, 1929), and its mechanism of action has been
extensively studied (Weiler et al., 1978, 1992; Linhardt et al., 1988;
Maillet et al., 1983). Heparin blocks the interaction between C1q and
complement activators and inhibits the assembly of C3 convertases in
the classical and alternative pathways. In addition, it may potentiate
C1 inhibitor-mediated inactivation of C1s, a mechanism shared by
heparin and related glycosaminoglycans (Wuillemin et al., 1997;
Kirschfink et al., 1997). A highly sulfated, low-molecular weight
heparin derivative has been shown to prevent complement-mediated
myocardial injury in the perfused rabbit heart (Gralinski et al.,
1997). Heparin-coated extracorporeal circuits inhibit complement
activation during cardiac surgery (te Velthuis et al., 1996). For more
information on naturally-occurring complement-inhibitory compounds,
refer to the references in table 6.
| |
VII. Complement Inhibition in Xenotransplantation |
|---|
|
TopPreviousNextReferences
|
|---|
Xenotransplantation, the ability to engraft organs across the
species barrier, would theoretically meet the demand for organ transplantation that has doubled since 1988 and is growing by 15% per
annum, requiring approximately 150,000 people worldwide to wait for
donor organs (Nainggolan, 1996). It is estimated that by the year 2010 the xenotransplantation market could be worth $6 billion (Nainggolan,
1996). In recent years there has been remarkable progress in prolonging
survival of xenogeneic organs in animal models of xenotransplantation,
and there is optimism in the scientific community about overcoming the
various immunological barriers to xenotransplantation (Auchincloss,
1997). However, there are serious obstacles to be overcome, and,
occasionally, we are reminded of the severe hurdles evolution has set
for xenotransplantation (Hammer, 1997). In an excellent review of
comparative physiology, biochemistry, and anatomy in the context of
xenotransplantation, Hammer (1997) concludes that "In the
pig-to-primate model, little convincing organ function has been
achieved. . . . Today's approaches are not convincing." The field of
xenotransplantation has been reviewed often and in great detail. The
recent volume edited by Cooper et al. (1997) provides an excellent
single source of information on this topic. The objective here is to
summarize briefly the main areas of research activity as they pertain
to complement inhibition in xenotransplantation.
Organ transplantation between widely disparate species is termed
"discordant" as opposed to "concordant" transplantation between closely related species (Calne, 1970). The hallmark of discordant xenotransplantation is the rapid and destructive rejection of the
xenograft, a process referred to as hyperacute rejection (HAR) (table
7). Activation of the complement system,
after recognition of the discordant organ by xenoreactive antibodies,
plays a crucial role in HAR (Baldwin et al., 1995; Sanfilippo, 1996;
Dalmasso, 1997). It is generally accepted that the relative importance
of the classical versus the alternative pathway in HAR depends on the
species combination studied. For example, the complete elimination of
natural antibodies from rats had little effect on their ability to
reject guinea pig hearts hyperacutely (Pruitt et al., 1993; Soares et
al., 1994). On the other hand, blocking or absorption of natural
antibodies in primates is an effective method of preventing HAR of
porcine hearts. Several other studies using different species combinations have shown that the alternative pathway of complement is
activated in HAR (Miyagawa et al., 1988; Wang et al., 1992; Forty et
al., 1992; Hengster et al., 1996). A recent study examined whether the
alternative and classical pathways can be activated independently in
HAR: human plasma was depleted of both C1q and factor D and then
reconstituted with purified C1q or factor D to restore the classical
and alternative pathways, respectively (Romanella et al., 1997). The
modified plasmas were tested in an ex vivo isolated mouse heart
perfusion model, and it was demonstrated that, in the mouse-to-human
species combination, both the classical and alternative pathways are
independently activated (Romanella et al., 1997).
|
Two main approaches have been used to prevent HAR (table
8). One method attempts to block the
interactions between native xenoreactive antibodies and the xenograft
endothelium. This strategy is aimed at the major xenoantigen
responsible for HAR, the -galactosyl epitope (Rother and Squinto,
1996; Oriol and Cooper, 1997). The other approach aims to block
complement activation using soluble complement inhibitors or transgenic
technology. Cobra venom factor (CVF) has been shown to deplete
complement and prolong graft survival (Leventhal et al., 1994).
