U.S. patent application number 13/426973 was filed with the patent office on 2012-09-20 for treatment of paroxysmal nocturnal hemoglobinuria patients by an inhibitor of complement.
This patent application is currently assigned to Alexion Pharmaceuticals, Inc.. Invention is credited to Leonard Bell, Russell P. Rother.
Application Number | 20120237515 13/426973 |
Document ID | / |
Family ID | 38222520 |
Filed Date | 2012-09-20 |
United States Patent
Application |
20120237515 |
Kind Code |
A1 |
Bell; Leonard ; et
al. |
September 20, 2012 |
TREATMENT OF PAROXYSMAL NOCTURNAL HEMOGLOBINURIA PATIENTS BY AN
INHIBITOR OF COMPLEMENT
Abstract
Eculizumab, a humanized monoclonal antibody against C5 that
inhibits terminal complement activation, showed activity in a
preliminary 12-week open-label trial in a small cohort of patients
with paroxysmal nocturnal hemoglobinuria (PNH). The present study
examined whether chronic eculizumab therapy could reduce
intravascular hemolysis, stabilize hemoglobin levels, reduce
transfusion requirements, and improve quality of life in a
double-blind, randomized, placebo-controlled, multi-center global
Phase III trial. It has been found that eculizumab stabilized
hemoglobin levels, decreased the need for transfusions, and
improved quality of life in PNH patients via reduced intravascular
hemolysis. Chronic eculizumab treatment appears to be a safe and
effective therapy for PNH.
Inventors: |
Bell; Leonard; (Woodbridge,
CT) ; Rother; Russell P.; (Oklahoma City,
OK) |
Assignee: |
Alexion Pharmaceuticals,
Inc.
Cheshire
CT
|
Family ID: |
38222520 |
Appl. No.: |
13/426973 |
Filed: |
March 22, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12225040 |
May 13, 2009 |
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PCT/US2007/006606 |
Mar 15, 2007 |
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13426973 |
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60783070 |
Mar 15, 2006 |
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Current U.S.
Class: |
424/135.1 ;
424/133.1; 424/158.1 |
Current CPC
Class: |
A61P 7/00 20180101; A61P
21/00 20180101; A61P 29/02 20180101; C07K 16/18 20130101; C07K
2317/565 20130101; A61K 9/0019 20130101; A61K 38/00 20130101; A61P
1/14 20180101; A61P 43/00 20180101; A61P 3/02 20180101; C07K
2317/51 20130101; A61P 3/00 20180101; A61K 2039/505 20130101; A61P
9/12 20180101; A61P 25/18 20180101; A61P 13/02 20180101; A61P 17/00
20180101; A61P 37/04 20180101; A61P 25/28 20180101; A61P 15/10
20180101; A61P 25/04 20180101; A61P 7/06 20180101; A61P 19/00
20180101; A61P 25/20 20180101; A61P 31/04 20180101; C07K 2317/24
20130101; C07K 2317/515 20130101; A61P 25/00 20180101; A61K 31/7105
20130101; A61P 25/26 20180101; A61P 7/02 20180101 |
Class at
Publication: |
424/135.1 ;
424/158.1; 424/133.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61P 7/06 20060101 A61P007/06; A61P 7/00 20060101
A61P007/00; A61P 37/04 20060101 A61P037/04; A61P 31/04 20060101
A61P031/04 |
Claims
1-90. (canceled)
91. A method for treating a patient that: (i) is afflicted with a
hemolytic disease and (ii) has been vaccinated against Neisseria
meningitides, the method comprising administering to the patient an
effective amount of an anti-C5 antibody that inhibits cleavage of
complement component C5 into fragments C5a and C5b.
92. The method according to claim 91, wherein the anti-C5 antibody
is a whole antibody or a C5-binding fragment thereof.
93. The method according to claim 91, wherein the anti-C5 antibody
is eculizumab.
94. The method according to claim 92, wherein the C5-binding
fragment is a Fab, a F(ab)', a F(ab').sub.2, an scFv, or a
diabody.
95. The method according to claim 91, wherein the anti-C5 antibody
is pexelizumab.
96. The method according to claim 91, wherein the antibody
comprises an altered constant region that exhibits decreased
effector function relative to the corresponding native constant
region.
97. The method according to claim 96, wherein the effector function
is selected from the group consisting of antibody-dependent
cell-mediated cytotoxicity (ADCC) and complement-dependent
cytotoxicity (CDC).
98. The method according to claim 91, wherein the anti-C5 antibody
is administered to the patient for at least 6 months.
99. The method according to claim 91, wherein the hemolytic disease
is paroxysmal nocturnal hemoglobinuria (PNH).
100. The method according to claim 99, wherein the patient has a
hemoglobin level less than (i) 14 g/dL if a man or (ii) 12 g/dL if
a woman.
101. The method according to claim 99, wherein the patient has a
hemoglobin level less than (i) 13 g/dL if a man or (ii) 11 g/dL if
a woman.
102. The method according to claim 99, wherein the patient has a
hemoglobin level less than (i) 12 g/dL if a man or (ii) 10 g/dL if
a woman.
103. The method according to claim 99, wherein the patient suffers
from hemolysis due to a C3b-mediated, extravascular clearance of
PNH erythrocytes through the reticuloendothelial system.
104. The method according to claim 99, wherein the patient is
anemic and the anemia results at least in part from extravascular
hemolysis, and wherein the patient remains anemic following
treatment with the anti-C5 antibody.
105. The method according to claim 99, wherein the patient is at
least 18 years of age and has: (i) a PNH type III erythrocyte
population of .gtoreq.10% and (ii) received at least four
transfusions during the 12 months immediately preceding the first
treatment with the anti-C5 antibody.
106. The method according to claim 91, wherein the patient has
aplastic anemia or myelodysplastic syndrome.
107. The method according to claim 92, wherein the method comprises
administering to the patient a pharmaceutical composition
comprising a single unit dosage form of the whole anti-C5 antibody
or C5-binding fragment thereof, and wherein the single unit dosage
form comprises 300 mg of the whole anti-C5 antibody or C5-binding
fragment thereof.
108. The method according to claim 107, wherein the single unit
dosage form has a volume of 30 mL.
109. The method according to claim 107, wherein the single unit
dosage form comprises a 10 mg/mL solution of the anti-C5 antibody
or C5-binding fragment thereof.
110. The method according to claim 107, wherein the single unit
dosage form is a preservative free formulation.
111. The method according to claim 91, wherein the anti-C5 antibody
is to be administered to the patient under the following treatment
schedule: (i) 600 mg of the antibody every 7.+-.2 days for the
first 4 weeks, (ii) 900 mg of the antibody for the fifth dose
7.+-.2 days after (i), and (iii) 900 mg of the antibody every
14.+-.2 days thereafter, and wherein administration of the antibody
to the patient occurs via 25 to 45 minute intravenous infusion.
112. A method for treating a patient afflicted with a hemolytic
disease, the method comprising: vaccinating the patient against
Neisseria meningitides; and administering to the patient an
effective amount of an anti-C5 antibody that inhibits cleavage of
complement component C5 into fragments C5a and C5b.
113. A method for treating a patient afflicted with a hemolytic
disease, the method comprising administering to the patient an
effective amount of an anti-C5 antibody that: (i) inhibits cleavage
of complement component C5 into fragments C5a and C5b and (ii)
comprises an altered constant region that exhibits decreased
effector function relative to the corresponding native constant
region.
114. The method according to claim 113, wherein the effector
function is selected from the group consisting of
antibody-dependent cell-mediated cytotoxicity (ADCC) and
complement-dependent cytotoxicity (CDC).
115. A method for treating a patient afflicted with a hemolytic
disease, the method comprising administering to the patient an
anti-C5 antibody under the following treatment schedule: (i) 600 mg
of the antibody every 7.+-.2 days for the first 4 weeks, (ii) 900
mg of the antibody for the fifth dose 7.+-.2 days after (i), and
(iii) 900 mg of the antibody every 14.+-.2 days thereafter, wherein
administration of the antibody to the patient is to occur via 25 to
45 minute intravenous infusion, and wherein the antibody inhibits
cleavage of complement component C5 into fragments C5a and C5b.
Description
BACKGROUND
[0001] Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired
hematologic disease that results from clonal expansion of
hematopoietic stem cells with somatic mutations in the X-linked
gene called PIG-A..sup.1,2 Mutations in PIG-A lead to an early
block in the synthesis of glycosylphosphatidylinositol
(GPI)-anchors, which are required to tether many proteins to the
cell surface. Consequently, PNH blood cells have a partial (type
II) or complete (type III) deficiency of GPI-anchored proteins.
[0002] Intravascular hemolysis is a prominent feature of PNH and a
direct result of the absence of the GPI-anchored complement
regulatory protein CD59..sup.3,4 Under normal circumstances, CD59
blocks the formation of the terminal complement complex (also
called the membrane attack complex) on the cell surface, thereby
preventing erythrocyte lysis and platelet activation..sup.5-8
Excessive or persistent intravascular hemolysis in PNH patients not
only results in anemia (normal ranges of hemoglobin are 14-18 g/dL
for men and 12-16 g/dL for women, and persons with lower levels are
considered to be anemic), but also hemoglobinuria and clinical
sequelae related to the release of the erythrocyte contents into
the circulation: fatigue, thrombosis, abdominal pain, dysphagia,
erectile dysfunction, and pulmonary hypertension..sup.9,10,21,22
Indeed, impaired quality of life in PNH is disproportionate to the
degree of anemia. Many PNH patients depend on blood transfusions to
maintain adequate erythrocyte hemoglobin levels. There have been no
therapies that effectively reduce intravascular hemolysis and
improve the associated clinical morbidities in PNH.
[0003] Eculizumab is a humanized monoclonal antibody directed
against the terminal complement protein C5:.sup.11 In a
preliminary, 12-week, open-label clinical study in 11 PNH patients,
eculizumab was shown to reduce intravascular hemolysis and
transfusion requirements..sup.12 However, this unblinded study
involved a small number of patients with no control arm and without
protocol-driven transfusion standards.
SUMMARY
[0004] The present pivotal, phase III study, Transfusion Reduction
Efficacy and Safety Clinical Investigation, Randomized,
Multi-Center, Double-Blind, Placebo-Controlled, Using Eculizumab in
Paroxysmal Nocturnal Hemoglobinuria (TRIUMPH), evaluated the effect
of eculizumab on the stabilization of hemoglobin levels and
transfusion requirements during 6 months of treatment in a cohort
of 87 transfusion-dependent PNH patients. Measures of intravascular
hemolysis and quality of life were also assessed. This is the first
placebo controlled study of a PNH patient population to control
hemolysis and to differentiate between the effects due to hemolysis
and the effects due to anemia.
[0005] It has been surprisingly discovered that certain aspects of
quality of life were unexpectedly improved by the treatment of PNH
patients with eculizumab. Furthermore, these improvements in the
quality of life were independent of transfusion. The improved
aspects include, e.g., global health status, physical functioning,
emotional functioning, cognitive functioning, role functioning,
social functioning, fatigue, pain, dyspnea, appetite loss and
insomnia. Improvement was also seen in nausea and vomiting,
diarrhea, constipation, and financial difficulties but did not
reach the level of statistical significance. Because the treated
patients remained anemic throughout their treatment, it was
unexpected that all of these improvements would have been seen
because they were previously thought to be a result of the patient
being anemic. Although not wishing to be bound by any theory, it
appears that some of the symptoms are likely due, at least in part,
to hemolysis and release of hemoglobin into the bloodstream and do
not result solely from the patient being anemic. The treatment with
eculizumab decreases the amount of lysis thereby limiting
hemoglobin release into the bloodstream, thereby apparently
resulting in the improvements seen in the treated patients' quality
of life. The results presented herein indicate that any treatment
that decreases hemolysis in a patient will result in an improvement
in the quality of life of said patient.
[0006] In certain aspects, the application provides a method to
improve at least one aspect of the quality of life of a patient
suffering from paroxysmal nocturnal hemoglobinuria, said method
comprising administering to said patient in need thereof a compound
which inhibits complement or inhibits formation of C5b-9.
[0007] In certain aspects, the application provides a method to
improve at least one aspect of the quality of life of a patient
suffering from paroxysmal nocturnal hemoglobinuria, said method
comprising administering to said patient in need thereof a compound
which inhibits intravascular hemolysis. In certain embodiments,
said method results in a greater than 30% reduction in LDH in said
patient.
[0008] In certain aspects, the application provides a method to
improve at least one aspect of the quality of life of an anemic
patient whose anemia results at least in part from hemolysis, said
method comprising administering to said patient in need thereof a
compound which inhibits intravascular hemolysis, wherein said
patient remains anemic. In certain embodiments, said method results
in a greater than 30% reduction in LDH in said patient.
