U.S. patent application number 14/453268 was filed with the patent office on 2015-03-19 for atypical hemolytic uremic syndrome biomarker proteins.
The applicant listed for this patent is Alexion Pharmaceuticals, Inc., Ryan Kitchel. Invention is credited to Krystin A. BEDARD, Roxanne COFIELL, Anjli KUKREJA, Susan Faas MCKNIGHT, Yan YAN.
Application Number | 20150079613 14/453268 |
Document ID | / |
Family ID | 51358124 |
Filed Date | 2015-03-19 |
United States Patent
Application |
20150079613 |
Kind Code |
A1 |
MCKNIGHT; Susan Faas ; et
al. |
March 19, 2015 |
ATYPICAL HEMOLYTIC UREMIC SYNDROME BIOMARKER PROTEINS
Abstract
The disclosure provides biomarker proteins, a change in the
concentration or activity level of which are associated with
atypical hemolytic uremic syndrome (aHUS) or clinically meaningful
treatment of aHUS with a complement inhibitor. Also provided are
compositions and methods for interrogating the concentration and/or
activity of one or more of the biomarker proteins in a biological
fluid. The compositions and methods are useful for, among other
things, evaluating risk for developing aHUS, diagnosing aHUS,
determining whether a subject is experiencing the first acute
presentation of aHUS, monitoring progression or abatement of aHUS,
and/or monitoring response to treatment with a complement inhibitor
or optimizing such treatment.
Inventors: |
MCKNIGHT; Susan Faas; (Old
Lyme, CT) ; COFIELL; Roxanne; (Glastonbury, CT)
; KUKREJA; Anjli; (Fairfield, CT) ; BEDARD;
Krystin A.; (US) ; YAN; Yan; (Cheshire,
CT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Kitchel; Ryan
Alexion Pharmaceuticals, Inc. |
Cheshire
Cheshire |
CT
CT |
US
US |
|
|
Family ID: |
51358124 |
Appl. No.: |
14/453268 |
Filed: |
August 6, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61913180 |
Dec 6, 2013 |
|
|
|
61863299 |
Aug 7, 2013 |
|
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Current U.S.
Class: |
435/7.92 ;
436/501 |
Current CPC
Class: |
G01N 2333/4716 20130101;
G01N 2333/485 20130101; C07K 16/18 20130101; C07K 2317/54 20130101;
A61P 13/12 20180101; C07K 2317/24 20130101; G01N 2333/7452
20130101; G01N 2333/5421 20130101; G01N 2333/7151 20130101; G01N
2333/70503 20130101; G01N 2333/70525 20130101; G01N 2333/8146
20130101; C07K 2317/76 20130101; G01N 33/6863 20130101; G01N
2333/775 20130101; G01N 2333/70539 20130101; C07K 2317/626
20130101; G01N 2333/5412 20130101; A61P 43/00 20180101; C07K
2317/622 20130101; G01N 2333/974 20130101; G01N 2800/347 20130101;
G01N 33/6893 20130101; G01N 2333/5434 20130101; C07K 2317/21
20130101; A61K 2039/505 20130101; G01N 2333/8139 20130101; G01N
2333/522 20130101; G01N 2333/57 20130101; C07K 2317/55 20130101;
G01N 2333/75 20130101; G01N 2800/226 20130101; G01N 2800/52
20130101; G01N 2800/50 20130101; G01N 2333/545 20130101 |
Class at
Publication: |
435/7.92 ;
436/501 |
International
Class: |
G01N 33/53 20060101
G01N033/53 |
Claims
1. A method for monitoring responsiveness of a subject to treatment
with an inhibitor of complement, the method comprising: determining
the concentration of at least two aHUS-associated biomarker
proteins in a biological fluid obtained from the subject, wherein
the aHUS-associated biomarker proteins are selected from the group
consisting of: CXCL10, MCP-1, TNFR1, IFN-.gamma., IL-6, a
proteolytic fragment of complement component factor B, soluble C5b9
(sC5b9), prothrombin fragment F1+2, D-dimer, thrombomodulin,
VCAM-1, von Willebrand Factor (vWF), complement component C5a,
.beta.2 microglobulin (.beta.2M), clusterin, cystatin C, NAG,
TIMP-1, NGAL, fatty acid binding protein 1 (FABP-1), albumin,
CXCL9, KIM-1, and CCL5, wherein the subject has, is suspected of
having, or is at risk for developing aHUS, wherein the subject has
been or is being treated with an inhibitor of complement, and
wherein: (a) a reduced concentration, as compared to the
concentration in a sample of biological fluid of the same type
obtained from the subject prior to treatment with the inhibitor, of
at least one of CXCL10, MCP-1, TNFR1, IFN-.gamma., IL-6, a
proteolytic fragment of complement component factor B, soluble C5b9
(sC5b9), prothrombin fragment F1+2, D-dimer, thrombomodulin,
VCAM-1, von Willebrand Factor (vWF), complement component C5a,
.beta.2 microglobulin (.beta.2M), clusterin, cystatin C, NAG,
TIMP-1, NGAL, fatty acid binding protein 1 (FABP-1), albumin,
CXCL9, and KIM-1; or (b) an increased concentration, as compared to
the concentration in a sample of biological fluid of the same type
obtained from the subject prior to treatment with the inhibitor, of
CCL5, indicates that the subject is responsive to treatment with
the inhibitor.
2-129. (canceled)
130. A method for diagnosing a subject as having or being at risk
for developing atypical hemolytic uremic syndrome (aHUS), the
method comprising: determining the concentration of at least two
aHUS-associated biomarker proteins in a biological fluid obtained
from a subject, wherein the aHUS-associated biomarker proteins are
selected from the group consisting of: a proteolytic fragment of
complement component factor B, soluble C5b9 (sC5b9),
thrombomodulin, VCAM-1, von Willebrand Factor (vWF), soluble CD40
ligand (sCD40L), prothrombin fragment F1+2, D-dimer, CXCL10, MCP-1,
TNFR1, IFN-.gamma., ICAM-1, IL-1 beta, IL-12 p70, complement
component C5a, .beta.2 microglobulin (.beta.2M), clusterin,
cystatin C, NAG, TIMP-1, NGAL, fatty acid binding protein 1
(FABP-1), CXCL9, KIM-1, IL-18, vascular endothelial cell growth
factor (VEGF), IL-6, albumin, IL-8, and CCL5, wherein an elevated
concentration, as compared to the concentration in a normal control
biological fluid of the same type, of at least one of a proteolytic
fragment of complement component factor B, soluble C5b9 (sC5b9),
thrombomodulin, VCAM-1, von Willebrand Factor (vWF), soluble CD40
ligand (sCD40L), prothrombin fragment F1+2, D-dimer, CXCL10, MCP-1,
TNFR1, IFN-.gamma., ICAM-1, IL-1 beta, IL-12 p70, complement
component C5a, .beta.2 microglobulin (.beta.2M), clusterin,
cystatin C, NAG, TIMP-1, NGAL, fatty acid binding protein 1
(FABP-1), CXCL9, KIM-1, IL-18, vascular endothelial cell growth
factor (VEGF), IL-6, albumin, IL-8, and CCL5, indicates that the
subject has, or is at risk for developing, aHUS.
131-138. (canceled)
139. A method for determining whether a subject is experiencing a
first acute atypical hemolytic uremic syndrome (aHUS)
manifestation, the method comprising: determining one or both of
the concentration of D-dimer and the concentration of fatty acid
binding protein 1 (FABP-1) in a biological fluid from said subject,
wherein i) an elevation in the D-dimer concentration, as compared
to the concentration of D-dimer in a normal control biological
fluid of the same type, ii) an elevation in the FABP-1
concentration, as compared to the concentration of FABP-1 in a
normal control biological fluid of the same type, or iii) both i
and ii indicates that the subject is experiencing a first acute
aHUS manifestation.
140-151. (canceled)
152. A method for diagnosing a patient as having atypical hemolytic
uremic syndrome (aHUS), the method comprising: (i) measuring in a
biological sample obtained from a patient suspected of having aHUS
or at risk of developing aHUS the concentration of each of at least
two aHUS-associated biomarkers selected from the group consisting
of: a proteolytic fragment of factor B, C5a, soluble C5b-9
(sC5b-9), soluble TNFR1 (sTNFR1), soluble VCAM-1 (sVCAM-1),
thrombomodulin, prothrombin fragments 1 and 2 (F1+2), D-dimer,
clusterin, TIMP-1, FABP-1, beta-2 microglobulin (b2m), and
cystatin-C, and (ii) diagnosing a patient as having aHUS if the
concentrations of at least two of the aHUS-associated biomarkers
are elevated as compared to normal control concentrations of the
same at least two biomarkers.
153. The method according to claim 152, wherein the at least two
aHUS-associated biomarker proteins are measured using an
immunoassay.
154. The method of claim 153, wherein the immunoassay is an
enzyme-linked immunosorbent assay (ELISA) or a radioimmunoassay
(RIA).
155. The method of claim 152, wherein the concentrations of at
least three aHUS-associated biomarker proteins are determined.
156. The method of claim 152, wherein the concentrations of at
least five aHUS-associated biomarker proteins are determined.
157. The method of claim 152, wherein the biological fluid is
blood.
158. The method of claim 152, wherein the biological fluid is a
blood fraction.
159. The method according to claim 158, wherein the blood fraction
is plasma or serum.
160. The method of claim 152, wherein the biological fluid is
urine.
161. The method of claim 152, wherein the concentration of at least
one aHUS-associated biomarker is measured in two or more types of
biological fluid.
162. The method of claim 152, wherein the concentration of a first
of the at least two aHUS biomarker proteins is measured in one type
of biological fluid and the concentration of a second of the at
least two aHUS biomarker proteins is measured in a second type of
fluid.
163-292. (canceled)
293. The method of claim 152, wherein the concentrations of two or
more of proteolytic fragment of factor B, C5a, and sC5b-9 are
measured.
