Discovery and development of the complement inhibitor eculizumab for the treatment of paroxysmal nocturnal hemoglobinuria.
ABSTRACT The complement system provides critical immunoprotective and immunoregulatory functions but uncontrolled complement activation can lead to severe pathology. In the rare hemolytic disease paroxysmal nocturnal hemoglobinuria (PNH), somatic mutations result in a deficiency of glycosylphosphatidylinositol-linked surface proteins, including the terminal complement inhibitor CD59, on hematopoietic stem cells. In a dysfunctional bone marrow background, these mutated progenitor blood cells expand and populate the periphery. Deficiency of CD59 on PNH red blood cells results in chronic complement-mediated intravascular hemolysis, a process central to the morbidity and mortality of PNH. A recently developed, humanized monoclonal antibody directed against complement component C5, eculizumab (Soliris; Alexion Pharmaceuticals Inc., Cheshire, CT, USA), blocks the proinflammatory and cytolytic effects of terminal complement activation. The recent approval of eculizumab as a first-in-class complement inhibitor for the treatment of PNH validates the concept of complement inhibition as an effective therapy and provides rationale for investigation of other indications in which complement plays a role.
- SourceAvailable from: Alberto Mella[Show abstract] [Hide abstract]
ABSTRACT: The identification of complement activity in serum and immunohistochemical samples represents a core element of nephropathology. On the basis of this observation, different experimental models and molecular studies have shown the role of this cascade in glomerular disease etiology, but the absence of inhibiting drugs have limited its importance. Since 2006, the availability of target-therapies re-defined this ancient pathway, and its blockage, as the new challenging frontier in renal disease treatment. In the graft, the complement cascade is able to initiate and propagate the damage in ischemia-reperfusion injury, C3 glomerulopathy, acute and chronic rejection, atypical hemolytic uremic syndrome and, probably, in many other conditions. The importance of complement-focused research is revealed by the evidence that eculizumab, the first complement-targeting drug, is now considered a valid option in atypical hemolytic uremic syndrome treatment but it is also under investigation in all the aforementioned conditions. In this review we evaluate the importance of complement cascade in renal transplantation diseases, focusing on available treatments, and we propose a speculative identification of areas where complement inhibition may be a promising strategy.World journal of transplantation. 09/2014; 4(3):168-75.
- [Show abstract] [Hide abstract]
ABSTRACT: Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by a hypercoagulable state associated with acute hemolysis. Eculizumab is used to reduce the intensity of intravascular hemolysis in PNH patients. The hemostatic status of three patients with PNH was assessed during eculizumab treatment by D-dimer assay and the global assays: thromboelastography (TEG), thrombin generation test (TGТ), and thrombodynamics (TD). In the state of hemolytic crisis before the therapy D-dimer concentration was increased in two patients accompanied by hypercoagulation changes in TEG parameter angle (α). TD parameter the clot growth velocity (V) revealed hypercoagulability while TGT parameter ETP was within the normal range in all patients. The lactate dehydrogenase (LDH) activity decreased during the 8months of eculizumab therapy. The physical health was improved, the frequency of hemolytic crisis decreased. Patients periodically exhibited hypercoagulable state: the mean values α=38±11° (with normal range 20-40°), ETP=1311±442nM·min (with normal range 800-1560nM·min), V=31±4μm/min (with normal range 20-29μm/min). During the eculizumab therapy two patients had the repeated clinical manifestation of acute hemolytic crisis, the parameters of the global tests were increased compared to the previous measurement. The global hemostasis tests TEG, TGT and TD revealed hypercoagulability in patients with PNH during eculizumab therapy. Copyright © 2014 Elsevier Inc. All rights reserved.Blood Cells Molecules and Diseases 12/2014; 54(2). · 2.33 Impact Factor
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ABSTRACT: Thrombotic microangiopathies (TMA) are disorders defined by the presence of a microangiopathic hemolytic anemia (with the characteristic hallmark of schistocytes in the peripheral blood smear), thrombocytopenia and organ malfunction of variable intensity. Thrombotic thrombocytopenic purpura and hemolytic uremic syndrome are the most important forms of TMA and, without the adequate treatment, they are associated with high morbimortality. In recent years, significant advances in the knowledge of the pathophysiology of TMA have occurred. Those advances have allowed us to move from a syndromic diagnosis with a similar treatment to all entities to the search of etiologic diagnosis which would lead to a specific treatment, finally leading to a better outcome of the patient. This document pretends to summarize the current status of knowledge of the pathophysiology of TMA and the therapeutic options available, and to offer a diagnostic and therapeutic practical tool to the professionals caring for the patients. Copyright © 2014 Elsevier España, S.L.U. All rights reserved.Medicina clinica. 11/2014;
VOLUME 25 NUMBER 11 NOVEMBER 2007 NATURE BIOTECHNOLOGY
Discovery and development of the complement
inhibitor eculizumab for the treatment of
paroxysmal nocturnal hemoglobinuria
Russell P Rother1, Scott A Rollins1, Christopher F Mojcik1, Robert A Brodsky2 & Leonard Bell1
The complement system provides critical immunoprotective
and immunoregulatory functions but uncontrolled complement
activation can lead to severe pathology. In the rare hemolytic
disease paroxysmal nocturnal hemoglobinuria (PNH), somatic
mutations result in a deficiency of glycosylphosphatidyl-
inositol-linked surface proteins, including the terminal
complement inhibitor CD59, on hematopoietic stem cells.
In a dysfunctional bone marrow background, these mutated
progenitor blood cells expand and populate the periphery.
Deficiency of CD59 on PNH red blood cells results in chronic
complement-mediated intravascular hemolysis, a process
central to the morbidity and mortality of PNH. A recently
developed, humanized monoclonal antibody directed against
complement component C5, eculizumab (Soliris; Alexion
Pharmaceuticals Inc., Cheshire, CT, USA), blocks the
proinflammatory and cytolytic effects of terminal complement
activation. The recent approval of eculizumab as a first-in-class
complement inhibitor for the treatment of PNH validates the
concept of complement inhibition as an effective therapy and
provides rationale for investigation of other indications in which
complement plays a role.