[0009] In certain aspects, the application provides a method of
prolonging the health-adjusted life expectancy of a patient
comprising administering to said patient in need thereof a compound
which inhibits formation of C5b-9. In certain embodiments, said
patient is anemic. In certain embodiments, said patient remains
anemic following treatment. In certain embodiments, said patient
has a hemoglobin level less than i) 14 g/dL if a man or ii) 12 g/dL
if a woman. In certain embodiments, said patient has a hemoglobin
level less than i) 13 g/dL if a man or ii) 11 g/dL if a woman. In
certain embodiments, said patient has a hemoglobin level less than
i) 12 g/dL if a man or ii) 10 g/dL if a woman. In certain
embodiments, said patient suffers from paroxysmal nocturnal
hemoglobinuria.
[0010] In certain aspects, the application provides a
pharmaceutical composition comprising an antibody that binds C5 or
an active antibody fragment thereof. In certain embodiments, the
antibody that binds C5 or an active antibody fragment thereof is
eculizumab. In certain embodiments, the antibody that binds C5 or
an active antibody fragment thereof is pexelizumab. In certain
embodiments, the pharmaceutical formulations of the application may
be administered to a subject, particularly a subject having
PNH.
[0011] In certain aspects, the application provides a method of
treating a patient suffering from paroxysmal nocturnal
hemoglobinuria by administering a pharmaceutical composition
comprising an antibody that binds C5 or an active antibody fragment
thereof. In certain embodiments, the antibody that binds C5 or an
active antibody fragment thereof is eculizumab. In certain
embodiments, the antibody that binds C5 or an active antibody
fragment thereof is pexelizumab. In certain embodiments, the
pharmaceutical formulations of the application may be administered
to a subject, particularly a subject having PNH.
[0012] In certain aspects, the application provides kits comprising
a pharmaceutical composition of the application. In some
embodiments, the kit further comprises at least one component of a
closed sterile system. Components of the closed sterile system
include, but are not limited to, needles, syringes, catheter based
syringes, needle based injection devices, needle-less injection
devices, filters, tubing, valves and cannulas. In a related
embodiment, the kit comprise components for the removal of a
preservative from the composition. Such components include filters,
syringes, vials, containers, tubing, etc.
[0013] In certain embodiments, said quality of life is measured by
a FACIT-Fatigue score. In certain embodiments, the FACIT-Fatigue
score increases by at least 3 points. In certain embodiments, the
FACIT-Fatigue score increases by .gtoreq.4 points.
[0014] In certain embodiments, said quality of life is measured by
an EORTC QLQ-C30 score. In certain embodiments, said EORTC QLQ-C30
score improves by .gtoreq.10% of the pretreatment score. In certain
embodiments, said aspect of the quality of life as measured by an
EORTC QLQ-C30 score is selected from the group consisting of a)
global health status, b) physical functioning, c) emotional
functioning, d) cognitive functioning, e) role functioning, f)
social functioning, g) fatigue, h) pain, i) dyspnea, j) appetite
loss, and k) insomnia. In certain embodiments, said aspect of
quality of life is fatigue.
[0015] In certain embodiments, said compound is selected from the
group consisting of CR1, LEX-CR1, MCP, DAF, CD59, Factor H, cobra
venom factor, FUT-175, complestatin, and K76 COOH. In certain
embodiments, said compound is a steroid that suppresses
complement.
[0016] In certain embodiments, said compound is selected from the
group consisting of antibodies, active antibody fragments, soluble
complement inhibitory compounds, proteins, soluble complement
inhibitors with a lipid tail, protein fragments, peptides, small
organic compounds, RNA aptamers, L-RNA aptamers, spiegelmers,
antisense compounds, serine protease inhibitors, double stranded
RNA, small interfering RNA, locked nucleic acid inhibitors, and
peptide nucleic acid inhibitors. In certain embodiments, said
compound is an antibody or an active antibody fragment. In certain
embodiments, said antibody or active antibody fragment is selected
from the group consisting of a) polyclonal antibodies, b)
monoclonal antibodies, c) single chain antibodies, d) chimeric
antibodies, e) humanized antibodies, f) Fabs, g) F(ab')s, h)
F(ab').sub.2s, i) Fvs, j) diabodies, and k) human antibodies.
[0017] In certain embodiments, said antibody or an active antibody
fragment thereof binds C5. In certain embodiments, said antibody or
active antibody fragment blocks C5 cleavage. In certain
embodiments, said antibody or active antibody fragment inhibits the
formation of C5b-9. In certain embodiments, said antibody is
eculizumab. In certain embodiments, said antibody or active
antibody fragment is administered for at least 6 months. In certain
embodiments, said patient has aplastic anemia or myelodysplastic
syndrome.
[0018] In certain embodiments, said antibody that binds C5 or an
active antibody fragment thereof is administered in a single unit
dosage form. In certain embodiments, the single unit dosage form is
a 300 mg unit dosage form. In certain embodiments, the single unit
dosage form is lyophilized. In certain embodiments, the single unit
dosage form is a sterile solution. In certain embodiments, the
single unit dosage form is a preservative free formulation. In
certain embodiments, the 300 mg single-use dosage form comprises 30
ml of a 10 mg/ml sterile, preservative free solution.
[0019] In certain embodiments, the antibody that binds C5 or an
active antibody fragment thereof comprises an altered constant
region, wherein said antibody or antigen-binding fragment exhibits
decreased effector function relative to an anti-CDCP1 antibody with
a native constant region. In certain embodiments, decreased
effector function comprises one or more properties of the following
group: a) decreased antibody-dependent cell-mediated cytotoxicity
(ADCC), and b) decreased complement dependent cytotoxicity (CDC),
compared to an anti-CDCP1 antibody with a native constant region.
In certain embodiments, the altered constant region comprises a
G2/G4 construct in place of the G1 domain.
[0020] In certain embodiments, the antibody that binds C5 or an
active antibody fragment thereof comprises a heavy chain variable
region and a light chain variable region, wherein the heavy chain
variable region comprises one or more CDR regions having an amino
acid sequence selected from the group consisting of SEQ ID NO:5,
SEQ ID NO:6, or SEQ ID NO:7, and wherein the light chain variable
region comprises one or more CDR regions having an amino acid
sequence selected from the group consisting of SEQ ID NO:8, SEQ ID
NO:9, or SEQ ID NO:10. In certain embodiments, the antibody that
binds C5 or an active antibody fragment thereof comprises a heavy
chain variable region and a light chain variable region, wherein
the heavy chain variable region consists of SEQ ID NO: 1 and the
light chain variable region consists of SEQ ID NO: 3. In certain
embodiments, the pharmaceutical composition comprises eculizumab.
In certain embodiments, the pharmaceutical composition comprises
pexelizumab. In certain embodiments, the antibody that binds C5 or
an active antibody fragment thereof comprises a heavy chain and a
light chain, wherein the heavy chain consists of SEQ ID NO: 2 and
the light chain consists of SEQ ID NO: 4.
[0021] In certain embodiments, said patient is anemic. In certain
embodiments, said patient remains anemic following treatment. In
certain embodiments, said patient has a hemoglobin level less than
1) 14 g/dL if a man or ii) 12 g/dL if a woman. In certain
embodiments, said patient has a hemoglobin level less than 1) 13
g/dL if a man or ii) 11 g/dL if a woman. In certain embodiments,
said patient has a hemoglobin level less than i) 12 g/dL if a man
or ii) 10 g/dL if a woman.
[0022] In certain embodiments, said health-adjusted life expectancy
is measured according to a unit selected from the group consisting
of Years of potential life lost, Disability-free life expectancy,
Health-adjusted life year, Quality adjusted life year, Healthy
years equivalents, Healthy days gained, Episode-free day, Q-TWiST,
Health Utilities Index, or Years of healthy life.
[0023] In certain embodiments, the health-adjusted life expectancy
in a subject is prolonged by at least one day. In certain
embodiments, the health-adjusted life expectancy in a subject is
prolonged by at least week. In certain embodiments, the
health-adjusted life expectancy in a subject is prolonged by at
least one month. In certain embodiments, the health-adjusted life
expectancy in a subject is prolonged by at least one year.
[0024] In certain embodiments, the pharmaceutical composition is in
a single unit dosage form. In certain embodiments, the single unit
dosage form is a 300 mg unit dosage form. In certain embodiments,
the pharmaceutical composition is lyophilized. In certain
embodiments, the pharmaceutical composition is a sterile solution.
In certain embodiments, the pharmaceutical composition is a
preservative free formulation. In certain embodiments, the
pharmaceutical composition comprises a 300 mg single-use
formulation of 30 ml of a 10 mg/ml sterile, preservative free
solution. In certain embodiments, the pharmaceutical composition
comprises an antibody that binds C5 or an active antibody fragment
thereof.
BRIEF DESCRIPTION OF THE FIGURES
[0025] FIGS. 1A-B show that eculizumab treatment decreases
intravascular hemolysis and increases PNH type III erythrocytes.
FIG. 1A shows the degree of intravascular hemolysis in PNH
patients, demonstrated by mean lactate dehydrogenase (LDH) levels.
FIG. 1B shows the mean proportion of PNH type III erythrocytes
assessed for placebo- and eculizumab-treated patients.
[0026] FIG. 2 shows the effect of eculizumab treatment on
transfusion requirements in PNH patients. This is a Kaplan-Meier
plot of time to first transfusion for eculizumab- and
placebo-treated patients from baseline through week 26. The P value
is from the log rank analysis.
[0027] FIG. 3 shows the effect of eculizumab on fatigue assessed by
the FACIT-Fatigue Instrument. Quality of Life scores were assessed
using the Functional Assessment of Chronic Illness Therapy-Fatigue
(FACIT-Fatigue) instrument. Values for change from baseline to 26
weeks represent least-square means. A positive change indicates an
improvement and a negative change indicates deterioration in the
FACIT-Fatigue measures of quality of life.
DETAILED DESCRIPTION
I. Definitions
[0028] The term "derived from" means "obtained from" or "produced
by" or "descending from".
[0029] The term "genetically altered antibodies" means antibodies
wherein the amino acid sequence has been varied from that of a
native antibody. Because of the relevance of recombinant DNA
techniques to this application, one need not be confined to the
sequences of amino acids found in natural antibodies; antibodies
can be redesigned to obtain desired characteristics. The possible
variations are many and range from the changing of just one or a
few amino acids to the complete redesign of, for example, the
variable or constant region. Changes in the constant region will,
in general, be made in order to improve or alter characteristics,
such as complement fixation, interaction with membranes and other
effector functions. Changes in the variable region will be made in
order to improve the antigen binding characteristics.
[0030] The term "an antigen-binding fragment of an antibody" refers
to any portion of an antibody that retains the binding utility to
the antigen. An exemplary antigen-binding fragment of an antibody
is the heavy chain and/or light chain CDR, or the heavy and/or
light chain variable region.
[0031] The term "homologous," in the context of two nucleic acids
or polypeptides refers to two or more sequences or subsequences
that have at least about 85%, at least 90%, at least 95%, or higher
nucleotide or amino acid residue identity, when compared and
aligned for maximum correspondence, as measured using the following
sequence comparison method and/or by visual inspection. In certain
embodiments, the "homolog" exists over a region of the sequences
that is about 50 residues in length, at least about 100 residues,
at least about 150 residues, or over the full length of the two
sequences to be compared.
[0032] Methods of determining percent identity are known in the
art. "Percent (%) sequence identity" with respect to a specified
subject sequence, or a specified portion thereof, may be defined as
the percentage of nucleotides or amino acids in the candidate
derivative sequence identical with the nucleotides or amino acids
in the subject sequence (or specified portion thereof), after
aligning the sequences and introducing gaps, if necessary to
achieve the maximum percent sequence identity, as generated by the
program WU-BLAST-2.0a19 (Altschul et al., J. Mol. Biol. 215:403-410
(1997); http://blast.wustl.edu/blast/READMahtm-1) with search
parameters set to default values. The HSP S and HSP S2 parameters
are dynamic values and are established by the program itself
depending upon the composition of the particular sequence and
composition of the particular database against which the sequence
of interest is being searched. A "% identity value" is determined
by the number of matching identical nucleotides or amino acids
divided by the sequence length for which the percent identity is
being reported.
II. Overview
[0033] The present disclosure relates to a method of treating
paroxysmal nocturnal hemoglobinuria ("PNH"), more specifically to
improving certain aspects of quality of life which are impaired in
PNH patients, and other hemolytic diseases in mammals.
Specifically, the methods of treating hemolytic diseases, which are
described herein, involve using compounds which bind to or
otherwise block the generation and/or activity of one or more
complement components. The present methods have been found to
provide surprising results. For instance, hemolysis rapidly ceases
upon administration of the compound which binds to or otherwise
blocks the generation and/or activity of one or more complement
components, with hemoglobinuria being significantly reduced after
treatment. Also, hemolytic patients can be rendered less dependent
on transfusions or transfusion-independent for extended periods
(twelve months or more), well beyond the 120 day life cycle of red
blood cells. In addition, type III red blood cell count can be
increased dramatically in the midst of other mechanisms of red
blood cell lysis (non-complement mediated and/or earlier complement
component mediated e.g., Cb3). Another example of a surprising
result is that symptoms resolved, indicating that NO serum levels
were increased enough even in the presence of other mechanisms of
red blood cell lysis. These and other results reported herein are
unexpected and could not be predicted from prior treatments of
hemolytic diseases.