294. The method of claim 152, wherein the concentrations of C5a and
sC5b-9 are measured.
295. The method of claim 152, wherein the concentrations of sVCAM-1
and thrombomodulin are measured.
296. The method of claim 152, wherein the concentrations of F1+2
and D-dimer are measured.
297. The method of claim 152, wherein the concentrations of two or
more of clusterin, TIMP-1, .beta.2m, FABP-1, and cystatin-C are
measured.
298. The method of claim 152, wherein the concentrations of at
least ten biomarker proteins are determined.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application Nos. 61/913,180 and 61/863,299, filed Dec. 6, 2013 and
Aug. 7, 2013, respectively. The entire contents of the
aforementioned application and any patents, patent applications,
and references cited throughout this specification are herein
incorporated by reference in their entireties.
TECHNICAL FIELD
[0002] The field of the invention is medicine, immunology,
molecular biology, and protein chemistry.
BACKGROUND
[0003] Hemolytic uremic syndrome (HUS) is characterized by
thrombocytopenia, microangiopathic hemolytic anemia, and acute
renal failure. HUS is classified as one of two types:
diarrheal-associated (D+ HUS; also referred to as shiga toxin
producing E. coli (STEC)-HUS or typical HUS) and non-diarrheal or
atypical HUS (aHUS). D+HUS is the most common form, accounting for
greater than 90% of cases and is caused by a preceding illness with
a shiga-like toxin-producing bacterium, e.g., E. coli O157:H7. aHUS
is rare and has a mortality rate of up to 25%. Many patients with
this disease will sustain permanent neurological or renal
impairment, e.g., at least 50% of aHUS patients progress to
end-stage renal failure (ESRF). See, e.g., Kavanagh et al. (2006)
British Medical Bulletin 77 and 78:5-22.
[0004] aHUS can be genetic, acquired, or idiopathic. Hereditable
forms of aHUS can be associated with mutations in a number of human
complement components including, e.g., complement factor H(CFH),
membrane cofactor protein (MCP), complement factor I (CFI),
C4b-binding protein (C4BP), complement factor B (CFB), and
complement component 3 (C3). See, e.g., Caprioli et al. (2006)
Blood 108:1267-1279. Certain mutations in the gene encoding CD55,
though not yet implicated in aHUS, are associated with the severity
of aHUS. See, e.g., Esparza-Gordillo et al. (2005) Hum Mol Genet
14:703-712.
[0005] Until recently, treatment options for patients with aHUS
were limited and often involved plasma infusion or plasma exchange.
In some cases, aHUS patients undergo uni- or bilateral nephrectomy
or renal transplantation (see Artz et al. (2003) Transplantation
76:821-826). However, recurrence of the disease in treated patients
is common. Recently, treatment of aHUS patients with the drug
Soliris.RTM. was approved in the United States of America and in
Europe. Despite finally having a useful drug for treatment of aHUS
patients, there is still a need to diagnose patients with aHUS, as
well as monitor the progression and abatement of aHUS.
SUMMARY
[0006] The present disclosure provides, among other things, a
variety of proteins whose activity and/or concentration in a
biological fluid is abnormal in patients afflicted with aHUS and/or
those aHUS patients receiving complement inhibitor therapy.
Hereinafter these proteins are referred to as "aHUS-associated
biomarker proteins" or "aHUS biomarker proteins". For example, the
inventors have observed that the concentrations and/or activities
of several proteins in the blood (e.g., serum and/or plasma) and
urine are abnormal in patients with aHUS. The inventors have also
observed that, following administration of an antagonist anti-C5
antibody (eculizumab) to a human, the concentrations of a subset of
these proteins change. In some instances, the concentration of one
or more of the proteins is normalized. While the disclosure is not
bound by any particular theory or mechanism of action, the
inventors believe that monitoring a patient treated with a
complement inhibitor (such as an anti-C5 antibody) for a change in
concentration of one or more of these proteins--aHUS biomarker
proteins--is useful for, e.g., diagnosing a patient as having or at
risk of developing aHUS. Monitoring the status of one or more of
these biomarker proteins can also be useful for determining whether
an aHUS patient is responding to therapy with a complement
inhibitor. Moreover, evaluating the status of one or more of the
biomarkers is also useful for identifying a dose--a threshold
dose--of a complement inhibitor, such as an anti-C5 antibody, that
by virtue of its effect on the concentration of one or more of the
aHUS biomarker proteins in the human is sufficient to achieve a
clinically-meaningful effect on the disease (i.e., sufficient to
treat a complement-associated disease such as aHUS).
[0007] Accordingly, in one aspect, the disclosure features a method
for monitoring or evaluating the status of atypical hemolytic
uremic syndrome (aHUS)-associated biomarker proteins in a subject
(e.g., a mammal such as a human) or a method for assessing one or
both of the concentration and activity level of at least one
atypical hemolytic uremic syndrome (aHUS)-associated biomarker
protein in a subject. The method comprises measuring in a
biological fluid obtained from the subject one or both of (i) the
concentration of at least one (e.g., at least two, three, four,
five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20) aHUS-associated biomarker proteins in the biological fluid,
wherein the aHUS-associated biomarker proteins are any of the
biomarkers set forth in Table 1, e.g., one selected from the group
consisting of: a proteolytic fragment of complement component
factor B (e.g., Ba or Bb), soluble C5b9 (sC5b9), thrombomodulin,
VCAM-1, von Willebrand Factor (vWF), soluble CD40 ligand (sCD40L),
prothrombin fragment F1+2, D-dimer, CXCL10, MCP-1, TNFR1,
IFN-.gamma., ICAM-1, IL-1 beta, IL-12 p70, complement component
C5a, .beta.2 microglobulin (.beta.2M), clusterin, cystatin C, NAG,
TIMP-1, NGAL, fatty acid binding protein 1 (FABP-1), CXCL9, KIM-1,
IL-18, vascular endothelial cell growth factor (VEGF), IL-6,
albumin, IL-8, and CCL5. The subject can be, e.g., a human having,
suspected of having, or at risk for developing, aHUS. The subject
can be one who has been (or is being) treated with an inhibitor of
complement (e.g., an inhibitor of complement component C5 such as
an anti-C5 antibody). The treatment can have occurred less than one
month (e.g., less than 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21,
20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2,
or 1 day) prior to obtaining the sample from the subject. The
method can further include the step of determining whether the
subject has or is at risk of developing aHUS. Where the subject has
been treated or is being treated with a complement inhibitor (e.g.,
an anti-C5 antibody) under a predetermined dosing schedule, the
method can further include determining whether the patient is
responsive (therapeutically) to the complement inhibitor
therapy.
[0008] In another aspect, the disclosure features a method for
monitoring or evaluating the status of atypical hemolytic uremic
syndrome (aHUS)-associated biomarker proteins in a subject (e.g., a
mammal such as a human) or a method for assessing one or both of
the concentration and activity level of at least one atypical
hemolytic uremic syndrome (aHUS)-associated biomarker protein in a
subject. The method comprises: (A) measuring in a biological fluid
obtained from the subject the concentration of at least one (e.g.,
at least two, three, four, five, six, seven, eight, nine, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20) aHUS-associated biomarker
proteins in the biological fluid, wherein the aHUS-associated
biomarker proteins are any of the biomarkers set forth in Table 1,
e.g., one selected from the group consisting of: a proteolytic
fragment of complement component factor B (e.g., Ba or Bb), soluble
C5b9 (sC5b9), thrombomodulin, VCAM-1, von Willebrand Factor (vWF),
soluble CD40 ligand (sCD40L), prothrombin fragment F1+2, D-dimer,
CXCL10, MCP-1, TNFR1, IFN-.gamma., ICAM-1, IL-1 beta, IL-12 p70,
complement component C5a, .beta.2 microglobulin (.beta.2M),
clusterin, cystatin C, NAG, TIMP-1, NGAL, fatty acid binding
protein 1 (FABP-1), CXCL9, KIM-1, IL-18, vascular endothelial cell
growth factor (VEGF), IL-6, albumin, IL-8, and CCL5; and (B)
recording (e.g., in an electronic patient record) the results of
the measurement(s) or communicating the results of the
measurement(s) to the subject, the subject's guardian, or a medical
professional in whose care the subject has been placed. The subject
can be, e.g., a human having, suspected of having, or at risk for
developing, aHUS. The subject can be one who has been (or is being)
treated with an inhibitor of complement (e.g., an inhibitor of
complement component C5 such as an anti-C5 antibody). The treatment
can have occurred less than one month (e.g., less than 31, 30, 29,
28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12,
11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day) prior to obtaining the
sample from the subject. The method can further include the step of
determining whether the subject has or is at risk of developing
aHUS. Where the subject has been treated or is being treated with a
complement inhibitor (e.g., an anti-C5 antibody) under a
predetermined dosing schedule, the method can further include
determining whether the patient is responsive (therapeutically) to
the complement inhibitor therapy.
[0009] In yet another aspect, the disclosure features a method for
monitoring or determining whether a patient is at risk for
developing thrombotic microangiopathy. The method includes (A)
measuring in a biological fluid obtained from the subject the
concentration of at least one (e.g., at least two, three, four)
biomarker protein associated with thrombosis or coagulation in the
biological fluid, wherein the biomarker proteins are any of such
biomarkers set forth in Table 1 or Table 11, e.g., F1+2 or D-dimer;
and (B) recording (e.g., in an electronic patient record) the
results of the measurement(s) or communicating the results of the
measurement(s) to the subject, the subject's guardian, or a medical
professional in whose care the subject has been placed. The subject
can be, e.g., a human having, suspected of having, or at risk for
developing, aHUS. The subject can be one who has been (or is being)
treated with an inhibitor of complement (e.g., an inhibitor of
complement component C5 such as an anti-C5 antibody). The treatment
can have occurred less than one month (e.g., less than 31, 30, 29,
28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12,
11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day) prior to obtaining the
sample from the subject. The method can further include the step of
determining whether the subject has or is at risk of developing
aHUS (or confirming a diagnosis of aHUS) using any of the methods
described herein. Where the subject has been treated or is being
treated with a complement inhibitor (e.g., an anti-C5 antibody)
under a predetermined dosing schedule, the method can further
include determining whether the patient is responsive
(therapeutically) to the complement inhibitor therapy, i.e., a
reduction in the concentration of one or more of the thrombosis or
coagulation-associated biomarkers occurs following treatment with
the complement inhibitor.