However, CVF achieves its effect by activating complement and
generating the anaphylatoxins C3a and C5a, which may cause endothelial
damage (Till et al., 1982; Schmid et al., 1997b). In addition, the
immunogenicity of CVF limits its usefulness. C1 inhibitor (C1-Inh), in
combination with heparin, blocks HAR mediated by the classical pathway
(Dalmasso and Platt, 1993). sCR1 has been shown to effectively delay
HAR in a variety of xenotransplantation models (reviewed in Baldwin et
al., 1995; Sanfilippo, 1996; Ryan, 1995; Levin et al., 1996; Marsh and
Ryan, 1997).
|
An alternative strategy for the suppression of HAR uses transgenic
technology for the production of animals expressing molecules of the
human RCA family (Cozzi and White, 1995; Platt and Logan, 1997; Squinto
and Fodor, 1997; Hancock, 1997). This is a promising area with
its own limitations (table 8). Disturbingly, the recent demonstration
that pig cell lines harbor endogenous retroviruses that could infect
human cells in vitro (Patience et al., 1997) raises the possibility
that pig retroviruses might infect human cells in xenotransplantation
(Chapman and Fishman, 1997; Allan, 1997; Murphy, 1996; Stoye, 1997;
Stoye and Coffin, 1995; Chapman et al., 1995; Patience et al., 1997;
Bach et al., 1998).
Clearly, there has been significant progress in our understanding of
the mechanisms of HAR, and it is reasonable to anticipate that this
principal immunological barrier to xenotransplantation, as well as
delayed rejection, will be overcome in the near future (Auchincloss,
1997). However, formidable obstacles to overcome chronic rejection of
xenografts remain (Hammer, 1997). Evidence indicates that chronic
rejection may be mediated by several complement-independent mechanisms,
including the activation of endothelium by IgM xenoreactive antibodies
(Platt et al., 1991; Blakely et al., 1994), activation of macrophages
or T cells (Blakely et al., 1994; Chen et al., 1992; Fryer et al.,
1994, 1995), activation of natural killer cells (Inverardi et al.,
1992; Arakawa et al., 1994), and antibody-dependent cellular
cytotoxicity (Schaapherder et al., 1994; Dennert, 1974; Lin et al.,
1997).
| |
VIII. Bispecific Antibodies for Immune Complex Removal |
|---|
|
TopPreviousNextReferences
|
|---|
The potential role of erythrocytes in the body's defense against
bacteria and viruses was recognized by Nelson (1953, 1955) who
demonstrated in vitro the binding of microorganisms to the erythrocyte
surface in the presence of antibody and complement and showed that the
immobilization of C3b-opsonized microbes on erythrocytes led to
increased phagocytosis of the adherent pathogens by leukocytes.
Erythrocyte-associated CR1 (Fearon, 1979) in primates plays a key role
in the elimination of antibody/antigen immune complexes (IC) by binding
C3b/C4b-opsonized IC in the circulation. The IC are then removed from
erythrocytes by macrophages for subsequent clearance in the liver and
spleen (Schifferli et al., 1986; Ahearn and Fearon, 1989). The
erythrocytes are returned to the circulation without lysis. Nelson's
original observations indicated that the erythrocyte-CR1 system could
possibly be manipulated for the removal of pathogens in human disease.
Thus, Taylor and colleagues (Taylor et al., 1991) speculated that if
targeted antigens could be bound to erythrocytes via CR1 in the absence
of complement, then it might be possible to use erythrocytes to treat a
variety of infectious diseases associated with blood-born pathogens.
This concept was systematically studied using bispecific, cross-linked
monoclonal antibodies (heteropolymers) with specificity for both
targeted antigen and the human CR1 (Taylor et al., 1991) (fig.