The complement system: a potential target for therapeutics
The complement system consists of more than 20 serum proteins that
interact in a precise series of enzymatic cleavage and membrane binding
events leading to the generation of products with immunoprotective,
immunoregulatory, proinflammatory and cytolytic properties1,2.
Proximal complement. The cascade proceeds through three distinct
pathways—alternative, classical and lectin—which all result in the gen-
eration of C3 convertase complexes that mediate the cleavage of C3 to
C3a and C3b1,3 (Fig. 1). Recent studies have delineated the structure
of C3 and C3 fragments and provided important insights into their
function4–7. The classical pathway is generally initiated by the interac-
tion of C1q with antigen-antibody complexes. The lectin pathway is
activated by the interaction of mannose-binding lectin (MBL) with
specific mannose-containing carbohydrate structures. The alternative
pathway is initiated by deposition of preformed C3b on a variety of
substrates such as bacteria and cell membranes, including erythro-
cytes. This C3b is continuously available due to the interaction of C3
with water, a process that represents an important part of the innate
immune system called complement ‘tick-over’. In addition to its role in
the initiation of alternative pathway activation, C3b is also necessary for
the amplification and progression of the complement cascade through
all pathways of activation and serves as a key immunoprotective and
The importance of proximal complement components in immunity is
illustrated by the clinical effects observed in people with genetic comple-
ment deficiencies. For example, individuals with C3 deficiency are at
particular risk of infection from polysaccharide-coated bacteria such as
Streptococcus pneumoniae, Haemophilus influenzae and Neisseria menin-
gitidis, and frequently die at a young age from overwhelming sepsis8. The
absence of C3 is generally thought to prevent the progression of comple-
ment through all pathways of activation, thereby reducing the body’s
ability to opsonize pathogens and mount an inflammatory response,
although some studies suggest that C5 cleavage can occur in the absence
of C3 by thrombin3,9. Individuals deficient in C1, C2 or C4 develop severe
autoimmune symptoms such as a lupus-like syndrome and are also sus-
ceptible to recurrent overwhelming sepsis. Thus, deficiencies in early
components of the complement cascade lead to impairment in the ability
to generate C3b, which is critical for immune complex clearance and is
the primary opsonin for many pathogenic microorganisms.
Terminal complement. All pathways of complement activation con-
verge at the cleavage of C5 into C5a and C5b by the C5 convertase
enzyme complexes. This cleavage event initiates the terminal comple-
ment cascade (Fig. 1). C5a is a potent anaphylatoxin that mediates
leukocyte chemotaxis, increases vascular permeability, alters smooth
muscle tone and induces secondary inflammatory mediators such as
hydrolytic enzymes, reactive oxygen species, arachidonic acid metabo-
lites and cytokines10. C5a receptors are present on a variety of cell types
that directly contribute to inflammation, including monocytes, mac-
rophages and neutrophils. The C5a receptor CD88 is a member of the
N-formyl peptide receptor family, which transduces signals through a G
protein–dependent pathway resulting in biochemical proinflammatory
1Alexion Pharmaceuticals, Inc., 352 Knotter Drive, Cheshire, Connecticut
06410, USA. 2Johns Hopkins University School of Medicine, Division of
Hematology, 720 Rutland Avenue, Ross Building Room 1025, Baltimore,
Maryland 21205, USA. Correspondence should be addressed to R.P.R.
Published online 7 November; corrected after print 12 December 2007;
NATURE BIOTECHNOLOGY VOLUME 25 NUMBER 11 NOVEMBER 2007
responses11. A second C5a receptor has been identified (C5L2) that
also facilitates C5a-mediated signaling in a variety of cell types12.
C5b recruits the terminal complement components C6, C7, C8 and
C9 to form C5b-9 or the terminal complement complex (TCC) on
the surface of cells. The generation of the TCC at sublytic concentra-
tions stimulates the release of many of the same proinflammatory
molecules described for C5a. Most importantly, unimpeded assembly
of the TCC on the cell surface results in cell lysis. Deposition of TCC
on erythrocytes results in the destruction of these cells in hemolytic
diseases such as PNH13,14.
The importance of terminal complement components in immu-
nity is demonstrated by individuals with genetic deficiencies of C5,
C6, C7, C8 or C9, who show an increased incidence of infections with
N. meningitidis, a bacterium that can cause meningitis8. Vaccines
against N. meningitidis are recommended for terminal comple-
ment–deficient individuals, although breakthrough infections have
been reported15. Interestingly, N. meningitidis infections in terminal
complement–deficient people are typically less severe than in normal
individuals, possibly due to the lack of TCC-mediated endotoxin
release from the bacteria15.
Targeted blockade of the complement cascade at C5. In the rational
design of a therapeutic complement inhibitor, C5 is an attractive
target1. Because C5 is common to all pathways of complement acti-
vation, blockade at this point stops the progression of the cascade
regardless of the stimuli. In addition, prevention of C5 cleavage effec-
tively blocks the generation of the potent proinflammatory mol-
ecule C5a and the cell lytic TCC. Importantly, C5 blockade preserves
the critical immunoprotective and immunoregulatory functions of
upstream components that culminate in C3b-mediated opsonization
and immune complex clearance.
Development of eculizumab
To generate potent inhibitors of complement
component C5, panels of murine anti-human
C5 monoclonal antibodies were generated
and initially screened for the ability to inhibit
TCC-mediated lysis of antibody-sensitized
chicken erythrocytes by human complement
in a standard hemolytic assay16. The most
potent candidates were cloned and purified,
and functional activity was further character-
ized to identify anti-C5 antibodies that could
also effectively block the generation of C5a. Of
~30,000 hybridomas screened, one monoclo-
nal antibody (mAb), designated murine 5G1.1
(m5G1.1 mAb), was identified that effectively
blocked both TCC-mediated hemolysis and
the generation of C5a, at a 0.5:1 molar ratio
of antibody to C5. Murine 5G1.1 mAb bind-
ing to human C5 mapped to the N-terminal
region of the alpha chain.