[0010] In another aspect, the disclosure features a method for
monitoring or evaluating the status of atypical hemolytic uremic
syndrome (aHUS)-associated biomarker proteins in a subject (e.g., a
mammal such as a human) or a method for assessing one or both of
the concentration and activity level of at least one atypical
hemolytic uremic syndrome (aHUS)-associated biomarker protein in a
subject. The method comprises: (A) measuring in a biological fluid
obtained from the subject the concentration of at least one (e.g.,
at least two, three, four, five, six, seven, eight, nine, 10, 11,
12, or 13) aHUS-associated biomarker proteins in the biological
fluid, wherein the aHUS-associated biomarker proteins are any of
the biomarkers set forth in Table 1, e.g., one selected from the
group consisting of: a proteolytic fragment of complement component
factor B (e.g., Ba or Bb), soluble C5b9 (sC5b9), C5a,
thrombomodulin, VCAM-1, prothrombin fragment F1+2, D-dimer, sTNFR1,
.beta.2 microglobulin (.beta.2M), clusterin, cystatin C, TIMP-1,
and fatty acid binding protein 1 (FABP-1); and (B) recording (e.g.,
in an electronic patient record) the results of the measurement(s)
or communicating the results of the measurement(s) to the subject,
the subject's guardian, or a medical professional in whose care the
subject has been placed. The subject can be, e.g., a human having,
suspected of having, or at risk for developing, aHUS. The subject
can be one who has been (or is being) treated with an inhibitor of
complement (e.g., an inhibitor of complement component C5 such as
an anti-C5 antibody). The treatment can have occurred less than one
month (e.g., less than 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21,
20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2,
or 1 day) prior to obtaining the sample from the subject. The
method can further include the step of determining whether the
subject has or is at risk of developing aHUS. Where the subject has
been treated or is being treated with a complement inhibitor (e.g.,
an anti-C5 antibody) under a predetermined dosing schedule, the
method can further include determining whether the patient is
responsive (therapeutically) to the complement inhibitor
therapy.
[0011] In some embodiments, any of the methods described herein can
further comprise determining whether the subject has or is at risk
for developing aHUS. In some embodiments, an elevated
concentration, as compared to the concentration in a normal control
biological fluid of the same type, of at least one of Ba, sC5b-9,
C5a, sCD40L, prothrombin fragment F1+2, D-dimer, thrombomodulin,
VCAM-1, vWF, FABP-1, .beta.2M, clusterin, cystatin C, TIMP-1,
albumin, NGAL, CXCL10, CXCL9, IL-18, TNFR1, VCAM-1, MCP-1, VEGF,
CCL5, IL-6, IFN.gamma., indicates that the subject has, or is at
risk for developing, aHUS.
[0012] In some embodiments, any of the methods described herein
include determining whether the subject has responded to treatment
with the complement inhibitor. In some embodiments, (a) a reduced
concentration, as compared to the concentration in a sample of
biological fluid of the same type obtained from the subject prior
to treatment with the inhibitor, of at least one of CXCL10, MCP-1,
TNFR1, IFN-.gamma., a proteolytic fragment of complement component
factor B (e.g., Ba or Bb), soluble C5b9 (sC5b9), prothrombin
fragment F1+2, d-dimer, thrombomodulin, VCAM-1, von Willebrand
Factor (vWF), complement component C5a, sC5b9, .beta.2
microglobulin (.beta.2M), clusterin, cystatin C, NAG, TIMP-1, NGAL,
fatty acid binding protein 1 (FABP-1), albumin, CXCL10, CXCL9, and
KIM-1; or (b) an increased concentration, as compared to the
concentration in a sample of biological fluid of the same type
obtained from the subject prior to treatment with the inhibitor, of
CCL5, indicates that the subject is responsive to treatment with
the inhibitor.
[0013] In another aspect, the disclosure features a method for
monitoring responsiveness of a subject (e.g., a mammal such as a
human) to treatment with an inhibitor of complement component C5.
The method includes: measuring the concentration of at least two
aHUS-associated biomarker proteins in a biological fluid, wherein
the aHUS-associated biomarker proteins are any of those set forth
in Table 1, e.g., one selected from the group consisting of: a
proteolytic fragment of complement component factor B (e.g., Ba or
Bb), soluble C5b9 (sC5b9), thrombomodulin, VCAM-1, von Willebrand
Factor (vWF), soluble CD40 ligand (sCD40L), prothrombin fragment
F1+2, D-dimer, CXCL10, MCP-1, TNFR1, IFN-.gamma., ICAM-1, IL-1
beta, IL-12 p70, complement component C5a, .beta.2 microglobulin
(.beta.2M), clusterin, cystatin C, NAG, TIMP-1, NGAL, fatty acid
binding protein 1 (FABP-1), CXCL9, KIM-1, IL-18, vascular
endothelial cell growth factor (VEGF), IL-6, albumin, IL-8, and
CCL5. The biological fluid is obtained from a subject: (i) having,
suspected of having, or at risk for developing, aHUS and (ii) who
is being (or who has been, e.g., recently) treated with an
inhibitor of complement component C5 under a predetermined dosing
schedule. In accordance with such methods, (a) a reduced
concentration, as compared to the concentration in a sample of
biological fluid of the same type obtained from the subject prior
to treatment with the inhibitor, of at least one of CXCL10, MCP-1,
TNFR1, IFN-.gamma., a proteolytic fragment of complement component
factor B (e.g., Ba or Bb), soluble C5b9 (sC5b9), prothrombin
fragment F1+2, d-dimer, thrombomodulin, VCAM-1, von Willebrand
Factor (vWF), complement component C5a, .beta.2 microglobulin
(.beta.2M), clusterin, cystatin C, NAG, TIMP-1, NGAL, fatty acid
binding protein 1 (FABP-1), albumin, CXCL10, CXCL9, and KIM-1; or
(b) an increased concentration, as compared to the concentration in
a sample of biological fluid of the same type obtained from the
subject prior to treatment with the inhibitor, of CCL5, indicates
that the subject is responsive to treatment with the inhibitor.
[0014] In some embodiments, any of the methods described herein
include determining whether the subject has responded to treatment
with the complement inhibitor. In some embodiments, a reduced
concentration, as compared to the concentration in a sample of
biological fluid of the same type obtained from the subject prior
to treatment with the inhibitor, of at least one of a proteolytic
fragment of complement component factor B (e.g., Ba or Bb), soluble
C5b9 (sC5b9), C5a, thrombomodulin, VCAM-1, prothrombin fragment
F1+2, D-dimer, sTNFR1, .beta.2 microglobulin (.beta.2M), clusterin,
cystatin C, TIMP-1, and fatty acid binding protein 1 (FABP-1).
[0015] In another aspect, the disclosure features a method for
monitoring responsiveness of a subject to treatment with an
inhibitor of complement, wherein the method comprises: determining
the concentration of at least two aHUS-associated biomarker
proteins in a biological fluid obtained from the subject, wherein
the aHUS-associated biomarker proteins are selected from the group
consisting of: CXCL10, MCP-1, TNFR1, IFN-.gamma., IL-6, a
proteolytic fragment of complement component factor B (e.g., Ba or
Bb), soluble C5b9 (sC5b9), prothrombin fragment F1+2, d-dimer,
thrombomodulin, VCAM-1, von Willebrand Factor (vWF), complement
component C5a, .beta.2 microglobulin (.beta.2M), clusterin,
cystatin C, NAG, TIMP-1, NGAL, fatty acid binding protein 1
(FABP-1), albumin, CXCL9, KIM-1, and CCL5. The subject has, is
suspected of having, or is at risk for developing aHUS and the
subject has been or is being treated with an inhibitor of
complement. (A) a reduced concentration, as compared to the
concentration in a sample of biological fluid of the same type
obtained from the subject prior to treatment with the inhibitor, of
at least one of CXCL10, MCP-1, TNFR1, IFN-.gamma., IL-6, a
proteolytic fragment of complement component factor B (e.g., Ba or
Bb), soluble C5b9 (sC5b9), prothrombin fragment F1+2, d-dimer,
thrombomodulin, VCAM-1, von Willebrand Factor (vWF), complement
component C5a, .beta.2 microglobulin (.beta.2M), clusterin,
cystatin C, NAG, TIMP-1, NGAL, fatty acid binding protein 1
(FABP-1), albumin, CXCL9, and KIM-1; or (B) an increased
concentration, as compared to the concentration in a sample of
biological fluid of the same type obtained from the subject prior
to treatment with the inhibitor, of CCL5, indicates that the
subject is responsive to treatment with the inhibitor.
[0016] In yet another aspect, the disclosure features a method for
reducing the number, frequency, or occurrence, likelihood of
occurrence, or risk of developing, TMA, using a complement
inhibitor in a manner sufficient to induce a physiological change
in at least two biomarker proteins associated with thrombosis or
coagulation. The method includes: (a) determining the concentration
of at least two biomarker proteins in a biological fluid obtained
from the subject, wherein the biomarker proteins are selected from
Table 1 or 11 and relate to thrombosis and/or coagulation (e.g.,
D-dimer or F1+2); and (b) administering to a subject having,
suspected of having, or at risk for developing, TMA an inhibitor of
complement in an amount and with a frequency sufficient to cause a
physiological change in at least each of two (2) of the biomarker
proteins, wherein the physiological change is a reduction in the
concentration of the at least two biomarker proteins relative to
the concentration of the markers in an equivalent biological sample
obtained from the subject prior to treatment with the complement
inhibitor. The method can include both measuring the concentration
of the biomarkers before and after treatment.