III The Complement System
[0034] The complement system, useful complement inhibitors, and use
of these inhibitors to treat PNH and other patients are more fully
described in PCT Patent Application PCT/US2005/003225 filed Feb. 3,
2005 and published as International Publication Number WO
2005/074607 A2 on Aug. 18, 2005, the contents of which are
incorporated herein by reference in their entirety.
[0035] The complement system acts in conjunction with other
immunological systems of the body to defend against intrusion of
cellular and viral pathogens. There are at least 25 complement
proteins, which are found as a complex collection of plasma
proteins and membrane cofactors. The plasma proteins make up about
10% of the globulins in vertebrate serum. Complement components
achieve their immune defensive functions by interacting in a series
of intricate but precise enzymatic cleavage and membrane binding
events. The resulting complement cascade leads to the production of
products with opsonic, immunoregulatory, and lytic functions.
[0036] The complement cascade progresses via the classical pathway
or the alternative pathway. These pathways share many components
and, while they differ in their initial steps, they converge and
share the same "terminal complement" components (C5 through C9)
responsible for the activation and destruction of target cells.
[0037] The classical complement pathway is typically initiated by
antibody recognition of and binding to an antigenic site on a
target cell. The alternative pathway is usually antibody
independent and can be initiated by certain molecules on pathogen
surfaces. Both pathways converge at the point where complement
component C3 is cleaved by an active protease (which is different
in each pathway) to yield C3a and C3b. Other pathways activating
complement attack can act later in the sequence of events leading
to various aspects of complement function.
[0038] C3a is an anaphylatoxin. C3b binds to bacterial and other
cells, as well as to certain viruses and immune complexes, and tags
them for removal from the circulation. C3b in this role is known as
opsonin. The opsonic function of C3b is considered to be the most
important anti-infective action of the complement system. Patients
with genetic lesions that block C3b function are prone to infection
by a broad variety of pathogenic organisms, while patients with
lesions later in the complement cascade sequence, i.e., patients
with lesions that block C5 functions, are found to be more prone
only to Neisseria infection, and then only somewhat more prone
(Fearon, 1983).
[0039] C3b also forms a complex with other components unique to
each pathway to form classical or alternative C5 convertase, which
cleaves C5 into C5a and C5b. C3 is thus regarded as the central
protein in the complement reaction sequence since it is essential
to both the alternative and classical pathways (Wurzner et al.,
1991). This property of C3b is regulated by the serum protease
Factor I, which acts on C3b to produce iC3b. While still functional
as opsonin, iC3b cannot form an active C5 convertase.
[0040] C5 is a 190 kDa beta globulin found in normal serum at
approximately 75 .mu.g/mL (0.4 .mu.M). C5 is glycosylated, with
about 13-3 percent of its mass attributed to carbohydrate. Mature
C5 is a heterodimer of a 999 amino acid 115 kDa alpha chain that is
disulfide linked to a 656 amino acid 75 kDa beta chain. C5 is
synthesized as a single chain precursor protein product of a single
copy gene (Haviland et al., 1991). The cDNA sequence of the
transcript of this gene predicts a secreted pro-C5 precursor of
1659 amino acids along with an 18 amino acid leader sequence.
[0041] The pro-C5 precursor is cleaved after amino acid 655 and
659, to yield the beta chain as an amino terminal fragment (amino
acid residues +1 to 655) and the alpha chain as a carboxyl terminal
fragment (amino acid residues 660 to 1658), with four amino acids
deleted between the two.
[0042] C5a is cleaved from the alpha chain of C5 by either
alternative or classical C5 convertase as an amino terminal
fragment comprising the first 74 amino acids of the alpha chain
(i.e., amino acid residues 660-733). Approximately 20 percent of
the 11 kDa mass of C5a is attributed to carbohydrate. The cleavage
site for convertase action is at or immediately adjacent to amino
acid residue 733. A compound that would bind at or adjacent to this
cleavage site would have the potential to block access of the C5
convertase enzymes to the cleavage site and thereby act as a
complement inhibitor.
[0043] C5 can also be activated by means other than C5 convertase
activity. Limited trypsin digestion (Minta and Man, 1977; Wetsel
and Kolb, 1982) and acid treatment (Yamamoto and Gewurz, 1978; Vogt
et al., 1989) can also cleave C5 and produce active C5b.
[0044] C5a is another anaphylatoxin. C5b combines with C6, C7, and
C8 to form the C5b-8 complex at the surface of the target cell.
Upon binding of several C9 molecules, the membrane attack complex
(MAC, C5b-9, terminal complement complex-TCC) is formed. When
sufficient numbers of MACs insert into target cell membranes the
openings they create (MAC pores) mediate rapid osmotic lysis of the
target cells. Lower, non-lytic concentrations of MACs can produce
other effects. In particular, membrane insertion of small numbers
of the C5b-9 complexes into endothelial cells and platelets can
cause deleterious cell activation. In some cases activation may
precede cell lysis.
[0045] As mentioned above, C3a and C5a are anaphylatoxins. These
activated complement components can trigger mast cell
degranulation, which releases histamine and other mediators of
inflammation, resulting in smooth muscle contraction, increased
vascular penneability, leukocyte activation, and other inflammatory
phenomena including cellular proliferation resulting in
hypercellularity. C5a also functions as a chemotactic peptide that
serves to attract pro-inflammatory granulocytes to the site of
complement activation.
[0046] The beneficial effect of anti-C5 mAb has previously been
reported in several experimental models including myocardial
reperfusion (Vakeva et al., 1998), systemic lupus erythematosus
(Wang et al., 1996) and rheumatoid arthritis (Wang et al, 1995); as
well as in human clinical trials (Kirschfink, 2001) of autoimmune
disease, cardiopulmonary bypass and acute myocardial
infarction.
IV Measures of Quality of Life
[0047] Various measurements exist to assess quality of life and the
effect of medical interventions on quality of life for example the
Mini-Mental State Examination (MMSE), the Short Test of Mental
Status, the European Organization for Research and Treatment of
Cancer (EORTC) Quality of Life Questionnaire, the FACIT
questionnaires and subscales including fatigue and anemia, the
Likert Scale, and Borg Scale (Tombaugh, et al., J. Am. Geriatr.
Soc. 40:922, 1992; Cummings, JAMA. 269(18):2420, 1993; Crum, et
al., JAMA. 269(18):2386, 1993; Folstein, et al., J. Psychiat. Res.
12:189, 1975; Kokmen, et al., Mayo Clin. Proc. 62:281, 1987;
Tang-Wai, et al., Arch. Neurol. 60:1777, 2003; Tamburini, Ann.
Oncol. 12(Suppl. 3):S7, 2001; Webster et al., Health and Quality of
Life Outcomes. 1:79, 2003, www.hqlo.com/content/I/I/79; Grant, et
al., Chest. 116:1208, 1999; and www.qolid.org). Any of these
measurements may be used to assess the change in quality of life
due to administration of a compound which inhibits complement or
inhibits formation of C5b-9.
[0048] In certain embodiments, improvement in quality of life due
to administration of a compound which inhibits complement or
inhibits formation of C5b-9 is measured by the Functional
Assessment of Chronic Illness Therapy (FACIT) Measurement System.
In certain embodiments, improvement in quality of life is measured
by: a) full scales; b) stand-alone subscales; and c) symptom
indices.
[0049] In certain embodiments, improvement in quality of life due
to administration of a compound which inhibits complement or
inhibits formation of C5b-9 is measured by a European Organization
for Research and Treatment of Cancer (EORTC) Quality of Life
Questionnaire. In certain embodiments, the EORTC questionnaire is
the QLQ-C30.
[0050] In certain embodiments, improvement in quality of life is
measured by the health-adjusted life expectancy (HALE) index as
described in Wilkins, R. and Adams, O B., Am J Public Health,
73:1073-1080 (1983). Health-adjusted life expectancy is an average
of the quality-adjusted life years (QALY) for a given population
and can be used to evaluate the therapeutic value of a medical
intervention. Quality-adjusted life years is a health index that
weighs each year of life on a scale from 1 to 0 (Weinstein M C and
Stason W B, N Engl J Med, 296:716-721 (1977)). Perfect health is
rated as 1, death is rated as 0, and disability and pain are rated
based on severity. QALY is determined by multiplying the number of
years at each health status.
[0051] In certain embodiments, improvement in quality of life is
measured by the following instruments: Years of potential life
lost, Disability-free life expectancy, Health-adjusted life year,
Quality adjusted life year, Healthy years equivalents, Healthy days
gained, Episode-free day, Q-TWiST, Health Utilities Index, and
Years of healthy life. These measurements account for both changes
in mortality as well as changes in morbidity and disability. Any of
these measurements may be used to assess the change in quality of
life due to administration of a compound which inhibits complement
or inhibits formation of C5b-9.
[0052] In one embodiment, the disclosed methods improve the quality
of life of a patient for at least one day, at least one week, at
least two weeks, at least three weeks, at least one month, at least
two months, at least three months, at least 6 months, at least one
year, at least 18 months, at least two years, at least 30 months,
or at least three years, or the duration of treatment.
[0053] In certain embodiments, the symptoms used to measure quality
of life are scaled for intensity. In certain embodiments, the
symptoms are scaled for frequency. In certain embodiments, the
symptoms are scaled for intensity and frequency.
[0054] In certain aspects, the application provides a method for
prolonging the health-adjusted life expectancy of a subject
comprising administering to the subject a compound which inhibits
complement or inhibits formation of C5b-9. The above measurements
account for both changes in mortality as well as changes in
morbidity and disability. Any of these measurements may be used to
assess the change in quality-adjusted life expectancy due to
administration of a compound which inhibits complement or inhibits
formation of C5b-9.
[0055] In one embodiment, the disclosed methods prolong the
health-adjusted life expectancy in a subject by at least one day,
at least one week, at least two weeks, at least three weeks, at
least one month, at least two months, at least three months, at
least 6 months, at least one year, at least 18 months, at least two
years, at least 30 months, or at least three years as measured by
the health-adjusted life expectancy (HALE) index as described in
Wilkins et al. Am J Public Health, 73:1073-1080 (1983).
Health-adjusted life expectancy is an average of the
quality-adjusted life years (QALY) for a given population and can
be used to evaluate the therapeutic value of a medical
intervention. Quality-adjusted life years is a health index that
weighs each year of life on a scale from 1 to 0 (Weinstein et al.,
N Engl J Med, 296:716-721 (1977)). Perfect health is rated as 1,
death is rated as 0, and disability and pain are rated based on
severity. QALY is determined by multiplying the number of years at
each health status.
V Inhibitors of the Complement Cascade
[0056] In certain embodiments, any compound which binds to or
otherwise blocks the generation and/or activity of one or more
complement components can be used in the present methods. In
certain embodiments, a complement inhibitor may be a small molecule
(up to 6,000 Da in molecular weight), a nucleic acid or nucleic
acid analog, a peptidomimetic, or a macromolecule that is not a
nucleic acid, a serine protease inhibitor, or a protein. These
agents include, but are not limited to, small organic molecules,
RNA aptamers including ARC187 (which is commercially available from
Archemix Corp., Cambridge, Mass.), L-RNA aptamers, Spiegelmers,
antisense compounds, molecules which may be utilized in RNA
interference (RNAi) such as double stranded RNA including small
interfering RNA (siRNA), locked nucleic acid (LNA) inhibitors,
peptide nucleic acid (PNA) inhibitors.
[0057] In certain embodiments, a complement inhibitor may be a
protein or protein fragment. Proteins are known which inhibit the
complement cascade, including CD59, CD55, CD46 and other inhibitors
of C8 and C9 (see, e.g., U.S. Pat. No. 6,100,443). Proteins known
as complement receptors and which bind complement are also known
(see, Published PCT Patent Application WO 92/10205 and U.S. Pat.
No. 6,057,131). Use of soluble forms of complement receptors, e.g.,
soluble CR1, can inhibit the consequences of complement activation
such as neutrophil oxidative burst, complement mediated neural
injury, and C3a and C5a production. In certain embodiments, a
complement inhibitor may be naturally occurring or soluble forms of
complement inhibitory compounds such as CR1, LEX-CR1, MCP, DAF,
CD59, Factor H, cobra venom factor, FUT-175, complestatin, and K76
COOH. Those of skill in the art recognize the above as some, but
not all, of the known methods of inhibiting complement and its
activation.
[0058] In certain embodiments, a complement inhibitor may be an
antibody capable of inhibiting complement, such as an antibody that
can block the formation of MAC. For example, an antibody complement
inhibitor may include an antibody that binds C5. Such anti-C5
antibodies may directly interact with C5 and/or C5b, so as to
inhibit the formation of and/or physiologic function of C5b.