Engineering. To reduce the potential for
immunogenicity, we cloned and grafted the
complementarity-determining regions of
m5G1.1 mAb into human heavy and light
chain antibody frameworks (Fig. 2)16,17. The
human heavy-chain framework was derived
from the H20C3H antibody variable region18,
which contained no amino acid changes rela-
tive to the original germline sequence. The
human light-chain framework was derived from the I.23 antibody
variable region19, which contained only one amino acid change from
the original germline sequence; the germline amino acid residue was
restored at this position. The use of purely germline framework accep-
tor sequences should further minimize potential immunogenicity of the
humanized antibody, because the immune system of every individual
should be tolerant to these sequences.
The use of antibodies as therapeutics in some clinical settings is com-
plicated by effector functions of such antibodies that serve to activate
complement and/or to bind to antibody receptors (Fc receptors) on
inflammatory cells after they have engaged their target. To reduce the
potential for eculizumab to elicit proinflammatory responses, we replaced
the heavy-chain constant region of the parental antibody with compo-
nents of both human IgG2 and IgG4 constant regions (Fig. 2)20. The
human IgG2 antibody isotype does not bind Fc receptors21,22, whereas
the human IgG4 isotype does not activate the complement cascade23,24.
The IgG2/IgG4 hybrid constant region of eculizumab includes the CH1
and hinge regions of human IgG2 fused to the CH2 and CH3 regions
of human IgG4, and lacks the ability to bind Fc receptor and to acti-
vate complement20. To avoid the generation of an antigenic site during
the fusion, we used a restriction endonuclease cleavage site common
to both IgG2 and IgG4 to join the two constant regions. Indeed, a total
of 31 amino acids flanking the fusion site are identical between IgG2
Binding characteristics. The humanized monoclonal antibody ecu-
lizumab maintained its ability to block the cleavage of C5 during
complement activation as the antibody potently inhibited both the gen-
eration of C5a and C5b-9–mediated human serum hemolytic activity16.
Eculizumab is highly species-restricted, with minimal activity against
any other primate or mammalian C5. No nonspecific or unexpected
C4 + C2
Immune complex and
M/O and mammalian
Factor B + D
Proximal complementTerminal complement
Figure 1 Targeted blockade of complement protein C5. The complement cascade can be activated
via the classical, lectin and alternative pathways. The proximal components of complement (proteins
upstream of C5) are essential for microbial opsonization and immune complex clearance. All pathways
of complement activation converge at the cleavage of the terminal complement protein C5 leading to
the generation of molecules with proinflammatory and cell lytic properties. Targeted blockade at C5
with eculizumab therefore prevents the deleterious properties of terminal complement activation while
preserving the immunoprotective and immunoregulatory functions of proximal complement. M/O,
microorganisms. TCC, terminal complement complex.
VOLUME 25 NUMBER 11 NOVEMBER 2007 NATURE BIOTECHNOLOGY
binding of eculizumab to any human tissue
examined in a tissue cross-reactivity study
was observed. In addition, the affinity of
eculizumab for human C5 (Kd = 120 pM) is
similar to that of the parental m5G1.1-mAb,
indicating that the binding properties of the
antibody were not compromised during the
The eculizumab pharmacokinetic profile was
analyzed using a one-compartmental model.
When administered by intravenous infusion,
eculizumab has a half-life of 272 ± 82 h, and its
distribution appears to be primarily limited to
the vascular space. Eculizumab serum concen-
trations appear to reach steady state after ~57
days. At steady state, the eculizumab accumu-
lation ratio (Racc) was calculated to be 1.075,
which is indicative of minimal accumulation
over time. Pharmacodynamic activity correlates
directly with eculizumab serum concentrations
and maintenance of trough levels above 35 µg/
ml results in essentially complete blockade of
hemolytic activity in vivo25.
Preclinical development. Because of the spe-
cies-restricted nature of eculizumab, pharma-
cological activities in vivo were explored with a
surrogate anti-mouse C5 antibody (BB5.1) that
demonstrated terminal complement–inhibitory
activity comparable to that of eculizumab. As no adequate mouse models
of PNH exist, in vivo studies targeting other therapeutic indications with
potential involvement of terminal complement activation were used. In
a collagen-induced model of arthritis, BB5.1 was shown to prevent the
onset of disease and to improve the course of previously established dis-
ease26. Treatment with BB5.1 also resulted in an improvement in renal
disease and in dramatic prolongation of survival in a lupus-like auto-
immune model27. These models provided substantial evidence that a
functionally blocking anti-C5 antibody could effectively and consistently
block terminal complement activation leading to a reduction in terminal
complement-mediated proinflammatory sequelae. The surrogate BB5.1
antibody was also used in mouse toxicity studies in which no compound-
related mortalities or significant abnormalities were observed.
Early clinical development. Initial human clinical studies of eculi-
zumab, and a single-chain antibody variant of eculizumab called pex-
elizumab, were performed in individuals with rheumatoid arthritis and
systemic lupus erythematosus28,29 and in individuals with coronary
artery bypass graft surgery and myocardial infarction, respectively30,31.