[0017] In yet another aspect, the disclosure features a method for
determining whether an aHUS patient treated with a complement
inhibitor under a predetermined dosing schedule is in need of: (i)
treatment with a different complement inhibitor or (ii) treatment
with the same complement inhibitor under a different dosing
schedule. The method comprises: (A) determining whether the aHUS
patient is responsive to treatment with the complement inhibitor
under the predetermined dosing schedule, wherein the determining
comprises: measuring in a biological fluid obtained from the
subject one or both of the concentration and activity of at least
two aHUS-associated biomarker proteins in the biological fluid,
wherein the aHUS-associated biomarker proteins are selected from
the group consisting of: a proteolytic fragment of complement
component factor B (e.g., Ba or Bb), soluble C5b9 (sC5b9),
thrombomodulin, VCAM-1, von Willebrand Factor (vWF), soluble CD40
ligand (sCD40L), prothrombin fragment F1+2, D-dimer, CXCL10, MCP-1,
TNFR1, IFN-.gamma., ICAM-1, IL-1 beta, IL-12 p70, complement
component C5a, .beta.2 microglobulin (.beta.2M), clusterin,
cystatin C, NAG, TIMP-1, NGAL, fatty acid binding protein 1
(FABP-1), CXCL9, KIM-1, IL-18, vascular endothelial cell growth
factor (VEGF), IL-6, albumin, IL-8, and CCL5, and wherein: (a) a
reduced concentration, as compared to the concentration in a sample
of biological fluid of the same type obtained from the subject
prior to treatment with the inhibitor, of at least one of CXCL10,
MCP-1, TNFR1, IFN-.gamma., a proteolytic fragment of complement
component factor B (e.g., Ba or Bb), soluble C5b9 (sC5b9),
prothrombin fragment F1+2, d-dimer, thrombomodulin, VCAM-1, von
Willebrand Factor (vWF), complement component C5a, sC5b9, .beta.2
microglobulin (.beta.2M), clusterin, cystatin C, NAG, TIMP-1, NGAL,
fatty acid binding protein 1 (FABP-1), albumin, CXCL10, CXCL9, and
KIM-1; or (b) an increased concentration, as compared to the
concentration in a sample of biological fluid of the same type
obtained from the subject prior to treatment with the inhibitor, of
CCL5, indicates that the subject is responsive to treatment with
the inhibitor; and (B) if the patient is not responsive to
treatment with the complement inhibitor, administering the patient
a different complement inhibitor or the same complement inhibitor
at a higher dose or more frequent dosing schedule as compared to
the predetermined dosing schedule.
[0018] In yet another aspect, the disclosure features a method for
determining whether an aHUS patient treated with a complement
inhibitor under a predetermined dosing schedule is in need of: (i)
treatment with a different complement inhibitor or (ii) treatment
with the same complement inhibitor under a different dosing
schedule. The method comprises: (A) determining whether the aHUS
patient is responsive to treatment with the complement inhibitor
under the predetermined dosing schedule, wherein the determining
comprises: measuring in a biological fluid obtained from the
subject one or both of the concentration and activity of at least
two aHUS-associated biomarker proteins in the biological fluid,
wherein the aHUS-associated biomarker proteins are selected from
the group consisting of: a proteolytic fragment of complement
component factor B (e.g., Ba or Bb), soluble C5b9 (sC5b9), C5a,
thrombomodulin, VCAM-1, prothrombin fragment F1+2, D-dimer, sTNFR1,
.beta.2 microglobulin (.beta.2M), clusterin, cystatin C, TIMP-1,
and fatty acid binding protein 1 (FABP-1), and wherein: (a) a
reduced concentration, as compared to the concentration in a sample
of biological fluid of the same type obtained from the subject
prior to treatment with the inhibitor, of at least one of a
proteolytic fragment of complement component factor B (e.g., Ba or
Bb), soluble C5b9 (sC5b9), C5a, thrombomodulin, VCAM-1, prothrombin
fragment F1+2, D-dimer, sTNFR1, .beta.2 microglobulin (.beta.2M),
clusterin, cystatin C, TIMP-1, and fatty acid binding protein 1
(FABP-1) indicates that the subject is responsive to treatment with
the inhibitor; and (B) if the patient is not responsive to
treatment with the complement inhibitor, administering the patient
a different complement inhibitor or the same complement inhibitor
at a higher dose or more frequent dosing schedule as compared to
the predetermined dosing schedule.
[0019] The concentration of one or more of the proteins can be
measured using, e.g., an immunoassay (e.g., enzyme linked
immunosorbent assay (ELISA), a radioimmunoassay (RIA), Western
blotting, or dot blotting) or cytometric bead array (CBA; see the
working examples). Such methods as well as kits useful for
performing the methods are described herein. Suitable methods for
measuring the activity of vWF are known in the art and described
herein.
[0020] In some embodiments of any of the methods described herein,
the concentrations of at least five individual aHUS-associated
biomarker proteins are measured. In some embodiments of any of the
methods described herein, the concentrations of at least ten
individual aHUS-associated biomarker proteins are measured. In some
embodiments of any of the methods described herein, the
concentrations of at least 15 individual aHUS-associated biomarker
proteins are measured. In some embodiments of any of the methods
described herein, the concentrations of at least 20 individual
aHUS-associated biomarker proteins are measured.
[0021] In some embodiments of any of the methods described herein,
the biological fluid is blood. In some embodiments, the biological
fluid is a blood fraction, e.g., serum or plasma. In some
embodiments, the biological fluid is urine. In some embodiments of
any of the methods described herein, all of the measurements are
performed on one biological fluid. In some embodiments of any of
the methods described herein, measurements are performed on at
least two different biological fluids obtained from the subject. In
some embodiments, the concentrations of at least two individual
aHUS-associated biomarker proteins are measured and the
concentration of the first aHUS-associated biomarker protein is
measured in one type of biological fluid and the second
aHUS-associated biomarker protein is measured in a second type of
biological fluid.
[0022] In some embodiments of any of the methods described herein,
the concentrations of at least two (e.g., at least three, four, or
all) of IFN-.gamma., ICAM-1, IL-1 beta, and IL-12 p70 are measured.
In some embodiments of any of the methods described herein, the
concentrations of both Ba and sC5b9 are measured. In some
embodiments of any of the methods described herein, the
concentrations of one or both of C5a and C5b9 are measured. In some
embodiments of any of the methods described herein, the
concentrations of at least two (e.g., at least three, four, five,
six, or all) of .beta.2M, clusterin, cystatin C, NAG, TIMP-1, NGAL,
and FABP-1 are measured. In some embodiments of any of the methods
described herein, the concentrations of CXCL10, CXCL9, and/or KIM-1
are measured. In some embodiments of any of the methods described
herein, the concentrations of one or both of D-dimer and F1+2 are
measured. In some embodiments of any of the methods described
herein, the concentrations of at least two (e.g., at least three,
four, or all) of sCD40L, prothrombin fragment F1+2, and D-dimer,
are measured. In some embodiments of any of the methods described
herein, the concentrations of thrombomodulin, VCAM-1, and/or vWF
are measured. In some embodiments of any of the methods described
herein, the concentrations of CXCL10, MCP-1, and/or TNFR1 are
measured. In some embodiments of any of the methods described
herein, the concentrations of at least two (e.g., at least three,
four, or all) of IFN-.gamma., ICAM-1, IL-1 beta, and IL-12 p70 are
measured.
[0023] In some embodiments of any of the methods described herein,
the concentrations of one or more of CXCL9, CXCL10, IL-1 beta,
IL-12 p70, IFN-.gamma., MCP-1, CCL5, sCD40L, and/or sTNFR1 is
measured in the serum of the subject. In some embodiments, the
concentrations of one or more of complement component C5a, sC5b9,
.beta.2 microglobulin (.beta.2M), clusterin, cystatin C, NAG,
TIMP-1, NGAL, fatty acid binding protein 1 (FABP-1), CXCL10, CXCL9,
and/or KIM-1 are measured in the urine of the subject. In some
embodiments of any of the methods described herein, the
concentrations of one or more of NGAL, a proteolytic fragment of
complement component factor B (e.g., Ba or Bb), soluble C5b9
(sC5b9), prothrombin fragment F1+2, D-dimer, thrombomodulin, and/or
von Willebrand Factor (vWF) are measured in the plasma of the
subject.
[0024] In some embodiments, the concentrations of two or more
(e.g., three, four, five, six, seven, eight, nine, 10, 11, 12, or
13) of a proteolytic fragment of complement component factor B
(e.g., Ba or Bb), soluble C5b9 (sC5b9), C5a, thrombomodulin,
VCAM-1, prothrombin fragment F1+2, D-dimer, sTNFR1, .beta.2
microglobulin (.beta.2M), clusterin, cystatin C, TIMP-1, and fatty
acid binding protein 1 (FABP-1) are measured.