[0059] Suitable anti-C5 antibodies are known to those of skill in
the art. Antibodies can be made to individual components of
activated complement, e.g., antibodies to C7, C9, etc. (see, e.g.,
U.S. Pat. No. 6,534,058; published U.S. patent application US
2003/0129187; and U.S. Pat. No. 5,660,825). U.S. Pat. No. 6,355,245
teaches an antibody which binds to C5 and inhibits cleavage into
C5a and C5b thereby decreasing the formation not only of C5a but
also the downstream complement components.
[0060] The concentration and/or physiologic activity of C5a and C5b
in a body fluid can be measured by methods well known in the art.
For C5a such methods include chemotaxis assays, RIAs, or ELISAs
(see, for example, Ward and Zvaifler, J Clin Invest. 1971 March;
50(3):606-16; Wurzner, et al., Complement Inflamm. 8:328-340,
1991). For C5b, hemolytic assays or assay; for soluble C5b-9 as
discussed herein can be used. Other assays known in the art can
also be used. Using assays of these or other suitable types,
candidate antibodies capable of inhibiting complement such as
anti-C5 antibodies, now known or subsequently identified, can be
screened in order to 1) identify compounds that are useful in the
practice of the application and 2) determine the appropriate dosage
levels of such compounds.
[0061] An antibody capable of inhibiting complement such as an
antibody that binds C5 affecting C5b is preferably used at
concentrations providing substantial reduction (i.e., reduction by
at least about 25% as compared to that in the absence of the
antibody that binds C5) in the C5b levels present in at least one
blood-derived fluid of the patient following activation of
complement within the fluid. Such concentrations can be
conveniently determined by measuring the cell-lysing ability (e.g.,
hemolytic activity) of complement present in the fluid or the
levels of soluble C5b-9 present in the fluid. Accordingly, a
specific concentration for an antibody that affects C5b is one that
results in a substantial reduction (i.e., a reduction by at least
about 25%) in the cell-lysing ability of the complement present in
at least one of the patient's blood-derived fluids. Reductions of
the cell-lysing ability of complement present in the patient's body
fluids can be measured by methods well known in the art such as,
for example, by a conventional hemolytic assay such as the
hemolysis assay described by Kabat and Mayer (eds), "Experimental
Immunochemistry, 2d Edition", 135-240, Springfield, Ill., CC Thomas
(1961), pages 135-139, or a conventional variation of that assay
such as the chicken erythrocyte hemolysis method described
below.
[0062] Specific antibodies capable of inhibiting complement, such
as an antibody that binds C5, are relatively specific and do not
block the functions of early complement components. In particular,
such specific agents will not substantially impair the opsonization
functions associated with complement component C3b, which functions
provide a means for clearance of foreign particles and substances
from the body.
[0063] C3b is generated by the cleavage of C3, which is carried out
by classical and/or alternative C3 convertases and results in the
generation of both C3a and C3b. Therefore, in order not to impair
the opsonization functions associated with C3b, specific antibodies
capable of inhibiting complement such as an antibody that binds C5
do not substantially interfere with the cleavage of complement
component C3 in a body fluid of the patient (e.g., serum) into C3a
and C3b. Such interference with the cleavage of C3 can be detected
by measuring body fluid levels of C3a and/or C3b, which are
produced in equimolar ratios by the actions of the C3 convertases.
Such measurements are informative because C3a and C3b levels will
be reduced (compared to a matched sample without the antibody
capable of inhibiting complement such as an antibody that binds C5)
if cleavage is interfered with by an antibody capable of inhibiting
complement such as an antibody that binds C5.
[0064] In practice, the quantitative measurement of such cleavage
is generally more accurate when carried out by the measurement of
body fluid C3a levels rather than of body fluid C3b levels, since
C3a remains in the fluid phase whereas C3b is rapidly cleared. C3a
levels in a body fluid can be measured by methods well known in the
art such as, for example, by using a commercially available C3a EIA
kit, e.g., that sold by Quidel Corporation, San Diego, Calif.,
according to the manufacturer's specifications. Particularly
specific antibodies capable of inhibiting complement such as an
antibody that binds C5 produce essentially no reduction in body
fluid C3a levels following complement activation when tested in
such assays.
[0065] Certain antibodies of the disclosure will prevent the
cleavage of C5 to form C5a and C5b, thus preventing the generation
of the anaphylatoxic activity associated with C5a and preventing
the assembly of the membrane attack complex associated with C5b. As
discussed above, in a particular embodiment, these anti-C5
antibodies will not impair the opsonization function associated
with the action of C3b.
[0066] A preferred method of inhibiting complement activity is to
use a monoclonal antibody which binds to complement C5 and inhibits
cleavage. This decreases the formation of both C5a and C5b while at
the same time allowing the formation of C3a and C3b which are
beneficial to the recipient. Such antibodies which are specific to
human complement are known (U.S. Pat. No. 6,355,245). These
antibodies disclosed in U.S. Pat. No. 6,355,245 include a preferred
whole antibody (now named eculizumab). A similar antibody against
mouse C5 is called BB5.1 (Frei et al., Mol. Cell. Probes. 1:141-149
(1987)). Antibodies to inhibit complement activity need not be
monoclonal antibodies. They can be, e.g., polyclonal antibodies.
They may additionally be antibody fragments. An antibody fragment
includes, but is not limited to, an Fab, F(ab'), F(ab').sub.2,
single-chain antibody, and Fv. Furthermore, it is well known by
those of skill in the art that antibodies can be humanized (Jones
et al., Nature 321:522-5 (1986)), chimerized, or deimmunized. The
antibodies to be used in the present disclosure may be any of
these. It is preferable to use humanized antibodies.
[0067] In specific embodiments, a therapeutic agent of the
disclosure comprises an antibody or antibody fragment. Antibodies
and fragments thereof may be made by any conventional method, such
as those methods described herein. Antibodies are found in multiple
forms, e.g., IgA, IgG, IgM, etc. Additionally, antibodies can be
engineered in numerous ways. They can be made as single-chain
antibodies (including small modular immunopharmaceuticals or
SMIPs.TM.), Fab and F(ab').sub.2 fragments, etc. Antibodies can be
humanized, chimerized, deimmunized, or fully human. Numerous
publications set forth the many types of antibodies and the methods
of engineering such antibodies. For example, see U.S. Pat. Nos.
6,355,245; 6,180,370; 5,693,762; 6,407,213; 6,548,640; 5,565,332;
5,225,539; 6,103,889; and 5,260,203.
[0068] This invention provides fragments of anti-C5 antibodies,
which may comprise a portion of an intact antibody, preferably the
antigen-binding or variable region of the intact antibody. Examples
of antibody fragments include Fab, Fab', F(ab').sub.2, and Fv
fragments; diabodies; linear antibodies (Zapata et al., Protein
Eng. 8:1057-1062 (1995)); single-chain antibody molecules; and
multispecific antibodies formed from antibody fragments.
[0069] Papain digestion of antibodies produces two identical
antigen-binding fragments, called "Fab" fragments, each with a
single antigen-binding site, and a residual "Fc" fragment, whose
name reflects its ability to crystallize readily. Pepsin treatment
of an antibody yields an F(ab').sub.2 fragment that has two
antigen-combining sites and is still capable of cross-linking
antigen.
[0070] "Fv" refers to the minimum antibody fragment that contains a
complete antigen-recognition and -binding site. This region
consists of a dimer of one heavy- and one light-chain variable
domain in tight, non-covalent association. It is in this
configuration that the three CDRs of each variable domain interact
to define an antigen-binding site on the surface of the
V.sub.H-V.sub.L dimer. Collectively, the six CDRs confer
antigen-binding specificity to the antibody. However, even a single
variable domain (or half of an Fv comprising only three CDRs
specific for an antigen) has the ability to recognize and bind
antigen, although likely at a lower affinity than the entire
binding site.
[0071] The Fab fragment also contains the constant domain of the
light chain and the first constant domain (CH1) of the heavy chain.
Fab' fragments differ from Fab fragments by the addition of a few
residues at the carboxy terminus of the heavy chain CH1 domain
including one or more cysteines from the antibody hinge region.
Fab'-SH is the designation herein for Fab' in which the cysteine
residue(s) of the constant domains bear a free thiol group.
F(ab').sub.2 antibody fragments originally were produced as pairs
of Fab' fragments that have hinge cysteines between them. Other
chemical couplings of antibody fragments are also known.
[0072] "Single-chain Fv" or "scFv" antibody fragments comprise the
V.sub.H and V.sub.L domains of an antibody, wherein these domains
are present in a single polypeptide chain. Preferably, the Fv
polypeptide further comprises a polypeptide linker between the
V.sub.H and V.sub.L domains that enables the scFv to form the
desired structure for antigen binding. For a review of scFv see
Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113,
Rosenburg and Moore, eds. (Springer-Verlag: New York, 1994), pp.
269-315.
[0073] SMIPs are a class of single-chain peptide engineered to
include a target binding region, effector domain (CH2 and CH3
domains). See, e.g., U.S. Patent Application Publication No.
20050238646. The target binding region may be derived from the
variable region or CDRs of an antibody, e.g., an antibody that
binds C5 of the application. Alternatively, the target binding
region is derived from a protein that binds C5.
[0074] The term "diabodies" refers to small antibody fragments with
two antigen-binding sites, which fragments comprise a heavy-chain
variable domain (V.sub.H) connected to a light-chain variable
domain (V.sub.L) in the same polypeptide chain (V.sub.H-V.sub.L).
By using a linker that is too short to allow pairing between the
two domains on the same chain, the domains are forced to pair with
the complementary domains of another chain and create two
antigen-binding sites. Diabodies are described more fully in, for
example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl.
Acad. Sci. USA, 90: 6444-6448 (1993).
[0075] It is well known that the binding to a molecule (or a
pathogen) of antibodies with an Fc region assists in the processing
and clearance of the molecule (or pathogen). The Fc portions of
antibodies are recognized by specialized receptors expressed by
immune effector cells. The Fc portions of IgG1 and IgG3 antibodies
are recognized by Fc receptors present on the surface of phagocytic
cells such as macrophages and neutrophils, which can thereby bind
and engulf the molecules or pathogens coated with antibodies of
these isotypes (C. A. Janeway et al., Immunobiology 5th edition,
page 147, Garland Publishing (New York, 2001)).
[0076] This disclosure also provides monoclonal anti-C5 antibodies.
A monoclonal antibody can be obtained from a population of
substantially homogeneous antibodies, i.e., the individual
antibodies comprising the population are identical except for
possible naturally-occurring mutations that may be present in minor
amounts. Monoclonal antibodies are highly specific, being directed
against a single antigenic site. Furthermore, in contrast to
conventional (polyclonal) antibody preparations that typically
include different antibodies directed against different
determinants (epitopes), each monoclonal antibody is directed
against a single determinant on the antigen. In addition to their
specificity, the monoclonal antibodies are advantageous in that
they are often synthesized by the hybridoma culture, uncontaminated
by other immunoglobulins. Monoclonal antibodies may also be
produced in transfected cells, such as CHO cells and NS0 cells. The
modifier "monoclonal" indicates the character of the antibody as
being obtained from a substantially homogeneous population of
antibodies and does not require production of the antibody by any
particular method. For example, the monoclonal antibodies to be
used in accordance with the present disclosure may be made by the
hybridoma method first described by Kohler et al., Nature
256:495-497 (1975), or may be made by recombinant DNA methods (see,
e.g., U.S. Pat. Nos. 4,816,567 and 6,331,415). The "monoclonal
antibodies" may also be isolated from phage antibody libraries
using the techniques described in Clackson et al., Nature
352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597
(1991), for example.
[0077] A description of the preparation of a mouse anti-human-C5
monoclonal antibody with specific binding characteristics is
presented in U.S. Patent Application Publication No. 20050226870.
Wurzner et al., Complement Inflamm. 8:328-340 (1991), describe the
preparation of other mouse anti-human-C5 monoclonal antibodies
referred to as N19-8 and N20-9.
[0078] Other antibodies specifically contemplated are "oligoclonal"
antibodies. As used herein, the term "oligoclonal" antibodies"
refers to a predetermined mixture of distinct monoclonal
antibodies. See, e.g., PCT publication WO 95/20401; U.S. Pat. Nos.
5,789,208 and 6,335,163. In one embodiment, oligoclonal antibodies
consisting of a predetermined mixture of antibodies against one or
more epitopes are generated in a single cell. In other embodiments,
oligoclonal antibodies comprise a plurality of heavy chains capable
of pairing with a common light chain to generate antibodies with
multiple specificities (e.g., PCT publication WO 04/009618).
Oligoclonal antibodies are particularly useful when it is desired
to target multiple epitopes on a single target molecule (e.g., C5).