The initial eculizumab studies served two purposes, to determine a
dosing schedule for future trials and to establish initial safety of termi-
nal complement inhibition in humans. Participants received a single
dose of eculizumab ranging from 0.1 mg/kg to 8 mg/kg; all doses were
observed to be safe and well-tolerated. The 8 mg/kg dose (~600 mg
total) provided complete complement blockade for 7–14 days. Studies
exploring multiple chronic dose regimens with eculizumab were also
performed in individuals with membranous nephritis and rheumatoid
PNH—a disease in search of a complement inhibitor
Etiology of PNH. PNH is a rare form of hemolytic anemia with an esti-
mated incidence of 1.3 cases per million per year and a prevalence of
15.9 cases per million34. The disease is manifest by an acquired genetic
deficiency of endogenous complement inhibitors on the surface of
blood cells; these cells are referred to as the PNH clone(s). PNH clones
are deficient in proteins that are normally linked to the cell surface
by a glycosylphosphatidylinositol (GPI)-anchor. The inability of
cells to attach proteins through this lipid moiety is caused by somatic
mutations occurring in the X-linked gene, phosphatidylinositol gly-
can–complementation class A (PIGA)35–37, a critical molecule in the
In individuals with PNH, cells deficient in GPI-anchored proteins
exist in all blood lineages including erythroid, myeloid and lymphoid
cells. These PNH cells are clonal and harbor matching PIGA mutations
indicating that the mutations occur in multipotent hematopoietic stem
cells38,39. Granulocytes from the blood of healthy controls also pos-
sess PIGA mutations at an exceedingly low frequency (~1 in 50,000),
suggesting that random mutations in this gene are important in the
pathogenesis of PNH40. Thus, it has been hypothesized that the devel-
opment of PNH requires two independent events to occur; hemato-
poietic stem cells must acquire a PIGA mutation and an immune attack
that preferentially targets normal stem cells must be present41,42. This
proposal presumes that the bone marrow insult targets a GPI-anchored
protein on the hematopoietic stem cell, thereby providing a survival
advantage to GPI-anchoring–deficient stem cells (that is, cells possess-
ing a PIGA mutation). It should be noted however that PIGA mutations
characterized in healthy controls appear to occur in more differenti-
ated colony-forming cells rather than multipotent hematopoietic stem
cells39. These colony-forming cells would not have self-renewal capacity
and therefore would not be propagated beyond their terminally dif-
There is clinical evidence to support a survival advantage of PIGA
mutated hematopoietic stem cell clones over normal stem cells in dam-
aged bone marrow. The mAb alemtuzumab (Campath) is used to treat
regions (murine origin)
Human IgG2 heavy chain
constant region 1 and hinge
Human IgG4 heavy chain
constant regions 2 and 3
Figure 2 Engineering of eculizumab to reduce immunogenicity and eliminate effector functions. To
minimize immunogenicity, we grafted murine complementarity-determining regions into human heavy
and light chain germline antibody framework sequences. Additionally, human IgG2 and IgG4 heavy
chain sequences were combined to form a hybrid constant region that is unable to bind Fc receptors
or to activate the complement cascade. Eculizumab exhibits high affinity for human C5, effectively
blocking its cleavage and downstream proinflammatory and cell lytic properties.
NATURE BIOTECHNOLOGY VOLUME 25 NUMBER 11 NOVEMBER 2007
chronic lymphocytic leukemia by targeting and destroying malignant
T- and B-cells that express the GPI-anchored protein CD52. In many
individuals, a PNH T-cell clone expands after alemtuzumab treatment.
In this case alemtuzumab supplies the bone marrow insult and the GPI-
anchoring deficient progenitor T-cells that lack CD52 escape immune
destruction and expand43.
Pathophysiology of PNH. Although PNH blood cells of all lineages are
missing the wide array of GPI-anchored proteins from their surface,
they are remarkably normal with regard to their physiological functions
(e.g., fighting infection, clotting, carrying oxygen). However, the PNH
red blood cell (RBC) is exquisitely sensitive to destruction because of
the inability to regulate autologous terminal complement assembly (Fig.
3). This defect is primarily triggered by the absence of terminal comple-
ment inhibitor function13,14,44,45. CD59 normally blocks formation of
the TCC on the RBC surface, thereby preventing intravascular hemoly-
sis46–48. Hemolysis in PNH is chronic because of a continuous state of
complement activation (tick-over; Fig. 1), but
brisk episodes of hemolysis (paroxysms) coin-
cident with increases in complement activa-
tion triggered by infections, surgery, strenuous
exercise, excessive alcohol intake and blood
transfusions frequently occur49.
Chronic hemolysis is central to the morbidi-
ties in PNH and contributes to mortality in
those with the disease (Fig. 3). It may result
in severe anemia requiring transfusion, and
refractory transfusion-dependent hemolytic
anemia has been considered a major complica-
tion of PNH warranting consideration of bone
marrow transplantation35. Fatigue in PNH
patients is tied directly to hemolysis, strikingly
disproportionate to the degree of anemia, fre-
quently debilitating and similar to the sever-
ity observed in anemic cancer patients25,49–52.