[0025] In some embodiments of any of the methods described herein,
the concentration of at least two of the group consisting of Ba,
sC5b-9, and C5a is measured. In some embodiments of any of the
methods described herein, the concentration of one or both of Ba
and sC5b9 is measured. In some embodiments of any of the methods
described herein, the concentration of one or both of C5a and C5b9
are measured. In some embodiments of any of the methods described
herein, the concentrations of at least two individual members of
the group consisting of .beta.2M, clusterin, cystatin C, albumin,
TIMP-1, NGAL, and FABP-1 are measured. In some embodiments of any
of the methods described herein, the concentrations of at least two
individual members of the group consisting of CXCL10, CXCL9, IL-18,
MCP-1, TNFR1, VEGF, IL-6, and IFN.gamma. are measured. In some
embodiments of any of the methods described herein, the
concentration of one or both of d-dimer and F1+2 is measured. In
some embodiments of any of the methods described herein, the
concentrations of at least two individual members of the group
consisting of sCD40L, prothrombin fragment F1+2, and d-dimer are
measured. In some embodiments of any of the methods described
herein, the concentration of thrombomodulin, VCAM-1, or vWF is
measured. In some embodiments of any of the methods described
herein, the concentration of TNFR1 is measured. In some embodiments
of any of the methods described herein, the concentrations of at
least two individual members of the group consisting of
IFN-.gamma., CXCL10, CXCL9, IL-18, TNFR1, VCAM-1, MCP-1, VEGF,
CCL5, and IL-6 are measured. In some embodiments of any of the
methods described herein, the concentration of at least one
aHUS-associated biomarker protein selected from the group
consisting of IFN-.gamma., CXCL10, CXCL9, IL-18, TNFR1, VCAM-1,
MCP-1, VEGF, and IL-6 is measured. In some embodiments of any of
the methods described herein, the concentration of at least one
aHUS-associated biomarker selected from the group consisting of
.beta.2 microglobulin (.beta.2M), clusterin, cystatin C, NAG,
TIMP-1, NGAL, fatty acid binding protein 1 (FABP-1), CXCL10, CXCL9,
albumin, and KIM-1 is measured. In some embodiments of any of the
methods described herein, the concentration of at least one
aHUS-associated biomarker protein selected from the group
consisting of: CXCL10, CXCL9, IL-18, MCP-1, TNFR1, VEGF, IL-6,
CCL5, IFN.gamma., IL-8, ICAM-1, IL-1 beta, and IL-12 p70 is
measured. In some embodiments of any of the methods described
herein, the concentration of CXCL9, CXCL10, IL-1 beta, IL-12 p70,
IFN-.gamma., MCP-1, CCL5, sCD40L, or sTNFR1 is measured in the
serum of the subject. In some embodiments of any of the methods
described herein, the concentration of at least one aHUS-associated
biomarker selected from the group consisting of .beta.2
microglobulin (.beta.2M), clusterin, cystatin C, NAG, TIMP-1, NGAL,
fatty acid binding protein 1 (FABP-1), CXCL10, CXCL9, albumin, and
KIM-1 is measured in the urine of the subject. In some embodiments
of any of the methods described herein, the concentration of NGAL,
a proteolytic fragment of complement component factor B (e.g., Ba
or Bb), soluble C5b9 (sC5b9), prothrombin fragment F1+2, D-dimer,
thrombomodulin, or von Willebrand Factor (vWF) is measured in the
plasma of the subject. In some embodiments of any of the methods
described herein, the concentration of Ba is measured (e.g., in the
plasma sample obtained from the subject).
[0026] In some embodiments of any of the methods described herein,
the method requires recording the measured value(s) of the
concentration of the at least one aHUS biomarker protein. The
recordation can be written or on a computer readable medium. The
method can also include communicating the measured value(s) of the
concentration of the at least one aHUS biomarker protein to the
subject and/or to a medical practitioner in whose care the subject
is placed.
[0027] In some embodiments, any of the methods described herein can
include the step of administering to the subject the complement
inhibitor at a higher dose or with an increased frequency of
dosing, relative to the predetermined dosing schedule, if the
subject is not responsive to treatment with the inhibitor under the
predetermined dosing schedule.
[0028] In some embodiments of any of the methods described herein,
the complement inhibitor is administered to the subject under a
predetermined dosing schedule based, in part, on the body weight of
the subject. For example, in the case of an antagonist anti-C5
antibody (e.g., eculizumab), for subjects having a body weight
greater than or equal to 40 kg, the antibody can be administered to
the subject for at least 7 weeks under the following schedule: at
least 800 mg of the antibody, once per week for four consecutive
weeks; at least 800 mg of the antibody once during the fifth week;
and at least 800 mg of the antibody bi-weekly thereafter. In some
embodiments, the antibody is administered to the subject for at
least 7 weeks under the following schedule: at least 900 mg of the
antibody, once per week for four consecutive weeks; at least 1200
mg of the antibody once during the fifth week; and at least 1200 mg
of the antibody bi-weekly thereafter.
[0029] In some embodiments of any of the methods described herein,
for subjects having a body weight less than 40 kg but greater than
or equal to 30 kg, the antibody can administered to the subject for
at least 7 weeks under the following schedule: at least 500 mg of
the antibody, once per week for two consecutive weeks; at least 700
mg of the antibody once during the third week; and at least 700 mg
of the antibody bi-weekly thereafter. In some embodiments, the
antibody is administered to the subject for at least 5 weeks under
the following schedule: at least 600 mg of the antibody, once per
week for two consecutive weeks; at least 900 mg of the antibody
once during the third week; and at least 900 mg of the antibody
bi-weekly thereafter.
[0030] In some embodiments of any of the methods described herein,
the body weight of the subject is less than 30 kg, but is greater
than or equal to 20 kg and the antibody is administered to the
subject for at least 5 weeks under the following schedule: at least
500 mg of the antibody, once per week for two consecutive weeks; at
least 500 mg of the antibody once during the third week; and at
least 500 mg of the antibody bi-weekly thereafter. In some
embodiments, the antibody is administered to the subject for at
least 5 weeks under the following schedule: at least 600 mg of the
antibody, once per week for two consecutive weeks; at least 600 mg
of the antibody once during the third week; and at least 600 mg of
the antibody bi-weekly thereafter.
[0031] In some embodiments of any of the methods described herein,
the body weight of the subject is less than 20 kg, but is greater
than or equal to 10 kg and the antibody is administered to the
subject for at least 4 weeks under the following schedule: at least
500 mg of the antibody once a week for one week; at least 200 mg of
the antibody once during the second week; and at least 200 mg of
the antibody bi-weekly thereafter. In some embodiments, the
antibody is administered to the subject for at least 4 weeks under
the following schedule: at least 600 mg of the antibody once a week
for one week; at least 300 mg of the antibody once during the
second week; and at least 300 mg of the antibody bi-weekly
thereafter.
[0032] In some embodiments of any of the methods described herein,
the body weight of the subject is less than 10 kg, but is greater
than or equal to 5 kg and the antibody is administered to the
subject for at least 5 weeks under the following schedule: at least
200 mg of the antibody, once per week for one week; at least 200 mg
of the antibody once during the second week; and at least 200 mg of
the antibody once every three weeks thereafter. In some
embodiments, the antibody is administered to the subject for at
least 5 weeks under the following schedule: at least 300 mg of the
antibody, once per week for one week; at least 300 mg of the
antibody once during the second week; and at least 300 mg of the
antibody every three weeks thereafter. Additional exemplary anti-C5
antibody dosing schedules (e.g., chronic dosing schedules) for aHUS
are described in International patent application publication no.
WO 2010/054403 (e.g., Tables 1 and 2 of WO 2010/054403), the
disclosure of which is incorporated herein by reference in its
entirety.
[0033] In some embodiments of any of the methods described herein,
the inhibitor is antibody or an antigen binding fragment thereof, a
small molecule, a polypeptide, a polypeptide analog, a
peptidomimetic, or an aptamer. In some embodiments, the inhibitor
can be one that inhibits one or more of complement components C1,
C2, C3, C4, C5, C6, C7, C8, C9, Factor D, Factor B, properdin, MBL,
MASP-1, MASP-2, or biologically active fragments of any of the
foregoing. In some embodiments of any of the methods described
herein, the complement inhibitor inhibits one or both of the
generation of the anaphylatoxic activity associated with C5a and/or
the assembly of the membrane attack complex associated with
C5b.
[0034] The compositions can also contain 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.
[0035] In some embodiments, the complement inhibitor can be a
complement receptor 2 (CR2)-factor H (FH) molecule comprising: a) a
CR2 portion comprising CR2 (e.g., human CR2) or a fragment thereof,
and b) a FH portion comprising a FH or a fragment thereof, wherein
the CR2-FH molecule or fragment thereof is capable of binding to a
CR2 ligand, and wherein the CR2-FH molecule is capable of
inhibiting complement activation of the alternative pathway.
Exemplary CR2-FH fusion proteins are described and exemplified in,
e.g., International patent application publication nos. WO
2007/149567 and WO 2011/143637, the disclosures of each of which
are incorporated herein by reference in their entirety. In some
embodiments, the complement inhibitor comprises a targeting domain
such as CR2 or an anti-C3d antibody as described in, e.g.,
International patent application publication no. WO 2011/163412,
the disclosure of which is incorporated herein by reference in its
entirety. Fusions of targeting domains with other complement
inhibitors such as CD59, CD55, and factor H-like molecules can be
used in the methods described herein as a complement inhibitor. See
WO 2011/163412, above.
[0036] In some embodiments of any of the methods described herein,
the inhibitor of complement is an antagonist antibody or
antigen-binding fragment thereof. The antibody or antigen-binding
fragment thereof can be selected from the group consisting of a
humanized antibody, a recombinant antibody, a diabody, a chimerized
or chimeric antibody, a monoclonal antibody, a deimmunized
antibody, a fully human antibody, a single chain antibody, an Fv
fragment, an Fd fragment, an Fab fragment, an Fab' fragment, and an
F(ab').sub.2 fragment.
[0037] In some embodiments of any of the methods described herein,
the antagonist antibody is an anti-C5 antibody such as eculizumab.
In some embodiments, the antagonist antibody is pexelizumab, a
C5-binding fragment of anti-C5 antibody.
[0038] In some embodiments of any of the methods described herein,
the inhibitor of complement is selected from the group consisting
of MB 12/22, MB12/22-RGD, ARC 187, ARC1905, SSL7, and OmCI.
[0039] In some embodiments of any of the methods described herein,
the subset of aHUS-associated biomarker proteins from which a
practitioner may determine the concentration of one or more (e.g.,
two, three, four, five, six, seven, eight, nine, 10, or more) of
can be: Ba, thrombomodulin, VCAM-1, TNFR1, F1+2, D-dimer, CXCL10,
IL-6, clusterin, TIMP-1, FABP-1, .beta.2M, and cystatin C.