In view of the assays and epitopes disclosed herein, those skilled
in the art can generate or select antibodies or mixtures of
antibodies that are applicable for an intended purpose and desired
need.
[0079] In certain embodiments that include a humanized and/or
chimeric antibody, one or more of the CDRs are derived from an
anti-human C5 antibody. In a specific embodiment, all of the CDRs
are derived from an anti-human C5 antibody. In another specific
embodiment, the CDRs from more than one anti-human C5 antibody are
mixed and matched in a chimeric antibody. For instance, a chimeric
antibody may comprise a CDR1 from the light chain of a first
anti-human C5 antibody combined with CDR2 and CDR3 from the light
chain of a second anti-human C5 antibody, and the CDRs from the
heavy chain may be derived from a third anti-human C5 antibody.
Further, the framework regions may be derived from one of the same
anti-human C5 antibodies, from one or more different antibodies,
such as a human antibody, or from a humanized antibody. Human or
humanized antibodies are specific for administration to human
patients.
[0080] In certain embodiments, single chain antibodies, and
chimeric, humanized or primatized (CDR-grafted) antibodies, as well
as chimeric or CDR-grafted single chain antibodies, comprising
portions derived from different species, are also encompassed by
the present disclosure as antigen-binding fragments of an antibody.
The various portions of these antibodies can be joined together
chemically by conventional techniques, or can be prepared as a
contiguous protein using genetic engineering techniques. For
example, nucleic acids encoding a chimeric or humanized chain can
be expressed to produce a contiguous protein. See, e.g., U.S. Pat.
Nos. 4,816,567 and 6,331,415; U.S. Pat. No. 4,816,397; European
Patent No. 0,120,694; WO 86/01533; European Patent No. 0,194,276
B1; U.S. Pat. No. 5,225,539; and European Patent No. 0,239,400 B1.
See also, Newman et al., BioTechnology 10:1455-1460 (1992),
regarding primatized antibody. See, e.g., Ladner et al., U.S. Pat.
No. 4,946,778; and Bird at al., Science 242:423-426 (1988),
regarding single chain antibodies.
[0081] In addition, functional fragments of antibodies, including
fragments of chimeric, humanized, primatized or single chain
antibodies, can also be produced. Functional fragments of the
subject antibodies retain at least one binding function and/or
modulation function of the full-length antibody from which they are
derived. Preferred functional fragments retain an antigen-binding
function of a corresponding full-length antibody (such as for
example, ability of antibody that binds C5 to bind C5).
[0082] General methods for the immunization of animals (in this
case with C5 and/or C5b, etc.), isolation of antibody producing
cells, fusion of such cells with immortal cells (e.g., myeloma
cells) to generate hybridomas secreting monoclonal antibodies,
screening of hybridoma supernatants for reactivity of secreted
monoclonal antibodies with a desired antigen (in this case the
immunogen or a molecule containing the immunogen), the preparation
of quantities of such antibodies in hybridoma supernatants or
ascites fluids, and for the purification and storage of such
monoclonal antibodies, can be found in numerous publications. These
include: Coligan, et al., eds. Current Protocols In Immunology,
John Wiley & Sons, New York, 1992; Harlow and Lane, Antibodies,
A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988;
Liddell and Cryer, A Practical Guide To Monoclonal Antibodies, John
Wiley & Sons, Chichester, West Sussex, England, 1991; Montz et
al., Cellular Immunol. 127:337-351 (1990); Wurzner et al.,
Complement Inflamm. 8:328-340 (1991); and Mollnes et al., Scand. J.
Immunol. 28:307-312 (1988).
VI Methods of Treatment
[0083] Methods of the application may be used to treat paroxysmal
nocturnal hemoglobinuria associated symptoms. Methods of the
application may be used to treat anemia associated symptoms.
Treatment of paroxysmal nocturnal hemoglobinuria and/or anemia may
be administered by standard means. Treatments of the application
may be used in combination with other treatments of the application
or known treatments for paroxysmal nocturnal hemoglobinuria and/or
anemia. Treatments of the application may be co-administered with
other treatments that treat symptoms of paroxysmal nocturnal
hemoglobinuria and/or anemia.
VII Pharmaceutical Formulations and Uses
[0084] Methods of administration of small molecules, proteins, and
nucleic acids are well-known to those of skill in the art. Methods
of administration of antibodies are well-known to those of skill in
the art. To achieve the desired inhibition, the antibodies can be
administered in a variety of unit dosage forms. The dose will vary
according to the particular antibody. For example, different
antibodies may have different masses and/or affinities, and thus
require different dosage levels. Antibodies prepared as Fab
fragments will also require differing dosages than the equivalent
intact immunoglobulins, as they are of considerably smaller mass
than intact immunoglobulins, and thus require lower dosages to
reach the same molar levels in the patient's blood. The dose will
also vary depending on the manner of administration, the particular
symptoms of the patient being treated, the overall health,
condition, size, and age of the patient, and the judgment of the
prescribing physician. Dosage levels of the antibodies for human
subjects are generally between about 1 mg per kg and about 100 mg
per kg per patient per treatment, and preferably between about 5 mg
per kg and about 50 mg per kg per patient per treatment. In terms
of plasma concentrations, the antibody concentrations are
preferably in the range from about 25 .mu.g/mL to about 500
.mu.g/mL. However, greater amounts may be required for extreme
cases and smaller amounts may be sufficient for milder cases.
[0085] In certain embodiments, the pharmaceutical composition is in
a single unit dosage form. In certain embodiments, the single unit
dosage form is a 300 mg unit dosage form. In certain embodiments,
the pharmaceutical composition is lyophilized. In certain
embodiments, the pharmaceutical composition is a sterile solution.
In certain embodiments, the pharmaceutical composition is a
preservative free formulation. In certain embodiments, the
pharmaceutical composition comprises a 300 mg single-use
formulation of 30 ml of a 10 mg/ml sterile, preservative free
solution. In certain embodiments, the antibody is administered
according to the following protocol: 600 mg via 25 to 45 minute IV
infusion every 7.+-.2 days for the first 4 weeks, followed by 900
mg for the fifth dose 7.+-.2 days later, then 900 mg every 14.+-.2
days thereafter. Antibody is administered via IV infusion over 25
to 45 minute.
[0086] Administration of the anti-C5 antibodies will generally be
performed by an intravascular route, e.g., via intravenous infusion
by injection. Other mutes of administration may be used if desired
but an intravenous route will be the most preferable. Formulations
suitable for injection are found in Remington's Pharmaceutical
Sciences, Mack Publishing Company, Philadelphia, Pa., 17th ed.
(1985). Such formulations must be sterile and non-pyrogenic, and
generally will include a pharmaceutically effective carrier, such
as saline, buffered (e.g., phosphate buffered) saline, Hank's
solution, Ringer's solution, dextrose/saline, glucose solutions,
and the like. The formulations may contain pharmaceutically
acceptable auxiliary substances as required, such as, tonicity
adjusting agents, wetting agents, bactericidal agents,
preservatives, stabilizers, and the like. In certain embodiments,
complement inhibitors such as eculizumab may be administered via IV
infusion and diluted to a final concentration of 5 mg/ml prior to
administration.
[0087] Administration of the antibodies capable of inhibiting
complement such as an antibody that binds C5 will generally be
performed by a parenteral route, typically via injection such as
intra-articular or intravascular injection (e.g., intravenous
infusion) or intramuscular injection. Other routes of
administration, e.g., oral (p.o.), may be used if desired and
practicable for the particular antibody capable of inhibiting
complement to be administered. Antibodies capable of inhibiting
complement such as an antibody that binds C5 can also be
administered in a variety of unit dosage forms and their dosages
will also vary with the size, potency, and in vivo half-life of the
particular antibody capable of inhibiting complement being
administered. Doses of antibodies capable of inhibiting complement
such as an antibody that binds C5 will also vary depending on the
manner of administration, the particular symptoms of the patient
being treated, the overall health, condition, size, and age of the
patient, and the judgment of the prescribing physician.
[0088] In certain embodiments, a typical therapeutic treatment
includes a series of doses, which will usually be administered
concurrently with the monitoring of clinical endpoints with the
dosage levels adjusted as needed to achieve the desired clinical
outcome. In certain embodiments, treatment is administered in
multiple dosages over at least a week. In certain embodiments,
treatment is administered in multiple dosages over at least a
month. In certain embodiments, treatment is administered in
multiple dosages over at least a year. In certain embodiments,
treatment is administered in multiple dosages over the remainder of
the patient's life.
[0089] The frequency of administration may also be adjusted
according to various parameters. These include the clinical
response, the plasma half-life of the therapeutic of the
disclosure, and the levels of the antibody in a body fluid, such
as, blood, plasma, serum, or synovial fluid. To guide adjustment of
the frequency of administration, levels of the therapeutic of the
disclosure in the body fluid may be monitored during the course of
treatment.
[0090] In certain embodiments, the frequency of administration may
be adjusted according to an assay measuring cell-lysing ability of
complement present in one or more of the patient's body fluids. The
cell-lysing ability can be measured as percent hemolysis in
hemolytic assays of the types described herein. A 10% or 25% or 50%
reduction in the cell-lysing ability of complement present in a
body fluid after treatment with the antibody capable of inhibiting
complement used in the practice of the application means that the
percent hemolysis after treatment is 90, 75, or 50 percent,
respectively, of the percent hemolysis before treatment.
[0091] For the treatment of hemolytic diseases such as PNH by
systemic administration of an antibody capable of inhibiting
complement such as an antibody that binds C5 (as opposed to local
administration), administration of a large initial dose is
specific, i.e., a single initial dose sufficient to yield a
substantial reduction, and more preferably an at least about 50%
reduction, in the hemolytic activity of the patient's serum. Such a
large initial dose is preferably followed by regularly repeated
administration of tapered doses as needed to maintain substantial
reductions of serum hemolytic titer. In another embodiment, the
initial dose is given by both local and systemic routes, followed
by repeated systemic administration of tapered doses as described
above.
[0092] Formulations particularly useful for antibody-based
therapeutic agents are also described in U.S. Patent App.
Publication Nos. 20030202972, 20040091490 and 20050158316. In
certain embodiments, the liquid formulations of the application are
substantially free of surfactant and/or inorganic salts. In another
specific embodiment, the liquid formulations have a pH ranging from
about 5.0 to about 7.0. In yet another specific embodiment, the
liquid formulations comprise histidine at a concentration ranging
from about 1 mM to about 100 mM. In still another specific
embodiment, the liquid formulations comprise histidine at a
concentration ranging from 1 mM to 100 mM. It is also contemplated
that the liquid formulations may further comprise one or more
excipients such as a saccharide, an amino acid (e.g., arginine,
lysine, and methionine) and a polyol. Additional descriptions and
methods of preparing and analyzing liquid formulations can be
found, for example, in PCT publications WO 03/106644, WO 04/066957,
and WO 04/091658.
[0093] Wetting agents, emulsifiers and lubricants, such as sodium
lauryl sulfate and magnesium stearate, as well as coloring agents,
release agents, coating agents, sweetening, flavoring and perfuming
agents, preservatives and antioxidants can also be present in the
pharmaceutical compositions of the application.
[0094] In certain embodiments, formulations of the subject
antibodies are pyrogen-free formulations which are substantially
free of endotoxins and/or related pyrogenic substances. Endotoxins
include toxins that are confined inside microorganisms and are
released when the microorganisms are broken down or die. Pyrogenic
substances also include fever-inducing, thermostable substances
(glycoproteins) from the outer membrane of bacteria and other
microorganisms. Both of these substances can cause fever,
hypotension and shock if administered to humans. Due to the
potential harmful effects, it is advantageous to remove even low
amounts of endotoxins from intravenously administered
pharmaceutical drug solutions. The Food & Drug Administration
("FDA") has set an upper limit of 5 endotoxin units (EU) per dose
per kilogram body weight in a single one hour period for
intravenous drug applications (The United States Pharmacopeial
Convention, Pharmacopeial Forum 26 (1):223 (2000)). When
therapeutic proteins are administered in amounts of several hundred
or thousand milligrams per kilogram body weight, as can be the case
with monoclonal antibodies, it is advantageous to remove even trace
amounts of endotoxin.
[0095] Formulations of the subject antibodies include those
suitable for oral, dietary, topical, parenteral (e.g., intravenous,
intraarterial, intramuscular, subcutaneous injection),
ophthalmologic (e.g., topical or intraocular), inhalation (e.g.,
intrabronchial, intranasal or oral inhalation, intranasal drops),
rectal, and/or intravaginal administration. Other suitable methods
of administration can also include rechargeable or biodegradable
devices and controlled release polymeric devices. Stents, in
particular, may be coated with a controlled release polymer mixed
with an agent of the application. The pharmaceutical compositions
of this disclosure can also be administered as part of a
combinatorial therapy with other agents (either in the same
formulation or in a separate formulation).