Hemolysis is also directly linked to diminished
health status and functioning25,49. Other dis-
abling morbidities evidenced by individuals
with PNH that are likely a direct result of
hemolysis include dysphagia, abdominal pain,
erectile dysfunction, pulmonary hypertension
and renal failure49,53,54. Thromboembolism,
the most feared complication in PNH, is also
directly associated with hemolysis and may
be induced by the release of free hemoglobin,
consumption of nitric oxide, and subsequent
Thrombosis is clinically evident in ~40% of
individuals with PNH and is the leading cause
of premature mortality64–68. Although venous
thrombosis is more typical in PNH, arterial
thrombosis, particularly in the cerebral cir-
culation, is not uncommon56,64. Thrombosis
is evident in individuals with small and large
PNH clone sizes and varying degrees of hemo-
lysis, although it appears to be more common
in those with larger clones56,57,66,68. Individuals
with hemolysis but without a history of trans-
fusion also experience thrombosis, including
fatal first thrombosis57. Subclinical thrombosis
has been reported in 60% of a cohort of PNH
patients, despite use of anticoagulation, and unrelated to transfusion
Complement inhibitors in PNH. As PNH is defined by the acquired
genetic deficiency of the endogenous terminal complement inhibitor
CD59 on the surface of blood cells, restoration of terminal complement
inhibition should effectively reduce hemolysis in PNH and abrogate
the serious clinical morbidities associated with this life-threatening
disease. Initial attempts to circumvent the functional defect in PNH
cells focused on replacing CD59 on the surface of PNH cells through
gene therapy. A recombinant, transmembrane form of CD59 (CD59-
TM) was generated and analyzed for the ability to regulate comple-
ment activity70. A GPI-anchoring deficient complement-sensitive B-cell
line derived from an individual with PNH was virally transduced with
CD59-TM, resulting in protection against classical complement-medi-
ated membrane damage. These data established that a functional trans-
membrane form of CD59 can be expressed on the surface of PNH cells
Smooth muscle dystonia
Impaired quality of life
Poor physical functioning
Liver, mesenteric, dermal, cerebral
cerebral vascular accident
Figure 3 Terminal complement deposition on the surface of PNH RBCs results in chronic hemolysis
and serious clinical morbidities. (a) Normal RBCs express complement regulatory proteins including the
terminal complement inhibitor CD59. CD59 is tethered to the RBC lipid bilayer of the cell membrane
by a GPI anchor. The presence of CD59 protects these cells from autologous complement-mediated
destruction. (b) PNH RBCs lack GPI-anchored proteins including CD59. Continuous complement
activation leads to formation of the terminal complement complex (TCC) that transverses the lipid
bilayer. These complexes form numerous pores in the RBC membrane, as shown by the electron
micrograph (inset). As pores form in the membrane, water enters the RBC (arrows), resulting from
osmotic pressure. (c) As the concentration of TCCs increases, the PNH RBC swells and ultimately
ruptures (hemolysis). Intravascular hemolysis causes the release of the RBC contents such as
hemoglobin into the plasma. Hemolysis in PNH is chronic and leads to multiple clinical sequelae,
including severe anemia requiring transfusions, disabling fatigue, dyspnea, impaired quality of life,
recurrent pain associated with smooth muscle dystonia, and thrombosis. Artwork by Edmond Alexander,
©2007 Edmond Alexander, printed with permission. Micrograph image is from E.R. Podack. Molecular
mechanisms of cytolysis by complement and cytolytic lymphocytes. J. Cell. Biochem. 30, 133–170
(1986). Copyright © 1986 Wiley-Liss. Reprinted with permission of Wiley-Liss, Inc., a subsidiary of
John Wiley & Sons, Inc.
VOLUME 25 NUMBER 11 NOVEMBER 2007 NATURE BIOTECHNOLOGY
through a gene therapy approach. Retroviral
transduction of PNH erythroid cells with a
functional PIGA gene has also been shown
to restore GPI-anchor protein expression on
the surface of these cells, thereby protecting
them from complement-mediated destruc-
tion71. However, to provide a renewable
source of complement-protected erythro-
cytes in individuals with PNH through a gene
therapy approach, efficient targeting of early
erythroid progenitor cells would be required,
which has not been achieved to date. This led
to the development of the complement inhibi-
tor eculizumab, which binds to and blocks the
cleavage of C5 in plasma, thereby reestablish-
ing terminal complement regulation in the plasma of individuals with
PNH. The potential effect of terminal complement blockade on hemo-
lysis in PNH was observed in a person with coexistent C9 deficiency
and PNH who had no signs of hemolysis72. PNH was diagnosed in this
individual only after the administration of a whole blood transfusion
during a routine surgery, which served to reconstitute C9 and trigger
Eculizumab in PNH: clinical trials
Eculizumab was evaluated in PNH in three separate parent studies—a
phase 2 pilot study and two phase 3 studies. All participants in these trials
were allowed to continue eculizumab in follow-on extension studies.
Pilot study. The first eculizumab PNH study (beginning in May 2002)
evaluated terminal complement inhibition in a 3-month open-label
phase 2 pilot study in 11 participants with hemolytic PNH at two sites
in the United Kingdom73. In this study, participants with at least four
transfusions in the previous 12 months received 600-mg infusions of
eculizumab every week for 4 weeks, followed 1 week later by a 900-mg
dose and then by 900 mg every other week through week 12. In this
study and all subsequent trials, participants were vaccinated against N.
meningitidis at least 2 weeks before the first dose of eculizumab. Clinical
and biochemical indicators of hemolysis were
measured throughout the trial. Mean lactate
dehydrogenase (LDH) levels decreased from
3111 U per liter before treatment to 594 U
per liter during treatment (P = 0.002; Table
1). The mean percentage of PNH erythrocytes
increased from 36.7% of the total erythrocyte
population to 59.2% (P = 0.005). The mean
and median transfusion rates decreased from
2.1 and 1.8 units per individual per month to
0.6 and 0.0 units per individual per month,
respectively (P = 0.003, for the comparison of
the median rates). Episodes of hemoglobin-
uria were reduced by 96% (P < 0.001), and
measurements of the quality of life substan-
tially improved. From this study, it appeared
that eculizumab was safe and well-tolerated in
individuals with PNH. This terminal comple-
ment inhibitor reduced hemolysis, hemoglo-
binuria, and the need for transfusion, with an
associated improvement in quality of life.
After completion of the initial 12-week study,
all 11 participants from the initial PNH study
chose to continue treatment in a 52-week exten-
sion study; all participants completed the study25. The dramatic reduc-
tions in hemolysis and the need for transfusion that were observed in
the pilot study were maintained throughout the extension study, and
significant improvements in quality-of-life measures also continued. In
this study, long-term eculizumab administration continued to be safe and
well-tolerated. Clinical breakthrough occurred in two individuals with
insufficient eculizumab levels; shortening the dosing interval (increasing
the total dose) resulted in rapid restoration of complement suppression,
abrogation of hemolysis, and resolution of disease signs and symptoms.
These data demonstrate the close relationship between sustained terminal
complement inhibition, hemolysis, and symptoms in individuals with
PNH. Further, dramatic clinical improvements in signs and symptoms
attributed specifically to hemolysis including hemoglobinuria, and the
smooth muscle dystonias—abdominal pain, dysphagia and erectile
dysfunction—were observed in these participants54. Ten of the original
11 participants have remained on eculizumab for more than 5 years of
Phase 3 pivotal program. The results from the initial open-label phase
2 pilot study were sufficiently compelling that discussions were initi-
ated with the FDA and the EMEA in order to plan phase 3 studies
for registration in the United States and Europe, respectively. In 2003,
Table 1 Efficacy results of the open-label phase 2 pilot study of eculizumab in
individuals with PNH
Units of packed RBCs transfused/patient/month
Mean ± s.e.m.