[0040] In yet another aspect, the disclosure features an array
comprising a plurality of binding agents, wherein each binding
agent of the plurality has a unique address on the array, wherein
the array comprises no more than 500 unique addresses, wherein each
binding agent of the plurality binds to a different biological
analyte protein, and wherein the array comprises binding agents
that bind to four or more analyte proteins set forth in Table 1,
e.g., selected from the group consisting of: a proteolytic fragment
of complement component factor B (e.g., Ba or Bb), soluble C5b9
(sC5b9), thrombomodulin, VCAM-1, von Willebrand Factor (vWF),
soluble CD40 ligand (sCD40L), prothrombin fragment F1+2, D-dimer,
CXCL10, MCP-1, TNFR1, IFN-.gamma., ICAM-1, IL-1 beta, IL-12 p70,
complement component C5a, .beta.2 microglobulin (.beta.2M),
clusterin, cystatin C, NAG, TIMP-1, NGAL, fatty acid binding
protein 1 (FABP-1), CXCL9, KIM-1, IL-18, vascular endothelial cell
growth factor (VEGF), IL-6, albumin, IL-8, and CCL5. The array is
useful in any of the methods described herein. In some embodiments,
the array is a protein chip. In some embodiments, each address of
the array is a well of an assay plate. In some embodiments, each
address of the array is a particle (e.g., a bead) having
immobilized thereupon a binding agent.
[0041] As used herein, the term "binding agent" includes any
naturally occurring, synthetic or genetically engineered agent,
such as protein, that binds an antigen (e.g., an aHUS biomarker
protein). Binding agents can be or be derived from
naturally-occurring antibodies. A binding protein or agent can
function similarly to an antibody by binding to a specific antigen
to form a complex. Binding agents or proteins can include isolated
antigen-binding fragments of antibodies.
[0042] In some embodiments, the array comprises antibodies that
bind to at least two (e.g., at least three, four, five, six, seven,
eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, or 25) of the analyte proteins. For example, the array can
comprise binding agents/antibodies that bind to at least two (e.g.,
three, four, five, six, seven, eight, nine, 10, 11, 12, or 13) of a
proteolytic fragment of complement component factor B (e.g., Ba or
Bb), soluble C5b9 (sC5b9), C5a, thrombomodulin, VCAM-1, prothrombin
fragment F1+2, D-dimer, sTNFR1, .beta.2 microglobulin (.beta.2M),
clusterin, cystatin C, TIMP-1, and fatty acid binding protein 1
(FABP-1).
[0043] In some embodiments, the array comprises no more than 200
(e.g., no more than 175, 150, 125, 100, 90, 80, 70, 60, 50, 45, 40,
35, 30, 25, or 20) unique addresses.
[0044] In yet another aspect, the disclosure features a diagnostic
kit comprising one or more of any of the arrays described herein
and, optionally, instructions for (a) obtaining and/or processing a
biological sample (e.g., a biological fluid) from a subject and/or
(b) measuring one or more analytes in a biological sample (e.g., a
biological fluid) from a subject.
[0045] In another aspect, the disclosure features a diagnostic kit
comprising: (a) an assay plate and (b) at least three binding
agents, each binding agent capable of binding to a different
biological analyte, wherein the analytes are those depicted in
Table 1, e.g., selected from the group consisting of: a proteolytic
fragment of complement component factor B (e.g., Ba or Bb), soluble
C5b9 (sC5b9), thrombomodulin, VCAM-1, von Willebrand Factor (vWF),
soluble CD40 ligand (sCD40L), prothrombin fragment F1+2, D-dimer,
CXCL10, MCP-1, TNFR1, IFN-.gamma., ICAM-1, IL-1 beta, IL-12 p70,
complement component C5a, .beta.2 microglobulin (.beta.2M),
clusterin, cystatin C, NAG, TIMP-1, NGAL, fatty acid binding
protein 1 (FABP-1), CXCL9, KIM-1, IL-18, vascular endothelial cell
growth factor (VEGF), IL-6, albumin, IL-8, and CCL5. In some
embodiments, the diagnostic kit comprises one or more means for
measuring the activity of vWF in human plasma.
[0046] In another aspect, the disclosure features a method for
diagnosing a subject as having, or being at risk for developing,
atypical hemolytic uremic syndrome (aHUS). The method includes:
measuring in a biological fluid the concentration of at least two
aHUS-associated biomarker proteins selected from the group
consisting of: a proteolytic fragment of complement component
factor B (e.g., Ba or Bb), soluble C5b9 (sC5b9), thrombomodulin,
VCAM-1, von Willebrand Factor (vWF), soluble CD40 ligand (sCD40L),
prothrombin fragment F1+2, D-dimer, CXCL10, MCP-1, TNFR1,
IFN-.gamma., ICAM-1, IL-1 beta, IL-12 p70, complement component
C5a, .beta.2 microglobulin (.beta.2M), clusterin, cystatin C, NAG,
TIMP-1, NGAL, fatty acid binding protein 1 (FABP-1), CXCL9, KIM-1,
IL-18, vascular endothelial cell growth factor (VEGF), IL-6,
albumin, IL-8, and CCL5. The biological fluid is one obtained from
a subject suspected of having or at risk for developing aHUS. In
accordance with the methods, an elevated concentration, as compared
to the concentration in a normal control biological fluid of the
same type, of at least one of Ba, sC5b-9, C5a, sCD40L, prothrombin
fragment F1+2, d-dimer, thrombomodulin, VCAM-1, vWF, FABP-1,
.beta.2M, clusterin, cystatin C, TIMP-1, albumin, NGAL, CXCL10,
CXCL9, IL-18, TNFR1, VCAM-1, MCP-1, VEGF, CCL5, IL-6, or
IFN.gamma., indicates that the subject has, or is at risk for
developing, aHUS. In some embodiments, the at least two
aHUS-associated biomarkers can be selected from Table 11, i.e., at
least two (e.g., three, four, five, six, seven, eight, nine, 10,
11, 12, or 13) of a proteolytic fragment of complement component
factor B (e.g., Ba or Bb), soluble C5b9 (sC5b9), C5a,
thrombomodulin, VCAM-1, prothrombin fragment F1+2, D-dimer, sTNFR1,
.beta.2 microglobulin (.beta.2M), clusterin, cystatin C, TIMP-1,
and fatty acid binding protein 1 (FABP-1).
[0047] As used herein, the term "normal," when used to modify the
term "individual" or "subject" refers to an individual or group of
individuals who does/do not have a particular disease or condition
(e.g., aHUS) and is also not suspected of having or being at risk
for developing the disease or condition. The term "normal" is also
used herein to qualify a biological specimen or sample (e.g., a
biological fluid) isolated from a normal or healthy individual or
subject (or group of such subjects), for example, a "normal control
sample" or "normal control biological fluid".
[0048] In yet another aspect, the disclosure features a method for
determining whether a patient is experiencing a first acute
atypical hemolytic uremic syndrome (aHUS) manifestation. The method
comprises: measuring one or both of the concentration of D-dimer
(e.g., the plasma concentration of d-dimer) and the concentration
of fatty acid binding protein 1 (FABP-1) (e.g., the urine
concentration of FABP-1), wherein an elevation in the d-dimer
concentration, relative to the concentration of d-dimer in a normal
control sample, and an elevation in the FABP-1 concentration,
relative to the concentration of FABP-1 in a normal control sample,
indicates that the aHUS patient is experiencing a first acute aHUS
manifestation. In some embodiments, the elevation of one or both of
d-dimer and FABP-1 can be significant elevations.
[0049] In another aspect, the disclosure features a method for
treating atypical hemolytic uremic syndrome (aHUS), the method
comprising administering to a subject having, suspected of having,
or at risk for developing, aHUS an inhibitor of complement (e.g.,
an inhibitor of complement component C5) in an amount and with a
frequency sufficient to effect a physiological change in at least
one (e.g., at least two, three, four, five, six, seven, eight,
nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,
or 25) aHUS-associated biomarker proteins, wherein the
physiological change is selected from the group consisting of: (a)
a reduced concentration, as compared to the concentration in a
sample of biological fluid of the same type obtained from the
subject prior to treatment with the inhibitor, of at least one of
CXCL10, MCP-1, TNFR1, IFN-.gamma., a proteolytic fragment of
complement component factor B (e.g., Ba or Bb), soluble C5b9
(sC5b9), prothrombin fragment F1+2, d-dimer, thrombomodulin,
VCAM-1, von Willebrand Factor (vWF), complement component C5a,
sC5b9, .beta.2 microglobulin (.beta.2M), clusterin, cystatin C,
NAG, TIMP-1, NGAL, fatty acid binding protein 1 (FABP-1), albumin,
CXCL10, CXCL9, and KIM-1; or (b) an increased concentration, as
compared to the concentration in a sample of biological fluid of
the same type obtained from the subject prior to treatment with the
inhibitor, of CCL5. In some embodiments, the at least one
aHUS-associated biomarker can be selected from Table 11, i.e., at
least one (e.g., two, three, four, five, six, seven, eight, nine,
10, 11, 12, or 13) of a proteolytic fragment of complement
component factor B (e.g., Ba or Bb), soluble C5b9 (sC5b9), C5a,
thrombomodulin, VCAM-1, prothrombin fragment F1+2, D-dimer, sTNFR1,
.beta.2 microglobulin (.beta.2M), clusterin, cystatin C, TIMP-1,
and fatty acid binding protein 1 (FABP-1).