3A).
|
The potential value of bispecific antibodies in this therapeutic
approach rests on their ability to facilitate antigen clearance in vivo
without destruction of erythrocytes. In studies designed to answer this
question, the injection into monkeys of sensitized erythrocytes
(containing 125I-labeled Ag attached to
51Cr-labeled monkey erythrocytes) led to rapid
clearance from the circulation of several different antigens with no
sequestration, lysis, or clearance of erythrocytes (Taylor et al.,
1992; Reist et al., 1993). Thus, large amounts of IgG can be bound via
CR1 to human or monkey erythrocytes without any phagocytic uptake by
mononuclear cells. In contrast, erythrocytes that bind comparable levels of IgG at sites other than CR1, are rapidly phagocytosed (Reinagel et al., 1997). The primary organs for uptake of the IC were
the liver and spleen (Reist et al., 1994). Similar studies in
experimental monkey models demonstrated the feasibility of using
bispecific antibodies to clear prototype viruses (Taylor et al.,
1997a,b) and autoantibodies (Ferguson et al., 1995a,b; Taylor and
Ferguson, 1995) from the circulation (fig. 3B), and, once again, the
cleared substrates were phagocytosed and destroyed in the liver (Taylor
et al., 1997a). In vitro studies using bispecific antibodies suggest
that a modification of this approach may be used to clear bacterial
pathogens from cystic fibrosis patients (McCormick et al., 1997). Mouse
monoclonal antibodies are inherently immunogenic in humans, but this
problem could be minimized through antibody engineering,
"humanization" methods, or transgenic technology for production of
completely human antibodies.
In contrast to the above approach that avoids complement activation,
bispecific antibodies have also been engineered to recruit complement
effector functions (Kontermann et al., 1997; Holliger et al., 1997). In
this case, human antibody fragments directed against human C1q were
isolated from a phage display library and coupled to lysozyme-specific
antibody fragments, creating bispecific antibodies (diabodies). These
were able to recruit C1q, effecting the lysis of lysozyme-coated sheep
erythrocytes (Kontermann et al., 1997). Other diabody constructions
were directed against the target antigen as well as against serum Ig
and were shown to recruit complement and promote cytotoxicity toward
colon carcinoma cells in conjunction with CD8+ T
cells (Holliger et al., 1997). Such bispecific antibodies may have
therapeutic utility in situations requiring complement activation.
| |
IX. Summary |
|---|
|
TopPreviousNextReferences
|
|---|
The use of powerful methodologies in molecular biology, biochemistry, and physiology in the last 2 decades has led to impressive progress in our understanding of the mechanisms of complement activation and its role as either a protective or a pathogenic factor in human disease. With respect to disease pathogenesis, the complexity of the complement cascade provides opportunities for several different therapeutic targets within the complement pathways. More than a century after complement was first described, we are about to witness in the near future the availability of a variety of complement inhibitors for specific therapies. Progress in the area of xenotransplantation has been substantial, but formidable obstacles remain to selective inhibition of the factors that block successful clinical xenotransplantation. Bispecific antibodies, designed to enhance rather than inhibit existing complement pathways, hold strong promise for the clearance of viral and bacterial pathogens from the circulation.
| |
Acknowledgments |
|---|
|
TopPreviousNextReferences
|
|---|
I am very grateful to Lloyd B. Klickstein, Alfred R. Rudolph, Robert D. Sindelar, and, especially, Ronald P. Taylor for their support and encouragement and for their painstaking comments and advice on the manuscript. I thank William M. Baldwin and Peter A. Ward for their valuable comments on the manuscript, as well. Any errors are solely my own responsibility. I thank Hedy Adari for help with the literature search.
| |
Footnotes |
|---|
a Address for correspondence: Savvas C. Makrides, PRAECIS Pharmaceuticals, Inc., 1 Hampshire St., Cambridge, MA 02139-1572.
| |
Abbreviations |
|---|
AHB, antisense homology boxes; C1qR, C1q receptor; C3aR, C3a receptor; C5aR, C5a receptor; CAB-2, complement activation blocker-2; cDNA, complementary deoxyribonucleic acid; CR2, complement receptor type 2; CVF, cobra venom factor; DAF-CD59, decay accelerating factor-CD59; GPI, glycosyl phosphatidyl inositol; HAR, hyperacute rejection; HEL, hen egg lysozyme; IC, immune complexes; Ig, immunoglobulins; LHR, long homologous repeat; mAb, monoclonal antibody; MAC, membrane attack complex; MCP-DAF, membrane cofactor protein-decay accelerating factor; P, properdin; RCA, regulators of complement activation; scFv, single chain Fv; SCR, short consensus repeat; sCR1, soluble complement receptor type 1; sCR1[desLHR-A], soluble complement receptor type 1 lacking long homologous repeat-A; sCR1-SLex, soluble complement receptor type 1-sialyl Lewisx; sDAF, soluble decay accelerating factor; sMCP, soluble membrane cofactor protein.