LDH (mean ± s.e.m.; U/L)a
2.1 ± 0.83 0.6 ± 0.67
3111 ± 598594 ± 320.002
PNH RBCs (proportions) 36.7 ± 5.959.2 ± 8.00.005
Days with hemoglobinuria/patient/month2.90.12<0.001
aThe normal range for LDH in this study was 150–480 U/L.
0 1020 30 40 50
Lactate dehydrogenase (U/L)
Eculizumab (n = 43)
Placebo (n = 44)
Figure 4 Reduction in hemolysis during treatment with eculizumab. Mean levels of lactate
dehydrogenase reflect the degree of hemolysis from baseline to week 52. The dashed line indicates the
upper limit of the normal range for lactate dehydrogenase (normal range, 103 to 223 U per liter). In
eculizumab-treated patients, the mean level of lactate dehydrogenase was rapidly reduced to just above
the upper limit of the normal range. In the placebo group, the mean level of lactate dehydrogenase
remained highly elevated. The arrow depicts the transition of placebo-treated patients in TRIUMPH to
eculizumab treatment in the phase 3 extension study at which time levels of lactate dehydrogenase
rapidly reduced to near normal values.
NATURE BIOTECHNOLOGY VOLUME 25 NUMBER 11 NOVEMBER 2007
eculizumab received orphan designation for PNH in both the United
States and Europe. The phase 3 eculizumab in PNH program consisted
of: TRIUMPH, the pivotal trial that examined eculizumab safety and
efficacy in a hemolytic PNH population with at least 4 transfusions
in 12 months; SHEPHERD, which examined safety and efficacy of
eculizumab in a broader and more heterogeneous hemolytic PNH
population in individuals with a history of transfusion; and a phase 3b
extension study to evaluate long-term safety and efficacy, including the
potential anti-thrombotic effect of eculizumab.
TRIUMPH was a double-blind, randomized, placebo-controlled, mul-
ticenter, phase 3 trial74. Individuals were first enrolled and treated in 2004
and received either placebo or eculizumab intravenously; eculizumab
was given at a dose of 600 mg weekly for 4 weeks, followed 1 week later
by a 900-mg dose and then 900 mg every 14 ± 2 days through week 26.
Eighty-seven participants (43 eculizumab, 44 placebo) at 34 international
sites were enrolled. The two primary endpoints were the stabilization of
hemoglobin levels and the number of units of packed red cells trans-
fused. Biochemical indicators of hemolysis and the participant’s fatigue
and quality of life were also assessed. After enrollment, participants were
observed for up to 13 weeks. Those who did not require a transfusion dur-
ing the observation period were considered ineligible for randomization.
Eighty-seven individuals underwent randomization. Every participant
treated with eculizumab experienced an objective response. Across all
treated participants, a significant reduction in hemolysis (as measured by
LDH) was observed after 1 week of treatment and maintained through-
out the 26 week study in the eculizumab group but not in placebo (Fig. 4).
There was an 85.8% lower median area under the curve for LDH plotted
against time (in days) in the eculizumab group, as compared with the pla-
cebo group (58,587 versus 411,822 U per liter, respectively; P < 1 × 10−6;
Table 2). After completion of the study, a similar reduction in hemolysis
was demonstrated with placebo participants transitioning to eculizumab.
Stabilization of hemoglobin levels in the absence of transfusions was
achieved in 49% (21 of 43) of the individuals assigned to eculizumab
and none (0 of 44) of those assigned to placebo (P < 1 × 10−6). During
the study, a median of 0 units of packed red cells was administered in the
eculizumab group, as compared with 10 units in the placebo group (P <
1 × 10−6). A total of 51% of the eculizumab group remained transfusion
independent during the entire 26-week study, whereas no one in the
placebo group achieved this status. Even among participants receiving
eculizumab in whom transfusion independence was not achieved, trans-
fusion requirements were reduced by 44% as these individuals also dem-
onstrated a clinically and statistically significant reduction in hemolysis.
The reduced but continued requirement for transfusion support in some
individuals may reflect more severe bone marrow aplasia and/or residual
low-level hemolysis through the extravascular compartment74. Clinically
and statistically significant improvements were
also found in fatigue, as measured by scores on
the Functional Assessment of Chronic Illness
Therapy-Fatigue (FACIT-Fatigue) instrument
(P = 6 × 10−6) and the European Organization
for Research and Treatment of Cancer Quality
of Life Questionnaire (EORTC QLQ-C30; P < 1
× 10−6). Participants that did not achieve com-
plete transfusion independence during eculi-
zumab treatment also experienced significant
improvement in fatigue and other measures of
quality of life. Of the 87 participants, 4 in the
eculizumab group and 9 in the placebo group
had serious adverse events, none of which were
considered to be treatment-related; all these
patients recovered without sequelae. The most
common adverse events reported in the eculizumab group were head-
ache, nasopharyngitis, back pain and nausea. Headache and back pain
occurred more frequently in the eculizumab group than in the placebo
group. Anti-eculizumab antibody responses were of low titer, transient,
and occurred at a frequency similar to that of placebo. The TRIUMPH
study demonstrated that eculizumab is a well-tolerated and effective
therapy for PNH.
After initiation of the TRIUMPH study, eculizumab was evaluated
in SHEPHERD, an open-label, non-placebo controlled, phase 3 clinical
safety and efficacy study in a more diverse PNH population includ-
ing patients with marked thrombocytopenia and minimal transfusion
requirements. Participants were first enrolled and treated in 2005, and
eculizumab was administered for a total of 52 weeks using a dosing
schedule identical to that of TRIUMPH. Ninety-seven individuals at 33
international sites were enrolled. Adverse events were similar to those
with placebo in the TRIUMPH study. Hemolysis was significantly
reduced leading to improved anemia with a reduction in or elimina-
tion of transfusion requirement, lessened fatigue and improved overall
health-related quality of life75. Compared to the overall population,
efficacy with eculizumab was similar in individuals who would be
expected to have less severe disease, including patient subgroups with
lower baseline hemolysis, mild anemia, and minimal or no pretreat-
ment transfusion requirement76.