[0096] The amount of the formulation which will be therapeutically
effective can be determined by standard clinical techniques. In
addition, in vitro assays may optionally be employed to help
identify optimal dosage ranges. The precise dose to be employed in
the formulation will also depend on the route of administration,
and the seriousness of the disease or disorder, and should be
decided according to the judgment of the practitioner and each
patient's circumstances. Effective doses may be extrapolated from
dose-response curves derived from in vitro or animal model test
systems. The dosage of the compositions to be administered can be
determined by the skilled artisan without undue experimentation in
conjunction with standard dose-response studies. Relevant
circumstances to be considered in making those determinations
include the condition or conditions to be treated, the choice of
composition to be administered, the age, weight, and response of
the individual patient, and the severity of the patient's symptoms.
For example, the actual patient body weight may be used to
calculate the dose of the formulations in milliliters (mL) to be
administered. There may be no downward adjustment to "ideal"
weight. In such a situation, an appropriate dose may be calculated
by the following formula: Dose (mL)=[patient weight (kg).times.dose
level (mg/kg)/drug concentration (mg/mL)]
[0097] To achieve the desired treatment results, anti-C5 antibodies
can be administered in a variety of unit dosage forms. The dose
will vary according to the particular antibody. For example,
different antibodies may have different masses and/or affinities,
and thus require different dosage levels. Antibodies prepared as
Fab' fragments or single chain antibodies will also require
differing dosages than the equivalent native immunoglobulins, as
they are of considerably smaller mass than native immunoglobulins,
and thus require lower dosages to reach the same molar levels in
the patient's blood.
[0098] Other therapeutics of the disclosure can also be
administered in a variety of unit dosage forms and their dosages
will also vary with the size, potency, and in vivo half-life of the
particular therapeutic being administered.
[0099] Doses of therapeutics of the disclosure will also vary
depending on the manner of administration, the particular symptoms
of the patient being treated, the overall health, condition, size,
and age of the patient, and the judgment of the prescribing
physician.
[0100] The formulations of the application can be distributed as
articles of manufacture comprising packaging material and a
pharmaceutical agent which comprises the antibody capable of
inhibiting complement and a pharmaceutically acceptable carrier as
appropriate to the mode of administration. The packaging material
may include a label which indicates that the formulation is for use
in the treatment of hemolytic diseases such as PNH. Although
antibodies are preferred, especially anti-C5 antibodies which have
already been shown to be safe and effective at decreasing the
accumulation of downstream complement components in persons, the
use of other complement inhibitors is also contemplated by this
disclosure. The pharmaceutical formulations and uses of the
disclosure may be combined with any known complement inhibitors or
hemolytic diseases treatments known in the art.
[0101] In certain aspects, the application provides kits comprising
a pharmaceutical composition of the application. In some
embodiments, the kit further comprises at least one component of a
closed sterile system. Components of the closed sterile system
include, but are not limited to, needles, syringes, catheter based
syringes, needle based injection devices, needle-less injection
devices, filters, tubing, valves and cannulas. In a related
embodiment, the kit comprise components for the removal of a
preservative from the composition. Such components include filters,
syringes, vials, containers, tubing, etc.
Exemplification
Methods
Patient Selection
[0102] The TRIUMPH trial consisted of a 2-week screening period, an
observation period of up to 3 months duration, and a 26-week
treatment period.
[0103] During the screening period, patients were evaluated with
respect to inclusion and exclusion criteria. Men and women, 18
years or older, diagnosed as having PNH with a type III erythrocyte
population of .gtoreq.10%, and who had received at least 4
transfusions in the previous 12 months were eligible. Concomitant
administration of erythropoietin, immunosuppressants,
corticosteroids, coumadin, low molecular weight heparin, iron
supplements, and folic acid were not reasons for exclusion,
provided the doses were steady prior to the first visit and
throughout the duration of the study. Because of the increased
frequency of neisserial infections in individuals genetically
deficient in terminal complement proteins, all patients were
vaccinated against Neisseria meningitides. Patients were to avoid
conception. The protocol was approved by an Investigational Review
Board at each clinical site and written informed consent was
obtained from all patients enrolled.
[0104] Patients transfused with a mean pre-transfusion hemoglobin
level >10.5 g/dL over the previous 12 months, and those who
showed evidence of having a suppressed immune response, complement
deficiency, or active bacterial infection, including any history of
meningococcal disease, were excluded from the study. Patients were
also not eligible if they had previously received a bone marrow
transplant or if they had participated in another trial or received
another investigational drug within 30 days of the first visit. An
individualized transfusion algorithm was calculated for each
patient based on their prior-12-month transfusion history; the
written algorithm documented the number of packed red blood cell
(PRBC) units transfused for given hemoglobin values and served as a
prospectively determined guide for transfusion during observation
and treatment periods.
[0105] Each patient considered eligible entered an observation
period of up to 13 weeks in order to confirm their PBRC transfusion
dependence. At least one transfusion--termed the "qualifying"
transfusion--during the 13-week observation period at a hemoglobin
value at or below 9 g/dL with symptoms, or at or below 7 g/dL with
or without symptoms, in accordance with the transfusion algorithm
indicated for each patient, was a requirement to proceed to
randomization. The hemoglobin value at which each individual's
qualifying transfusion was administered, was defined as the
hemoglobin "set point" for that individual for the purpose of the
primary efficacy variable. A platelet count .gtoreq.100,000/mL and
a LDH level .gtoreq.1.5 times the upper limit of the normal range
were also required either at screening or during the observation
period for eligibility.
Study Design
[0106] Patients were randomly assigned on a one-on-one basis to
receive either placebo or eculizumab (Soliris.TM., Alexion
Pharmaceuticals, Inc.) within 10 days of the qualifying
transfusion. Study medication was dosed in a blinded fashion as
follows: 600 mg eculizumab for patients randomly assigned to active
drug, or placebo for those patients randomly assigned to placebo,
respectively via IV infusion every 7.+-.1 days for 4 doses;
followed by 900 mg eculizumab, or placebo, respectively, via IV
infusion 7.+-.1 day later; followed by a maintenance dose of 900 mg
eculizumab, or placebo, respectively, via IV infusion every 14.+-.2
days for a total of 26 weeks of treatment.
Measures of Clinical Efficacy
[0107] There were two co-primary endpoints in the study: (1)
stabilization of hemoglobin levels, defined as a hemoglobin value
maintained above the individual hemoglobin set point in the absence
of transfusions for the entire 26-week treatment period, and (2)
reduction in units of PRBCs transfused during the 26-week treatment
phase of the study. The trigger for transfusion during the study
period remained unchanged for each patient, as compared with their
care before entry into the study: patients received blood
transfusions when they had symptoms resulting from anemia and
reached their individualized, pre-determined "set point".
Pre-specified secondary endpoints included transfusion avoidance,
hemolysis as measured by LDH area under the curve from baseline to
26 weeks, and QoL changes as measured from baseline to 26 weeks
using the Functional Assessment of Chronic Illness Therapy-Fatigue
(FACIT-Fatigue) instrument..sup.13 Pre-specified exploratory
analyses included assessment of the EORTC QLQ-C30
instrument,.sup.14 the change in LDH from baseline through week 26,
and thrombosis. Other pre-specified measurements included
pharmacokinetics, pharmacodynamics, and immunogenicity of
eculizumab. Time to first transfusion during the 26-week treatment
phase and the proportion of PNH type III blood cells were also
assessed.
Safety Assessments
[0108] Treatment-emergent adverse events, clinical laboratory tests
(e.g., serum chemical analyses and complete blood counts),
electrocardiogram data, and vital signs were assessed. Adverse
events were defined using the MedDRA preferred terms and tabulated
as incidence rates per treatment group.
Statistical Analysis
[0109] For co-primary endpoints, analyses were performed according
to the intention to treat using the data from all patients who were
randomized and received study drug; stabilization of hemoglobin
levels was analyzed using the Fisher's exact test and total PRBC
units transfused were analyzed with the Wilcoxon's rank sum test.
For comparison of treatment effect on transfusion avoidance, the
Fisher's exact test was used on the incidence and the log rank test
was used for time to first transfusion. For LDH area under the
curve the Wilcoxon's rank sum test was used.
[0110] Quality of life measure of fatigue was assessed using the
scoring guidelines for the FACIT-Fatigue instrument..sup.15
Assessment of quality of life measures based on the EORTC QLQ-C30
instrument was conducted in accordance with the appropriate scoring
guidelines..sup.16 The changes of FACIT-Fatigue and EORTC QLQ-C30
scores from baseline through 26 weeks were analyzed using a mixed
model, with baseline as covariate, treatment and time as fixed
effects, and patient as a random effect. Changes in LDH levels and
PNH type III erythrocytes from baseline through 26 weeks were
analyzed using the same mixed model. Two-sided tests were used for
all analyses. The adverse events and long-term safety checklist
were tabulated separately and compared between treatments using the
Fisher's exact test. A p-value .ltoreq.0.05 was considered to be
statistically significant.
Results
Characteristics of Patients
[0111] A total of 115 PNH patients were screened. Six patients did
not meet the inclusion/exclusion criteria during the screening
period. Twenty-one other patients did not receive a qualifying
transfusion and were not randomized into the treatment phase. One
patient who did not meet the inclusion criteria was inadvertently
randomized, but did not receive study medication. Thus 87 hemolytic
PNH patients (35 men and 52 women) were enrolled and randomized to
receive either eculizumab (N=43) or placebo (N=44), exceeding the
original target of 75 randomized patients.
[0112] Patient characteristics were similar in the eculizumab- and
placebo-treated cohorts: median age, 41 (range 20-85) and 35 (range
18-78) years; median duration of PNH, 4.2 (range 0.8 to 29.7) and
9.2 (range 0.4 to 38.3) years; patients with history of aplastic
anemia, 4 and 11; history of myelodysplastic syndrome, 1 and 0; and
history of thrombosis, 9 (16 events) and 8 (11 events). Stable
usage of concomitant medications at baseline in the eculizumab- and
placebo-treated groups included the following: erythropoietin, 3
patients and 0 patients; cyclosporine, 1 and 1; anticoagulants
(coumarins or heparins) 21 and 11; and steroids (glucocorticoids or
androgenic steroids), 12 and 12, respectively.
[0113] Of the 87 patients randomized, 85 completed the trial. Two
patients who did not complete the trial had been randomized to the
eculizumab arm: one patient discontinued due to the inconvenience
of travel to the study site and the second patient became pregnant.
Ten patients in the placebo-treatment group discontinued infusions,
in all cases due to perceived lack of efficacy, but they remained
in the study for monitoring purposes.
Pharmacokinetics/Pharmacodynamics
[0114] In 42 of the 43 eculizumab-treated patients the levels of
drug during the maintenance period (900 mg every 2 weeks.+-.2 days)
were sufficient to completely block serum hemolytic activity (mean
trough value at week 26 of 101.8 .mu.g/mL). A single patient did
not sustain therapeutic trough levels of eculizumab and
demonstrated a breakthrough in complement blockade during the last
few days of each dosing interval. These breakthroughs were
clinically manageable and quickly resolved following the next
dose.
Hemolytic Efficacy Variables
[0115] The impact of terminal complement inhibition with eculizumab
on chronic intravascular hemolysis in PNH patients was demonstrated
in this study by an immediate (one week) and sustained decrease in
mean levels of LDH (FIG. 1A). The median LDH area under the curve
during the 26-week study period was reduced 85.8% in
eculizumab-relative to placebo-treated patients (P<0.001). The
mean LDH level decreased from 2199.7.+-.157.7 IU/L at baseline to
327.3.+-.67.6 IU/L by 26 weeks in eculizumab-treated patients while
levels in placebo-treated patients remained consistently elevated
with values of 2259.0.+-.158.5 IU/L at baseline and 2418.9.+-.140.3
IU/L at 26 weeks (P<0.001, for eculizumab versus placebo). A
second biochemical measure of hemolysis, serum aspartate
aminotransferase (AST), also showed a statistically significant
improvement following eculizumab- versus placebo-treatment (data
not shown). Haptoglobin levels were statistically significantly
increased in eculizumab- as compared to placebo-treated patients
but mean levels of haptoglobin were still below normal levels in
eculizumab-treated patients (data not shown).
[0116] FIG. 1A shows the degree of intravascular hemolysis in PNH
patients, demonstrated by mean lactate dehydrogenase (LDH) levels
(.+-.standard error) from baseline (study week 0) to week 26 for
both eculizumab- and placebo-treated patient populations. Screening
occurred up to 3 months prior to study week 0. The upper limit of
the normal range (103-223 IU/L) for LDH is indicated by a dashed
line. LDH was reduced to a mean level just above the upper limit of
normal at week 26 for eculizumab-treated patients; 15 of 41
patients who completed the study demonstrated LDH levels within the
normal range. All placebo-treated patients remained at least 5
times above the upper limit of normal at week 26. The P value is
based on a mixed model analysis from baseline through week 26. FIG.