[0050] In yet another aspect, the disclosure features a method for
treating atypical hemolytic uremic syndrome (aHUS) using a
complement inhibitor in a manner sufficient to induce a
physiological change in at least two aHUS-associated biomarker
proteins. The method includes: (a) determining the concentration of
at least two aHUS-associated biomarker proteins in a biological
fluid obtained from the subject, wherein the aHUS-associated
biomarker proteins are selected from the group consisting of:
CXCL10, MCP-1, TNFR1, IFN-.gamma., IL-6, a proteolytic fragment of
complement component factor B (e.g., Ba or Bb), soluble C5b9
(sC5b9), prothrombin fragment F1+2, d-dimer, thrombomodulin,
VCAM-1, von Willebrand Factor (vWF), complement component C5a,
.beta.2 microglobulin (.beta.2M), clusterin, cystatin C, NAG,
TIMP-1, NGAL, fatty acid binding protein 1 (FABP-1), albumin,
CXCL9, KIM-1, and CCL5; and (b) administering to a subject having,
suspected of having, or at risk for developing, aHUS an inhibitor
of complement in an amount and with a frequency sufficient to cause
a physiological change in at least each of two (2) aHUS-associated
biomarker proteins, wherein the physiological change is selected
from the group consisting of: (a) a reduced concentration, as
compared to the concentration in a sample of biological fluid of
the same type obtained from the subject prior to treatment with the
inhibitor, of at least one of CXCL10, MCP-1, TNFR1, IFN-.gamma.,
IL-6, a proteolytic fragment of complement component factor B
(e.g., Ba or Bb), soluble C5b9 (sC5b9), prothrombin fragment F1+2,
d-dimer, thrombomodulin, VCAM-1, von Willebrand Factor (vWF),
complement component C5a, .beta.2 microglobulin (.beta.2M),
clusterin, cystatin C, NAG, TIMP-1, NGAL, fatty acid binding
protein 1 (FABP-1), albumin, CXCL9, or KIM-1; and (b) an increased
concentration in a biological fluid of obtained from the subject,
as compared to the concentration in a sample of biological fluid of
the same type obtained from the subject prior to treatment with the
inhibitor, of CCL5. The method can also include determining whether
the physiological changes occurred. In some embodiments, the at
least two aHUS-associated biomarkers can be selected from Table 11,
i.e., at least two (e.g., three, four, five, six, seven, eight,
nine, 10, 11, 12, or 13) of a proteolytic fragment of complement
component factor B (e.g., Ba or Bb), soluble C5b9 (sC5b9), C5a,
thrombomodulin, VCAM-1, prothrombin fragment F1+2, D-dimer, sTNFR1,
.beta.2 microglobulin (.beta.2M), clusterin, cystatin C, TIMP-1,
and fatty acid binding protein 1 (FABP-1).
[0051] In some embodiments, the methods can further include the
step of measuring the concentrations of at least two individual
aHUS-associated biomarker proteins in a biological fluid, wherein
the aHUS-associated biomarker proteins are selected from the group
consisting of: a proteolytic fragment of complement component
factor B (e.g., Ba or Bb), soluble C5b9 (sC5b9), thrombomodulin,
VCAM-1, von Willebrand Factor (vWF), soluble CD40 ligand (sCD40L),
prothrombin fragment F1+2, D-dimer, CXCL10, MCP-1, TNFR1,
IFN-.gamma., ICAM-1, IL-1 beta, IL-12 p70, complement component
C5a, .beta.2 microglobulin (.beta.2M), clusterin, cystatin C, NAG,
TIMP-1, NGAL, fatty acid binding protein 1 (FABP-1), CXCL9, KIM-1,
IL-18, vascular endothelial cell growth factor (VEGF), IL-6,
albumin, IL-8, and CCL5. The biological fluid is obtained from the
subject. In some embodiments, the at least two aHUS-associated
biomarkers can be selected from Table 11, i.e., at least two (e.g.,
three, four, five, six, seven, eight, nine, 10, 11, 12, or 13) of a
proteolytic fragment of complement component factor B (e.g., Ba or
Bb), soluble C5b9 (sC5b9), C5a, thrombomodulin, VCAM-1, prothrombin
fragment F1+2, D-dimer, sTNFR1, .beta.2 microglobulin (.beta.2M),
clusterin, cystatin C, TIMP-1, and fatty acid binding protein 1
(FABP-1).
[0052] In some embodiments, any of the methods described herein can
include determining whether the at least two (e.g., at least three,
four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, or 25) physiological changes have
occurred. In some embodiments, the concentrations of at least two
of IFN-.gamma., ICAM-1, IL-1 beta, and IL-12 p70 are reduced. In
some embodiments, the concentrations of both Ba and sC5b9 are
reduced. In some embodiments, the concentration (e.g., the urine
concentration) of each of C5a and sC5b9 is reduced. In some
embodiments of any of the methods described herein, the
concentrations (e.g., the urine concentration) of at least two
(e.g., at least three, four, five, six, or all) of .beta.2M,
clusterin, cystatin C, NAG, TIMP-1, NGAL, and FABP-1 are reduced.
In some embodiments, the concentrations (e.g., the urine
concentration) of CXCL10, CXCL9, and/or KIM-1 are reduced. In some
embodiments, the concentrations (e.g., plasma concentration) of one
or both of D-dimer and F1+2 are reduced. In some embodiments, the
concentrations (e.g., the serum and/or plasma concentrations) of at
least two (e.g., at least three, or all) of sCD40L, prothrombin
fragment F1+2, and D-dimer are reduced. In some embodiments, the
concentrations of thrombomodulin, VCAM-1, and/or vWF are reduced.
In some embodiments, the concentrations (e.g., the serum
concentrations) of CXCL10, MCP-1, and TNFR1 are reduced. In some
embodiments, the concentrations (e.g., the serum concentrations) of
at least two (e.g., at least three, four, or all) of IFN-.gamma.,
ICAM-1, IL-1 beta, and IL-12 p70 are reduced. In some embodiments,
the at least two physiological changes can be a reduction in
concentration of at least two aHUS-associated biomarkers selected
from Table 11, i.e., at least two (e.g., three, four, five, six,
seven, eight, nine, 10, 11, 12, or 13) of a proteolytic fragment of
complement component factor B (e.g., Ba or Bb), soluble C5b9
(sC5b9), C5a, thrombomodulin, VCAM-1, prothrombin fragment F1+2,
D-dimer, sTNFR1, .beta.2 microglobulin (.beta.2M), clusterin,
cystatin C, TIMP-1, and fatty acid binding protein 1 (FABP-1).
[0053] In some embodiments of any of the methods described herein,
the Ba concentration (e.g., plasma Ba concentration) is reduced by
at least 10% by week 6 post-initiation of treatment. In some
embodiments of any of the methods described herein, the Ba
concentration (e.g., plasma Ba concentration) is reduced by at
least 30% by week 12 post-initiation of treatment. In some
embodiments of any of the methods described herein, the C5a
concentration (e.g., urinary C5a concentration) is reduced by at
least 40% by week 3 post-initiation of treatment. In some
embodiments of any of the methods described herein, the C5a
concentration (e.g., urinary C5a concentration) is reduced by at
least 70% by week 6 post-initiation of treatment. In some
embodiments of any of the methods described herein, the C5b-9
concentration (e.g., urinary or plasma C5b-9 concentration) is
reduced by at least 50% by week 3 post-initiation of treatment. In
some embodiments of any of the methods described herein, the F1+2
concentration (e.g., the plasma concentration of F1+2) is reduced
by at least 20% by week 6 post-initiation of treatment. In some
embodiments of any of the methods described herein, the d-dimer
concentration (e.g., the plasma concentration of d-dimer) is
reduced by at least 40% by week 6 post-initiation of treatment. In
some embodiments of any of the methods described herein, the
thrombomodulin concentration (e.g., the serum concentration of
thrombomodulin) is reduced by at least 20% by week 12
post-initiation of treatment. In some embodiments of any of the
methods described herein, the VCAM-1 concentration (e.g., the serum
concentration of VCAM-1) is reduced by at least 20% by week 12
post-initiation of treatment.
[0054] In some embodiments of any of the methods described herein,
the inhibitor of complement is administered to the subject in an
amount and with a frequency sufficient to effect a physiological
change in three or more aHUS-associated biomarkers. In some
embodiments, the inhibitor of complement is administered to the
subject in an amount and with a frequency sufficient to effect a
physiological change in at least four aHUS-associated biomarkers.
In some embodiments, the inhibitor of complement is administered to
the subject in an amount and with a frequency sufficient to effect
a physiological change in at least five aHUS-associated biomarkers.
In some embodiments, the inhibitor of complement is administered to
the subject in an amount and with a frequency sufficient to effect
a physiological change in at least 10 aHUS-associated biomarkers.
In some embodiments, the inhibitor of complement component C5 is
administered to the subject in an amount and with a frequency
sufficient to effect a physiological change in 15 or more
aHUS-associated biomarkers.
[0055] In some embodiments, a physiological change in at least two
(e.g., at least three, four, five, six, seven, eight, nine, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 or more)
aHUS-associated biomarker proteins occurs within two days, three
days, four days, five days, six days, one week, two weeks, three
weeks, four weeks, six weeks, two months, nine weeks, or three
months or more after administration (e.g., chronic administration)
of the inhibitor.
[0056] In some embodiments, the concentration of at least one
(e.g., at least two, three, four, five, six, seven, eight, nine,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25)
aHUS-associated biomarker protein is reduced by at least 5 (e.g.,
at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70) %
following administration of the inhibitor.
[0057] In some embodiments, the concentration of at least one
(e.g., at least two, three, four, five, six, seven, eight, nine,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25)
aHUS-associated biomarker protein is reduced to within 50 (e.g.,
49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33,
32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16,
15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1) % of the
normal concentration of the biomarker protein following
administration of one or more doses of the inhibitor.