Of the 195 individuals entering the pilot, TRIUMPH or SHEPHERD
studies, 187 participants completed these studies and elected to receive
eculizumab in a common 104-week phase 3b open-label extension
study. Overall, there was no evidence of an increased incidence of
infection across PNH studies with eculizumab as compared to pla-
cebo, including serious infections, severe infections or multiple infec-
tions. Two individuals vaccinated against N. meningitidis experienced
meningococcal sepsis; they were treated and recovered without clini-
cal sequelae. Serious hemolysis was not observed in any of the initial
16 participants who discontinued treatment. Long-term eculizumab
treatment was associated with a reduction in hemolysis up to more
than 54 months of continuous treatment. Importantly, the design of
the phase 3b extension study specified that the thrombosis rate for each
participant would be compared before and during eculizumab treat-
ment. Early evaluation shows that the 7.37 thrombotic events per 100
patient-years experienced before eculizumab was reduced to 1.07 events
per 100 patient-years with eculizumab treatment (P < 1 × 10−6)77.
Approval of eculizumab. After completion of TRIUMPH and after the
pre-specified interim 26-week analysis in SHEPHERD, applications
for marketing authorization of eculizumab in the treatment of PNH
were submitted to the FDA and EMEA in September 2006. The FDA
Table 2 Primary and secondary endpoints of the phase 3 pivotal efficacy study
<0.000001Stabilization of hemoglobin levels
(percent of patients)a
Mean ± s.e.m.11.0 ± 0.833.0 ± 0.67
Transfusion Avoidance (percent of patients)
FACIT-Fatigue score (change from baseline)– 4.0 ± 1.76.4 ± 1.20.000006
LDH (area under the curve; U/L × day)411,82258,587<0.000001
VOLUME 25 NUMBER 11 NOVEMBER 2007 NATURE BIOTECHNOLOGY
accepted the application for priority review and in March 2007, less
than 6 months after the completion of SHEPHERD, FDA approved
eculizumab for the treatment of patients with PNH to reduce hemo-
lysis. The EMEA granted review of the eculizumab application under
the Accelerated Assessment procedure, the fastest evaluation time
frame for full approval awarded by EMEA. In June 2007, eculizumab
became the first medicinal product to receive approval by the European
Commission within the Accelerated Assessment procedure and it is
indicated for the treatment of patients with PNH.
Conclusions and perspectives
Clinical development of eculizumab spanned a full decade before its
first approval in the rare, disabling and life-threatening, clonal blood
disorder PNH (Fig. 5). The studies before initiation of the PNH clinical
program provided an important assessment of the safety profile and
immunomodulatory effects of eculizumab. Once initiated, the PNH
program has been one of the most extensive pre-approval clinical pro-
grams for an ‘ultra-orphan’ disease; the design of the multinational
trials enrolling 195 participants allowed sequential, controlled and
long-term evaluation of both biochemical and clinically validated mea-
surements in the PNH population. This paradigm of careful evaluation
may be used in the consideration of eculizumab in other uncommon,
debilitating and life-threatening diseases for which there is evidence
suggesting that terminal complement may significantly contribute
to the pathophysiology of the disease, including antibody-mediated
transplant rejection78, myasthenia gravis and other peripheral neu-
ropathies79–83, fulminant lupus27,29, catastrophic anti-phospholipid
syndrome84,85 and pattern 2 multiple sclerosis86. The understanding
of critical pharmacokinetic and pharmacodynamic properties in PNH
patients and the accomplishment of large-scale commercial manufac-
turing of eculizumab may also permit the evaluation of new formula-
tions of eculizumab in future development programs for still other
diseases with evidence suggesting complement-mediated pathology,
including asthma87 and age-related macular degeneration88.
Clinical trials have demonstrated that eculizumab dramatically and
reproducibly abrogates intravascular hemolysis, the primary clinical
manifestation of PNH. These trials have shown that the reduction in
hemolysis is independently associated with improvements in major
morbidities in individuals with PNH including anemia, fatigue and
health-related quality of life, as well as fewer thrombotic events. Because
of the extremely rare nature of the disease and the ethical dilemma
posed by the efficacy evident with eculizumab in the treatment of PNH,
a prospective randomized controlled clinical trial is not a feasible means
to examine the long-term impact of eculizumab on survival in indi-
viduals with PNH. However, given the evident anti-hemolytic effect
and attendant clinical benefit of eculizumab administration, it is pos-
sible that eculizumab could beneficially impact several of the leading
causes of premature mortality in PNH including venous and arterial
thrombosis, hemorrhage that may be worsened by platelet consump-
tion, renal failure and liver failure. In a post hoc analysis of the survival
of eculizumab-treated PNH patients compared to historical controls64,
~15 deaths would have been expected during the 2-year period of ecu-
lizumab treatment; however, two deaths were observed during this
period, or 87% fewer than expected (P = 0.0005). This preliminary
analysis suggests that long-term eculizumab treatment may beneficially
affect survival in individuals diagnosed with PNH.
The humanization strategy used to construct eculizumab has resulted
in only very rare evidence of responses to eculizumab, with an incidence
similar to that found with placebo treatment. There is no evidence of
tolerance or a neutralizing antibody response in any treated individual
to date and infusion-related immune effects are similar in frequency
to placebo. It is interesting to speculate that the selection of purely
germline framework acceptor regions, seen as native by all recipients,
has allowed for this important safety benefit.