1B shows the mean proportion (.+-.standard error) of PNH type III
erythrocytes assessed for placebo- and eculizumab-treated patients.
The screening visit occurred up to 3 months prior to study week 0.
The P value is based on a mixed model analysis from baseline
through week 26.
[0117] A corollary to the reduction in intravascular hemolysis
during eculizumab treatment was an observed increase in the PNH
type III erythrocyte population (FIG. 1B). The mean proportions of
type III erythrocytes increased from 28.1.+-.2.0% at baseline to
56.9.+-.3.6% by week 26 for eculizumab-treated patients while
proportions in the placebo group remained constant with mean values
of 35.7.+-.2.8% before treatment to 35.5.+-.2.8% at 26 weeks
(P<0.001, for eculizumab versus placebo). By contrast, the
proportions of PNH type III granulocytes and monocytes did not
change significantly between the treatment groups during the
treatment period and were greater than 90% at week 26.
Clinical Efficacy
Co-Primary Endpoints
[0118] The co-primary efficacy endpoints in the TRIUMPH trial were
stabilization of hemoglobin levels and reduction in PRBC units
transfused. At the end of the treatment period, 48.8% of
eculizumab-treated patients had maintained levels of hemoglobin
above the pre-specified set-point (median set-point value of 7.7
g/dL for both treatment groups) in the absence of transfusions,
whereas stabilization of hemoglobin did not occur in any of the
patients in the placebo group (P<0.001; Table 1). By week 26,
the median of PRBC units transfused per patient was 0 in the
eculizumab group and 10.0 in the placebo cohort (P<0.001), while
the mean of PRBC units transfused was 3.0 and 11.0 in the
eculizumab and placebo cohorts, respectively. In the 6-month period
prior to the study, the median of PRBC units transfused per patient
was 9.0 in the eculizumab cohort and 8.5 in placebo patients while
the mean of PRBC units transfused was 9.6.+-.0.6 and 9.7.+-.0.7,
respectively. Mean hemoglobin levels were similar between the
treatment groups at baseline (10.0.+-.1.8 g/dL in
eculizumab-treated patients and 9.7.+-.1.8 g/dL in placebo-treated
patients) and did not substantially change by week 26 (10.1.+-.2.5
g/dL and 8.9.+-.2.2 g/dL in eculizumab and placebo cohorts,
respectively).
[0119] The median time to first transfusion was not reached during
the study period in eculizumab-treated patients (it was greater
than 26 weeks) while the placebo group reached the median time to
first transfusion in only 4 weeks (P<0.001; FIG. 2). Transfusion
avoidance was achieved in 51.2% and 0% of the eculizumab and
placebo cohorts, respectively (P<0.001). By the end of the
26-week treatment period, the total PRBC units transfused were 131
in eculizumab-treated patients versus 482 in the placebo group
(Table 1). By contrast, in the 6-month period prior to the study,
total PRBC units transfused in the eculizumab- and placebo-cohorts
were 413 and 417, respectively.
TABLE-US-00001 TABLE 1 Stabilization of Hemoglobin Levels and
Reduction in Transfusion Requirements during Eculizumab Treatment
PRE-TREATMENT HISTORY* TREATMENT PERIOD Placebo Eculizumab Placebo
Eculizumab P Value .dagger.Stabilization of Hemoglobin NA NA 0 48.8
<0.001.dagger-dbl. Levels in the absence of transfusions
(percent of patients) .dagger.Units Transfused per Patient Median
8.5 9.0 10 0 <0.001.sctn. Mean .+-. SE 9.7 .+-. 0.7 9.6 .+-. 0.6
11.0 .+-. 0.83 3.0 .+-. 0.67 Total Units Transfused per Group 417
413 482 131 *Twelve month historical transfusion data normalized to
6 months .dagger.Co-primary endpoints .dagger-dbl.Base on 2-sided
Fisher's exact test .sctn.Based on Wilcoxon's rank sum test NA, not
applicable
Improvements in Quality of Life Measures
[0120] Assessments of quality of life in PNH patients during
eculizumab treatment were performed using two different
instruments, FACIT-Fatigue and the EORTC QLQ-C30.
Eculizumab-treated patients showed a mean increase (improvement) in
the FACIT-Fatigue score of 6.4.+-.1.2 points from baseline to
26-weeks while the mean score in placebo patients decreased by
4.0.+-.1.7 points, a total difference between the treatment groups
of 10.4 points (FIG. 3). Mixed model analysis of covariance
demonstrated a statistically significant difference between
treatment groups (P<0.001).
[0121] For the EORTC instrument, improvements were observed with
eculizumab-treatment in each subscale. Statistically significant
improvements with eculizumab-compared with placebo-treated groups
were observed in the following quality of life subscales (Table 2):
global health status (P<0.001), physical functioning
(P<0.001), emotional functioning (P=0.008), cognitive
functioning (P=0.002), role functioning (P<0.001), social
functioning (P=0.003), fatigue (P<0.001), pain (P=0.002),
dyspnea (P<0.001), appetite loss (P<0.001), and insomnia
(P=0.014). The improvements with eculizumab treatment in the other
scales, including nausea and vomiting, diarrhea, constipation, and
financial difficulties, did not reach statistical significance.
TABLE-US-00002 TABLE 2 Effect of Eculizumab Treatment on Quality of
Life Assessed by the EORTC QLQ-C30 Instrument Mean Change from
Baseline to Week 26* Absolute P Placebo Eculizumab Difference
Value.dagger. Global Health -8.5 10.9 19.4 <0.001 Status
Functional Role -6.9 17.9 24.8 <0.001 Social 2.0 16.7 14.6
=0.003 Cognitive -6.1 7.9 14.0 =0.002 Physical -3.5 9.4 13.0
<0.001 Emotional -3.7 7.5 11.2 =0.008 Symptoms/Single Items
Fatigue 10.0 -16.9 27.0 <0.001 Pain 5.3 -12.3 17.6 =0.002
Dyspnea 8.9 -7.9 16.9 <0.001 Appetite Loss 3.3 -10.3 13.6
<0.001 Insomnia 4.9 -7.9 12.8 =0.014 Financial 0.0 -10.3 10.3
=0.186 Difficulties Constipation 0.0 -6.3 6.3 =0.199
Nausea/vomiting 2.8 -0.4 3.2 =0.056 Diarrhea 5.7 4.8 0.9 =0.147 *A
positive change indicates an improvement in global health status
and functional scales and a negative change indicates an
improvement in symptom and single item scales. .dagger.Based on a
mixed analysis-of-covariance model with visit as a fixed effect,
patient as a random effect and baseline as a covariate.
Relationship Between FACIT-Fatigue Quality of Life and
Intravascular Hemolysis
[0122] In order to determine if there was a treatment independent
relationship between the FACIT-Fatigue quality of life instrument
and intravascular hemolysis, an analysis was performed whereby the
mean LDH level (through the 26 week study period) for each TRIUMPH
patient was analyzed as a function of the patient's respective mean
change in FACIT-Fatigue score from baseline (through the 26 week
study period) (see Table 3). For this analysis, mean levels of LDH
were divided into 4 groups that included: normal levels, 1-2 times
the upper limit of normal (ULN), 2-10 times the upper limit of
normal, and greater than 10 times the upper limit of normal. The
analysis demonstrated that patients who maintained normal LDH
levels throughout the study experienced significant improvements in
fatigue when compared to patients who had incrementally higher
levels of LDH throughout the study (p=0.0048). These data establish
a clear link between increasing intravascular hemolysis as measured
by LDH levels and decreased quality of life as measured by the
FACIT-Fatigue quality of life instrument.
TABLE-US-00003 TABLE 3 Relationship between FACIT-Fatigue and
Intravascular Hemolysis Change of FACIT Treatment LDH from Baseline
Group Category <4 >=4 P Value Combined Normal 4 (30.77%) 9
(69.23%) .0048 1-2 .times. ULN 16 (61.54%) 10 (38.46%) 2-10 .times.
ULN 18 (72.00%) 7 (28.00%) >10 .times. ULN 16 (80.00%) 4
(20.00%)
Safety
[0123] There were no deaths in the study. Serious adverse events
(SAEs) were reported for 13 patients, of which 4 occurred in the
eculizumab-treated cohort and 9 were in the placebo-treated cohort
(see Table 4). All patients recovered without sequelae.
[0124] The most common AEs reported for eculizumab-treated patients
were headache, nasopharyngitis, back pain, and upper respiratory
tract infection. Headache and back pain occurred more commonly in
the eculizumab-treatment group compared with the placebo group.
However, the increase in headaches was limited to the first 2 weeks
of therapy and was mild to moderate. There were no statistically
significant differences in incidents rates between treatment groups
for any AEs reported.
[0125] One episode of thrombosis (Budd-Chiari) occurred in a
placebo-treated patient. There were no thromboses in
eculizumab-treated patients.
[0126] Only one patient showed a detectable level of
anti-eculizumab antibodies in the eculizumab-treated cohort; this
response was weak (did not titrate), occurred at only one time
point and did not result in a disruption of complement
blockade.
TABLE-US-00004 TABLE 4 Adverse Event Reporting Placebo Eculizumab
Patients Patients n (percent) n (percent) SERIOUS ADVERSE EVENTS*
Total 9 (20.5) 4 (9.3) Eculizumab treatment emergent Exacerbation
of PNH 3 (6.8) 1 (2.3) Renal colic 0 1 (2.3) Lumbar sacral disc
prolapse 0 1 (2.3) Alpha streptococcal bacteremia 0 1 (2.3) Central
line infection and UTI 1 (2.3) 0 (0) Upper respiratory tract
infection 1 (2.3) 0 (0) Probable viral infection 1 (2.3) 0 (0)
Neutropenia 1 (2.3) 0 (0) Cellulitis/folliculitis/ 1 (2.3) 0 (0)
neutropenia Anemia and pyrexia 1 (2.3) 0 (0) MOST FREQUENT ADVERSE
EVENTS*.dagger. Headache.sctn. 12 (27.3) 19.dagger-dbl. (44.2)
Nasopharyngitis 8 (18.2) 10 (23.3) Upper respiratory tract
infection 10 (22.7) 6 (14) Back pain 4 (9.1) 8 (18.6) Nausea 5
(11.4) 7 (16.3) Cough 4 (9.1) 5 (11.6) Diarrhea 5 (11.4) 4 (9.3)
Arthralgia 5 (11.4) 3 (7.0) Abdominal pain 5 (11.4) 2 (4.7)
Dizziness 5 (11.4) 2 (4.7) Vomiting 5 (11.4) 2 (4.7) Fatigue 1
(2.3) 5 (11.6) Viral infection 5 (11.4) 1 (2.3) *By preferred terms
.dagger.Occurring in 10% or more of patients .dagger-dbl.Sixteen of
19 patients experienced headache within 48 hours of infusion
.sctn.Following the first 2 weeks of dosing, 20.9% of eculizumab-
and 22.7% of placebo-treated patients experienced headache
Discussion
[0127] Chronic intravascular hemolysis with periods of acute
exacerbation are the classical manifestations of PNH, frequently
resulting in anemia, the need for transfusions to sustain
hemoglobin levels, and deterioration in quality of life. In the
phase III pivotal TRIUMPH study, we examined the effect of terminal
complement inhibition with eculizumab on hemoglobin levels and
transfusion requirements in patients with PNH. Forty-nine percent
of patients treated with eculizumab over the 6-month period
demonstrated stabilization of hemoglobin in the absence of
transfusions compared to no patients in the placebo arm of the
trial. Over 50% of eculizumab-treated patients were transfusion
independent during the entire study compared to none in the placebo
arm, and the overall mean transfusion rate was reduced by 73%.
Moreover, even in patients who did not achieve transfusion
independence, eculizumab treatment was associated with a 44%
reduction in the rate of transfusion (data now shown).
[0128] Lactate dehydrogenase, a biochemical marker of hemolysis in
PNH,.sup.9 was immediately and consistently decreased in all
eculizumab-treated patients, while patients in the placebo cohort
continued to hemolyze with levels of LDH exceeding 5 times the
upper limit of the normal range in all patients at the study end.
Levels of LDH were reduced into the normal range in approximately
one-third of eculizumab-treated patients, while the remainder
stabilized at a level just above the upper limit of normal
suggesting residual low level hemolysis in some patients. Levels of
haptoglobin, a more sensitive marker of the presence of cell free
hemoglobin in the circulation, were undetectable in most patients.
Low level hemolysis in a subset of eculizumab-treated patients is
possibly due to an inherent decrease in survival of these cells or
C3b-mediated, extravascular clearance of PNH erythrocytes through
the reticuloendothelial system..sup.17
[0129] Before eculizumab treatment, hemoglobin levels in study
patients were artificially maintained by frequent transfusion.