| |
References |
|---|
|
TopPrevious |
|---|
-amyloid peptides initiate the complement cascade without producing a comparable effect on the terminal pathway in vitro.
Exp Neurol
146:
388-394[Medline].
monoclonal antibodies from a novel strain of minilocus transgenic mice.
Nat Biotechnol
14:
845-851.[Medline]-chain is crucial for C4b binding and factor I-cofactor function.
Biochem J
323:
469-475.-Amyloid activates complement by binding to a specific region of the collagen-like domain of the C1q A chain.
J Immunol
152:
5050-5059[Abstract].-Gal epitopes in transgenic pig by introduction of human 1-2 fucosyltransferase.
Transplant Proc
29:
894[Medline].(1,2)-fucosyltransferase and its effect on -Gal epitopes in transgenic pig.
Xenotransplantation
3:
81-86.Gal antibodies.
Transplantation
61:
851-855[Medline].gal antibodies with monoclonal anti-idiotypic antibodies, in
Xenotransplantation: The Transplantation of Organs and Tissues Between Species 2nd ed (Cooper DKC,
Kemp E,
Platt JL andWhite DJG eds) pp 377-386,
Springer, Berlin.-glycyrrhetinic acid.
Immunology
90:
115-120[Medline].1,3Gal epitope.
Transplantation
63:
1673-1682[Medline].2-glycoprotein I.
Proc Natl Acad Sci USA
81:
3640-3644-amyloid protein activation of complement in vitro.
Brain Res
749:
135-138[Medline]., dramatically alter the mortality from Zymosan-induced multiple organ dysfunction syndrome (MODS): C5a contributes to mods while MIP-1 has a protective role.
Mol Immunol
33:
1135-1137[Medline].-amyloid in Alzheimer disease.
Proc Natl Acad Sci USA
89:
10016-10020-galactosyl natural antibody.
J Exp Med
182:
1345-1355-galactosyl epitope: A sugar coating that makes viruses and cells unpalatable.
Cell
86:
185-188[Medline].(1-3)gal reaction to avoid hyperacute rejection: Molecular genetic approaches, in
Xenotransplantation: The Transplantation of Organs and Tissues Between Species 2nd ed (Cooper DKC,
Kemp E,
Platt JL andWhite DJG eds) pp 683-700,
Springer, Berlin.1,3)Gal in transgenic mice and pigs by the expression of an (1,2)fucosyltransferase.
Proc Natl Acad Sci USA
93:
7190-7195(1,3)GAL by transfection of N-acetylglucosaminyl transferase III (GnT-III) gene.
Transplant Proc
29:
891-892[Medline].(1,3)Gal by N-acetylglucosaminyltransferase III (GnT-III) in transgenic mice.
Transplant Proc
29:
895-896[Medline].(1,3)Gal-binding proteins.
Xenotransplantation
3:
18-23.-protein is critical for classical complement pathway activation: Implications for Alzheimer's disease pathogenesis.
Nat Med
3:
77-79[Medline]. peptide.
J Neurochem
69:
388-398[Medline].