Rigorous evaluation of eculizumab in PNH has helped tease out
the role of hemolysis in mediating critical patient-reported outcomes,
including fatigue and patient functioning. The reproducible demonstra-
tion that patient improvement with eculizumab, due to the abrogation
shown to be
PNH shown to
be caused by
mutations in the
form of CD59
shown to protect
PNH cells from
Phase 1 safety
First PNH patient
dosed in an open-
label phase 2 pilot
study in the UK
Phase 2 pilot
study published in
in the US for the
patients with PNH
Case study of a
generation of the
and cell lytic
FDA and EMEA
status in PNH
BLA and MAA
submitted to FDA
Figure 5 Eculizumab in PNH development timeline. BLA, biologic license application; MAA, marketing authorization application.
NATURE BIOTECHNOLOGY VOLUME 25 NUMBER 11 NOVEMBER 2007
of the underlying hemolysis, is distinct from associated improvements
in anemia may have applications to understanding the potential
multifactorial etiology of fatigue in individuals with anemia in other
disease settings, such as cancer. Similarly, evident improvement in
smooth muscle dystonias and thrombosis with eculizumab treatment
in PNH has helped to establish new and important clinical paradigms,
particularly with regard to the role of cell-free plasma hemoglobin and
nitric oxide scavenging in thrombosis, smooth muscle dystonia and
vasomotor abnormalities53. In this case, focus on a rare disease may
not only lead to benefit for afflicted individuals but may also contribute
to the overall foundation of knowledge. Such knowledge may provide
insight into more common diseases with disordered nitric oxide regula-
tion, including systemic and pulmonary hypertension, diabetes, sickle
cell disease, and other genetic or acquired hemolytic anemias.
In summary, data from six clinical studies, enrolling a total of 195
participants, demonstrate the robust efficacy of eculizumab for the
treatment of individuals with PNH. Every individual treated with ecu-
lizumab to date has shown an objective improvement in hemolysis.
Treatment with eculizumab modifies the clinical course of PNH and
markedly reduces the serious morbidities associated with the disease. In
particular, therapy with eculizumab reduces anemia, blood transfusion
requirements, and the risk for life-threatening thrombosis. Eculizumab
also improves other morbidities that are often serious, including fatigue,
pain, dyspnea and impaired overall quality of life. Treated individuals
will continue to be monitored in a global registry. Overall, the therapeu-
tic benefit and safety of eculizumab in the treatment of patients with
the rare blood disease PNH validates the utility of terminal complement
inhibitor therapeutics for the treatment of medical conditions where
complement activation is involved.
The authors wish to acknowledge the Alexion research team for their involvement
in the identification and characterization of eculizumab, the Alexion clinical and
drug development teams for the eculizumab clinical program, the clinicians and
patients for their participation in the trials, and Kerry Quinn-Senger and Rebecca
Baker for assistance with the preparation of this manuscript.
COMPETING INTERESTS STATEMENT
The authors declare competing financial interests: details accompany the full-text
HTML version of the paper at http://www.nature.com/naturebiotechnology/.
Published online at http://www.nature.com/naturebiotechnology/
Reprints and permissions information is available online at http://npg.nature.com/
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VOLUME 25 NUMBER 12 DECEMBER 2007 NATURE BIOTECHNOLOGY
Erratum: Funding crunch forces stem cell company to abandon therapies
Nat. Biotechnol. 25, 951–952, 2007; published online 3 September 2007
In the version of this article originally published, in the penultimate paragraph, the name of the Monash University scientist was misspelled; the
correct spelling is Colin Pouton. In addition, the statement in paragraph 2 “the SSCC, which is being supported by the same group that funded
ESI, the Agency for Science, Technology and Research (A*STAR)” is incorrect. ESI is not funded by A*STAR. Its major investor is Bio-One Capital,
a venture capital fund controlled through the Singapore Government’s Economic Development Board. The SSCC is funded by A*STAR.
Erratum: Mice with a human touch
Christopher Thomas Scott
Nat. Biotechnol. 25, 1075–1077, 2007; published online 6 October 2007
In the version of this article initially published, it was incorrectly stated on p. 1076, paragraph 2, line four from bottom, that “Aya Jakobovits,
originally at Cell Genesys and later at Abgenix, shepherded the XenoMouse through its entire development.” Five of the six XenoMouse strains
developed at Abgenix were initiated after Jakobovits left in 1997.
Erratum: Discovery and development of the complement inhibitor
eculizumab for the treatment of paroxysmal nocturnal hemoglobinuria
Russell P Rother, Scott A Rollins, Christopher F Mojcik, Robert A Brodsky & Leonard Bell
Nat. Biotechnol. 25, 1256–1264 (2007); published online 7 November 2007; corrected after print 7 December 2007
In the version of this article initially published, on p. 1258, paragraph 2, the incorrect serum concentrations to reach steady state and the incorrect
accumulation ratio (Racc) for eculizumab were given. The corrected sentence reads “Eculizumab serum concentrations appear to reach steady state
after ~57 days. At steady state, the eculizumab accumulation ratio (Racc) was calculated to be 1.075.” In addition, in Figure 5, in the PDF version only,
the word “assessment” was cut off the bottom of the box beginning “September 2006.” The final sentence should read “BLA and MAA submitted
to FDA and EMEA, respectively; receive accelerated assessment.” And in Table 1, the heading “Units of packed RBCs transfused/patient/month”
now appears in a separate row. The errors have been corrected in the HTML and PDF versions of the article.
Corrigendum: A diverse family of thermostable cytochrome P450s created
by recombination of stabilizing fragments
Yougen Li, D Allan Drummond, Andrew M Sawayama, Christopher D Snow, Jesse D Bloom & Frances H Arnold
Nat. Biotechnol. 25, 1051–1056 (2007); published online 26 August 2007; corrected after print 7 December 2007
In the version of this article initially published, the sentence starting with “The most thermostable 450 P450 chimera” contains an extra “450.” The
correct sentence should start as follows “The most thermostable P450 chimera”. The error has been corrected in the HTML and PDF versions of
ERRATA AND CORRIGENDA
© 2007 Nature Publishing Group http://www.nature.com/naturebiotechnology