Therefore, stabilization of hemoglobin levels with a concomitant
cessation of or reduction in transfusions represents a net increase
in endogenous hemoglobin levels. Our data suggest that resolution
of hemolysis with eculizumab results in a new steady state
hemoglobin level determined by a balance between the extent of the
underlying bone marrow dysfunction, the number of PNH erythrocytes
that are preserved by eculizumab therapy and the new level (if any)
of transfusion requirement.
[0130] Patients with PNH generally experience markedly impaired
quality of life characterized by fatigue, anemia, thrombosis, and
pulmonary hypertension as well as smooth muscle dystonia including
abdominal pain, dysphagia, and erectile dysfunction..sup.9,10,18
These symptoms have been attributed to both excessive intravascular
hemolysis and downstream scavenging of nitric oxide by cell free
hemoglobin in plasma. The reduction of intravascular hemolysis in
eculizumab-treated patients in the current study was associated
with significant improvements in the fatigue component of quality
of life relative to placebo-treated patients as assessed via the
FACIT-Fatigue instrument. Further, eculizumab therapy was
associated with a median increase of 6.4 points over baseline
values established before treatment. It has previously been
demonstrated that an increase of 3 or more points from baseline
represents a clinically important difference in this quality of
life instrument..sup.19 Patients who received eculizumab also
experienced a significant improvement in most domains of the EORTC
QLQ-30 relative to the placebo-treated cohort including global
health status, physical functioning, emotional functioning,
cognitive functioning, role functioning, social functioning,
fatigue, pain, dyspnea, appetite loss, and insomnia. Improvement in
the fatigue component of the EORTC QLQ-30 provides support for the
improvement demonstrated in the FACIT-Fatigue instrument during
eculizumab therapy. Importantly, these improvements in quality of
life in the eculizumab-treated patients occurred despite similar
levels of erythrocyte hemoglobin in the two treatment groups,
further supporting the contribution of hemolysis per se, as opposed
to anemia, in mediating the poor quality of life in PNH patients.
Clinical assessment of additional life quality-related symptoms of
PNH, including abdominal pain, dysphagia, and erectile dysfunction,
have also been reported to improve during eculizumab
therapy..sup.20
[0131] Eculizumab was safe and well-tolerated. There were no deaths
in the study and only a single thrombotic event which occurred in a
placebo patient in a site (the hepatic veins) which is typical of
the thrombosis in PNH. The relative brief duration of this study
was not sufficient to address the relevant issue of a possible
protection from thrombosis by terminal complement inhibition with
eculizumab.
[0132] Adverse events were generally mild with headache occurring
at increased frequency in the eculizumab-treated patients; however,
this increased frequency did not persist following the first two
doses of therapy. There were 4 SAEs in the eculizumab treatment
group and 9 SAEs in the placebo group. There was no evidence of
increased infection risk in eculizumab-treated patients during the
study period. One eculizumab-treated patient showed a low level of
anti-eculizumab antibodies at one time point during the study which
did not persist and did not result in a disruption of complement
blockade. There were no AEs associated with eculizumab withdrawal
in the 2 eculizumab-treated patients who did not complete the
trial. Additional safety assessments, as well as efficacy measures,
are being examined in an ongoing multi-center, open-label Phase III
safety trial of eculizumab (SHEPHERD) in approximately 95 patients
with PNH.
[0133] Results from the current randomized, double-blind,
placebo-controlled, global study show that terminal complement
inhibition with eculizumab appears to be a safe and effective
therapy for patients with the rare disorder PNH. Treatment with
eculizumab reduced intravascular hemolysis, and stabilized
hemoglobin levels despite a reduction of transfusions, to the point
where most PNH patients were rendered transfusion independent.
Substantial and clinically meaningful improvements in fatigue and
other key quality of life parameters were also demonstrated. All of
the 85 patients who completed the study elected to receive
eculizumab in an open-label extension study and all currently
remain on drug. The results of the TRIUMPH study indicate that
terminal complement inhibition with eculizumab safely and
effectively addresses an important consequence of the underlying
genetic defect in PNH hematopoietic stem cells by providing a
therapeutic replacement for the terminal complement inhibitor
deficiency.
[0134] The present invention provides among other things treatment
with an inhibitor of complement. Many variations of the invention
will become apparent to those skilled in the art upon review of
this specification. The full scope of the invention should be
determined by reference to the claims, along with their full scope
of equivalents, and the specification, along with such
variations.
[0135] All publications and patents mentioned herein, including
those items listed below, are hereby incorporated by reference in
their entirety as if each individual publication or patent was
specifically and individually indicated to be incorporated by
reference. In case of conflict, the present application, including
any definitions herein, will control. [0136] 1. Takeda J, Miyata T,
Kawagoe K et al. Deficiency of the GPI anchor caused by a somatic
mutation of the PIG-A gene in paroxysmal nocturnal hemoglobinuria.
Cell 1993; 73:703-11. [0137] 2. Bessler M, Mason P J, Hillmen P et
al. Paroxysmal nocturnal haemoglobinuria (PNH) is caused by somatic
mutations in the PIG-A gene. EMBO J 1994; 13:110-7. [0138] 3.
Yamashina M, Ueda E, Kinoshita T et al. Inherited complete
deficiency of 20-kilodalton homologous restriction factor (CD59) as
a cause of paroxysmal nocturnal hemoglobinuria. N Engl J Med 1990;
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Paroxysmal nocturnal hemoglobinuria due to hereditary nucleotide
deletion in the HRF20 (CD59) gene. Eur Immunol 1992; 22:2669-73.
[0140] 5. Holguin M H, Fredrick L R, Bernshaw N J, Wilcox L A,
Parker C J. Isolation and characterization of a membrane protein
from normal human erythrocytes that inhibits reactive lysis of the
erythrocytes of paroxysmal nocturnal hemoglobinuria. J Clin Invest
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complement-inhibitory activity of CD59 resides in its capacity to
block incorporation of C9 into membrane C5b-9. J Immunol 1990;
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control of complement on blood platelets. Modulation of platelet
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complex. J Biol Chem 1989; 264:19228-35. [0143] 8. Wiedmer T, Hall
S E, Ortel T L, Kane W H, Rosse W F, Sims P J. Complement-induced
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The clinical sequelae of intravascular hemolysis and extravascular
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hemoglobinuria. Hoffman. New York: Churchill Livingstone, 2000:
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Inhibition of complement activity by humanized antibody that binds
C5 and single-chain Fv. Mol Immunol 1996; 33:1389-401. [0147] 12.
Hillmen P, Hall C, Marsh J C et al. Effect of eculizumab on
hemolysis and transfusion requirements in patients with paroxysmal
nocturnal hemoglobinuria. N Engl J Med 2004; 350:552-9. [0148] 13.
Yellen S B, Cella D F, Webster K, Blendowski C, Kaplan E. Measuring
fatigue and other anemia-related symptoms with the Functional
Assessment of Cancer Therapy (FACT) measurement system. J Pain
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Bergman B et al. The European Organization for Research and
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TABLE-US-00005 [0158] SEQUENCES SEQ ID NO: 1-Eculizumab V.sub.H
QVQLVQSGAEVKKPGASVKVSCKASGYIFSNYWIQWVRQAPGQGLEWMGE
ILPGSGSTEYTENFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARYF
FGSSPNWYFDVWGQGTLVTVSSA SEQ ID NO: 2-Eculizumab Heavy chain
QVQLVQSGAEVKKPGASVKVSCKASGYIFSNYWIQWVRQAPGQGLEWMGE
ILPGSGSTEYTENFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARYF
FGSSPNWYFDVWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCL
VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGT
QTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKD
TLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNST
YRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVY
TLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK SEQ ID NO:
3-Eculizumab V.sub.L
MDMRVPAQLLGLLLLWLRGARCDIQMTQSPSSLSASVGDRVTITCGASEN
IYGALNWYQQKPGKAPKLLIYGATNLADGVPSRFSGSGSGTDFTLTISSL
QPEDFATYYCQNVLNTPLTFGQGTKVEIKRT SEQ ID NO: 4-Eculizumab Light chain
MDMRVPAQLLGLLLLWLRGARCDIQMTQSPSSLSASVGDRVTITCGASEN
IYGALNWYQQKPGKAPKLLIYGATNLADGVPSRFSGSGSGTDFTLTISSL
QPEDFATYYCQNVLNTPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSG
TASVVCLLNNFYFREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST
LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 5-Eculizumab CDRH1
NYWIQ SEQ ID NO: 6-Eculizumab CDRH2 EILPGSGSTEYTENFKD SEQ ID NO:
7-Eculizumab CDRH3 YFFGSSPNWYFDV SEQ ID NO: 8-Eculizumab CDRL1
GASENIYGALN SEQ ID NO: 9-Eculizumab CDRL2 GATNLAD SEQ ID NO:
10-Eculizumab CDRL3 QNVLNTPLT
Sequence CWU 1
1
101123PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 1Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly
Tyr Ile Phe Ser Asn Tyr 20 25 30Trp Ile Gln Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Glu Ile Leu Pro Gly Ser Gly
Ser Thr Glu Tyr Thr Glu Asn Phe 50 55 60Lys Asp Arg Val Thr Met Thr
Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser Ser
Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Tyr Phe
Phe Gly Ser Ser Pro Asn Trp Tyr Phe Asp Val Trp 100 105 110Gly Gln
Gly Thr Leu Val Thr Val Ser Ser Ala 115 1202448PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
2Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5
10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ile Phe Ser Asn
Tyr 20 25 30Trp Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Glu Ile Leu Pro Gly Ser Gly Ser Thr Glu Tyr Thr
Glu Asn Phe 50 55 60Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Thr
Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Tyr Phe Phe Gly Ser Ser Pro
Asn Trp Tyr Phe Asp Val Trp 100 105 110Gly Gln Gly Thr Leu Val Thr
Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125Ser Val Phe Pro Leu
Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr 130 135 140Ala Ala Leu
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr145 150 155
160Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
165 170 175Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val Thr 180 185 190Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr
Cys Asn Val Asp 195 200 205His Lys Pro Ser Asn Thr Lys Val Asp Lys
Thr Val Glu Arg Lys Cys 210 215 220Cys Val Glu Cys Pro Pro Cys Pro
Ala Pro Pro Val Ala Gly Pro Ser225 230 235 240Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro 260 265 270Glu
Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280
285Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val
290 295 300Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
Glu Tyr305 310 315 320Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser
Ser Ile Glu Lys Thr 325 330 335Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu 340 345 350Pro Pro Ser Gln Glu Glu Met
Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp385 390 395
400Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser
405 410 415Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His
Glu Ala 420 425 430Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Leu Gly Lys 435 440 4453131PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 3Met Asp Met Arg Val Pro
Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp1 5 10 15Leu Arg Gly Ala Arg
Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser 20 25 30Leu Ser Ala Ser
Val Gly Asp Arg Val Thr Ile Thr Cys Gly Ala Ser 35 40 45Glu Asn Ile
Tyr Gly Ala Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys 50 55 60Ala Pro
Lys Leu Leu Ile Tyr Gly Ala Thr Asn Leu Ala Asp Gly Val65 70 75
80Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
85 90 95Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln
Asn 100 105 110Val Leu Asn Thr Pro Leu Thr Phe Gly Gln Gly Thr Lys
Val Glu Ile 115 120 125Lys Arg Thr 1304236PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
4Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp1 5
10 15Leu Arg Gly Ala Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser 20 25 30Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Gly
Ala Ser 35 40 45Glu Asn Ile Tyr Gly Ala Leu Asn Trp Tyr Gln Gln Lys
Pro Gly Lys 50 55 60Ala Pro Lys Leu Leu Ile Tyr Gly Ala Thr Asn Leu
Ala Asp Gly Val65 70 75 80Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly
Thr Asp Phe Thr Leu Thr 85 90 95Ile Ser Ser Leu Gln Pro Glu Asp Phe
Ala Thr Tyr Tyr Cys Gln Asn 100 105 110Val Leu Asn Thr Pro Leu Thr
Phe Gly Gln Gly Thr Lys Val Glu Ile 115 120 125Lys Arg Thr Val Ala
Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 130 135 140Glu Gln Leu
Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn145 150 155
160Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
165 170 175Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser
Lys Asp 180 185 190Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser
Lys Ala Asp Tyr 195 200 205Glu Lys His Lys Val Tyr Ala Cys Glu Val
Thr His Gln Gly Leu Ser 210 215 220Ser Pro Val Thr Lys Ser Phe Asn
Arg Gly Glu Cys225 230 23555PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 5Asn Tyr Trp Ile Gln1
5617PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 6Glu Ile Leu Pro Gly Ser Gly Ser Thr Glu Tyr Thr
Glu Asn Phe Lys1 5 10 15Asp713PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 7Tyr Phe Phe Gly Ser Ser Pro
Asn Trp Tyr Phe Asp Val1 5 10811PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 8Gly Ala Ser Glu Asn Ile
Tyr Gly Ala Leu Asn1 5 1097PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 9Gly Ala Thr Asn Leu Ala Asp1
5109PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 10Gln Asn Val Leu Asn Thr Pro Leu Thr1 5
* * * * *
References