0031-6997/98/501-0059$03.00/0 PHARMACOLOGICAL REVIEWS
Copyright © 1998 by The American Society for Pharmacology and Experimental Therapeutics
This article has been cited by other articles:
|
|
 |
|
  R. M. Brodbeck, D. N. Cortright, A. P. Kieltyka, J. Yu, C. O. Baltazar, M. E. Buck, R. Meade, G. D. Maynard, A. Thurkauf, D.-S. Chien, et al. Identification and Characterization of NDT 9513727 [N,N-bis(1,3-Benzodioxol-5-ylmethyl)-1-butyl-2,4-diphenyl-1H-imidazole-5-methanamine], a Novel, Orally Bioavailable C5a Receptor Inverse Agonist J. Pharmacol. Exp. Ther., December 1, 2008; 327(3): 898 - 909. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G Girardi and N Mackman Tissue factor in antiphospholipid antibody-induced pregnancy loss: a pro-inflammatory molecule Lupus, October 1, 2008; 17(10): 931 - 936. [Abstract] [PDF]
|
|
|
|
 |
|
 M. Katragadda, D. Morikis, and J. D. Lambris Thermodynamic Studies on the Interaction of the Third Complement Component and Its Inhibitor, Compstatin J. Biol. Chem., December 31, 2004; 279(53): 54987 - 54995. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. K. Ganesh, S. A. Smith, G. J. Kotwal, and K. H. M. Murthy Structure of vaccinia complement protein in complex with heparin and potential implications for complement regulation PNAS, June 15, 2004; 101(24): 8924 - 8929. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Yazdanbakhsh, S. Kang, D. Tamasauskas, D. Sung, and A. Scaradavou Complement receptor 1 inhibitors for prevention of immune-mediated red cell destruction: potential use in transfusion therapy Blood, June 15, 2003; 101(12): 5046 - 5052. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. L. Shields, J. Lai, R. Keck, L. Y. O'Connell, K. Hong, Y. G. Meng, S. H. A. Weikert, and L. G. Presta Lack of Fucose on Human IgG1 N-Linked Oligosaccharide Improves Binding to Human Fcgamma RIII and Antibody-dependent Cellular Toxicity J. Biol. Chem., July 19, 2002; 277(30): 26733 - 26740. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Rehrig, S. D. Fleming, J. Anderson, J. M. Guthridge, J. Rakstang, C. E. McQueen, V. M. Holers, G. C. Tsokos, and T. Shea-Donohue 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., November 15, 2001; 167(10): 5921 - 5927. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Abe, K. Shibata, H. Akatsu, N. Shimizu, N. Sakata, T. Katsuragi, and H. Okada Contribution of Anaphylatoxin C5a to Late Airway Responses After Repeated Exposure of Antigen to Allergic Rats J. Immunol., October 15, 2001; 167(8): 4651 - 4660. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. P. Larsen, R. R. Regal, and J. F. Regal Trimellitic Anhydride-Induced Allergic Response in the Guinea Pig Lung Involves Antibody-Dependent and -Independent Complement System Activation J. Pharmacol. Exp. Ther., April 13, 2001; 296(2): 284 - 292. [Abstract] [Full Text]
|
|
|
|
 |
|
 M. J. Walport Complement- Second of Two Parts N. Engl. J. Med., April 12, 2001; 344(15): 1140 - 1144. [Full Text] [PDF]
|
|
|
|
 |
|
 F. A. De Wolf and G. M. Brett Ligand-Binding Proteins: Their Potential for Application in Systems for Controlled Delivery and Uptake of Ligands Pharmacol. Rev., June 1, 2000; 52(2): 207 - 236. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Caliezi, W. A. Wuillemin, S. Zeerleder, M. Redondo, B. Eisele, and C. E. Hack C1-Esterase Inhibitor: An Anti-Inflammatory Agent and Its Potential Use in the Treatment of Diseases Other Than Hereditary Angioedema Pharmacol. Rev., March 1, 2000; 52(1): 91 - 112. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Heller, M. Hennecke, U. Baumann, J. E. Gessner, A. Meyer zu Vilsendorf, M. Baensch, F. Boulay, A. Kola, A. Klos, W. Bautsch, et al. Selection of a C5a Receptor Antagonist from Phage Libraries Attenuating the Inflammatory Response in Immune Complex Disease and Ischemia/Reperfusion Injury J. Immunol., July 15, 1999; 163(2): 985 - 994. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. M. Blom, J. Webb, B. O. Villoutreix, and B. Dahlback A Cluster of Positively Charged Amino Acids in the C4BP alpha -Chain Is Crucial for C4b Binding and Factor I Cofactor Function J. Biol. Chem., July 2, 1999; 274(27): 19237 - 19245. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
 |
 |
 |
|
 |
 |
 |