[0058] In some embodiments of any of the methods described herein,
the concentration of FABP-1 (e.g., urinary FABP-1) is reduced by at
least 80% (e.g., 85, 90, 95, or up to 100%) following
administration of an inhibitor of human complement (e.g., an
anti-C5 antibody). In some embodiments of any of the methods
described herein, the concentration of cystatin-C (e.g., urinary
cystatin-C) is reduced by at least 80% (e.g., 85, 90, 95, 99, or up
to 100%) following administration of an inhibitor of human
complement (e.g., an anti-C5 antibody). In some embodiments of any
of the methods described herein, the concentration of clusterin
(e.g., urinary clusterin) is reduced by at least 80% (e.g., 85, 90,
95, 98, or up to 100%) following administration of an inhibitor of
human complement (e.g., an anti-C5 antibody). In some embodiments
of any of the methods described herein, the concentration of a
proteolytic fragment of factor B (e.g., Ba) is reduced by at least
10% (e.g., 15, 20, 25, 30, or 40%) following administration of an
inhibitor of human complement (e.g., an anti-C5 antibody). In some
embodiments of any of the methods described herein, the
concentration of sTNFR1 is reduced by at least 80% (e.g., 85, 90,
or more %) following administration of an inhibitor of human
complement (e.g., an anti-C5 antibody). In some embodiments of any
of the methods described herein, the concentration of
thrombomodulin or sVCAM-1 is reduced by at least 80% (e.g., 85, 90,
95, or up to 100%) following administration of an inhibitor of
human complement (e.g., an anti-C5 antibody). In some embodiments
of any of the methods described herein, the concentration of one or
both of F1+2 or D-dimer is reduced by at least 80% (e.g., 85, 90,
95, or more %) following administration of an inhibitor of human
complement (e.g., an anti-C5 antibody).
[0059] In some embodiments of any of the methods described herein,
the concentration of at least one (e.g., at least two, three, four,
five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 21, 22, 23, 24, or 25) of the aHUS-associated biomarker
proteins is normalized following administration of the inhibitor.
In some embodiments, the concentrations (e.g., the urine
concentrations) of at least three of .beta.2 microglobulin
(.beta.2M), clusterin, cystatin C, NAG, TIMP-1, NGAL, fatty acid
binding protein 1 (FABP-1), CXCL10, CXCL9, and KIM-1 are
normalized.
[0060] As used herein, the term "normalized" or like grammatical
terms, when used in the context of the effect of a complement
inhibitor therapy on the concentration or activity of an aHUS
biomarker protein, refers to a concentration or activity measured
in a biological fluid of a biomarker protein that has been brought
within 50 (e.g., 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38,
37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21,
20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2,
or 1) % of the average concentration or activity range of the aHUS
biomarker protein as measured in a sample of the same type of
biological fluid obtained from a group of healthy individuals
(normal individuals). For example, treatment of an aHUS patient
with a complement inhibitor can normalize an elevated urine
clusterin concentration to within, e.g., 20% of the normal average
urine concentration range of clusterin. In some embodiments,
treatment with the complement inhibitor would restore the urine
concentration of clusterin to within the normal average urine
concentration range of clusterin.
[0061] In some embodiments of any of the methods described herein,
the subject has received dialysis at least once (e.g., at least
twice, thrice, four times, or five times or more) within the three
months (e.g., 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 week(s)) prior
to treatment with the inhibitor. For example, in some embodiments
the subject received dialysis one time two months before receiving
the complement inhibitor therapy. In another example, the subject
may be one who has received dialysis three times within the three
month period just prior to receiving the complement inhibitor
therapy. In some embodiments of any of the methods described
herein, relative to the concentration in a healthy subject, the
concentrations of one or more of TNFR1, Ba, thrombomodulin fragment
F1+2, and sC5b9 are elevated. In some embodiments of any of the
methods described herein, relative to the concentrations (e.g., the
urine concentrations) in a healthy human, the concentrations of one
or more of .beta.2M, sC5b9, C5a, cystatin C, clusterin, TIMP-1, and
NGAL are elevated.
[0062] In some embodiments of any of the methods described herein,
the subject (e.g., a human subject) is experiencing a first acute
aHUS manifestation. For example, prior to treatment with the
complement inhibitor, the subject can have elevated concentrations,
relative to the normal concentrations, of one or both of D-dimer
and FABP-1.
[0063] In some embodiments of any of the methods described herein,
the subject (e.g., a human subject) is one having aHUS, but deemed
to be in clinical remission (e.g., the subject is one having normal
levels of platelets or other hematologic markers such as LDH or
haptoglobin). In some embodiments, such a subject is one having
elevated levels of one or more of the aHUS biomarkers described
herein including, but not limited, one or more of Ba, D-dimer,
VCAM-1, and prothrombin fragments 1+2.
[0064] It is understood that for any of the methods described
herein, the concentration and/or activity of one or more aHUS
biomarker proteins can be determined. For example, in some
embodiments, a practitioner may measure the activity of vWF in a
biological sample obtained from the subject as a proxy for the
concentration of vWF (or other biomarker proteins) in the sample.
Methods for assessing relative activity of the aHUS biomarker
proteins set forth in Table 1 are known in the art.
[0065] As discussed in detail herein (for example, in the working
examples), aHUS is a genetic, life threatening disease involving
chronic complement dysregulation. Patients afflicted with the
disease suffer from, among other things, thrombotic microangiopathy
(TMA), which can result in stroke and kidney failure. Eculizumab,
an antagonist anti-C5 antibody, has been shown to dramatically
reduce TMA, normalize platelet levels, and improve renal function
of aHUS patients. Yet, even with the clear and robust clinical
benefit of complement inhibitor therapy for aHUS patients, some
patients still experience elevated levels of several aHUS biomarker
proteins in the face of treatment. For example, the inventors have
discovered that, in some patients, a proteolytic fragment of
complement component factor B (e.g., Ba or Bb) levels (e.g., in
plasma) do not normalize following treatment with an antagonist
anti-C5 antibody. In addition, for some patients, levels of
prothrombin fragment 1+2, D-dimer, thrombomodulin, VCAM-1, TNFR1,
and CXCL10 levels are reduced but do not normalize over time. While
the disclosure is not bound by any particular theory or mechanism
of action, these observations suggest that, for some patients, low
levels of inflammation and coagulopathy may persist even with
complement inhibitor therapy. Thus, the disclosure contemplates
methods in which a complement inhibitor is administered in
combination with a second therapy to address the low level of
persistent inflammation in some patients with aHUS.
[0066] Thus, in yet another aspect, the disclosure features a
method for treating atypical hemolytic uremic syndrome (aHUS). The
method comprises administering (e.g., chronically administering) to
a subject (e.g., a human subject) having, suspected of having, or
at risk for developing, aHUS a therapeutically effective amount of
an inhibitor of complement (e.g., an inhibitor of complement
component C5) and a therapeutically effective amount of: (i) an
anti-coagulant, (ii) a fibrinolytic agent; (iii) an
anti-inflammatory agent; or (iv) an inhibitor of IL-6, IL-8,
CXCL-9, IL-18, or VEGF. In some embodiments, two inhibitors of
complement can be used (e.g., an inhibitor of C5 and an inhibitor
of C3, such as, an anti-Factor B antibody, an anti-C3 antibody, or
an anti-C3b antibody). In some embodiments, at the time of
discontinuing therapy with an inhibitor of C5, an inhibitor of
complement component C3 can be administered to the patient for a
time sufficient to reduce upstream alternative pathway
activation.
[0067] In some embodiments, the methods can include monitoring the
status of one or more aHUS biomarkers and determining whether to
start a second therapy (in addition to complement inhibitor
therapy) or modify the dosing regimen of one or more second
therapies being administered to an aHUS patient. For example,
during treatment (e.g., chronic treatment) with a complement
inhibitor, the concentration of one or more aHUS associated
biomarker proteins can be measured in one or more biological fluids
obtained from the subject. If the concentration of one or more of
the biomarker proteins has not normalized and/or remains elevated,
a medical practitioner may elect to administer to the subject one
or more additional secondary agents (e.g., anti-inflammatories) to
address any pathophysiological effects resulting from the elevated
biomarkers.
[0068] The complement inhibitor can be any of those described
herein. In some embodiments of any of the methods described herein,
the inhibitor is antibody or an antigen binding fragment thereof, a
small molecule, a polypeptide, a polypeptide analog, a
peptidomimetic, or an aptamer. In some embodiments, the inhibitor
can be one that inhibits one or more of complement components C1,
C2, C3, C4, C5, C6, C7, C8, C9, Factor D, Factor B, properdin, MBL,
MASP-1, MASP-2, or biologically active fragments of any of the
foregoing. In some embodiments of any of the methods described
herein, the complement inhibitor inhibits one or both of the
generation of the anaphylatoxic activity associated with C5a and/or
the assembly of the membrane attack complex associated with
C5b.
[0069] The compositions can also contain 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.
[0070] In some embodiments of any of the methods described herein,
the inhibitor of complement is an antagonist antibody or
antigen-binding fragment thereof. The antibody or antigen-binding
fragment thereof can be selected from the group consisting of a
humanized antibody, a recombinant antibody, a diabody, a chimerized
or chimeric antibody, a monoclonal antibody, a deimmunized
antibody, a fully human antibody, a single chain antibody, an Fv
fragment, an Fd fragment, an Fab fragment, an Fab' fragment, and an
F(ab').sub.2 fragment.
[0071] In some embodiments of any of the methods described herein,
the antagonist antibody is an anti-C5 antibody such as eculizumab.
In some embodiments, the antagonist antibody is pexelizumab, a
C5-binding fragment of anti-C5 antibody.
[0072] In some embodiments of any of the methods described herein,
the inhibitor of complement is selected from the group consisting
of MB 12/22, MB12/22-RGD, ARC 187, ARC1905, SSL7, and OmCI.
[0073] In some embodiments, the anti-coagulant is selected from the
group consisting of: a coumarin, heparin, a factor Xa inhibitor,
and a thrombin inhibitor. Examples of anti-coagulants include,
e.g., warfarin (Coumadin), aspirin, heparin, phenindione,
fondaparinux, idraparinux, and thrombin inhibitors (e.g.,
argatroban, lepirudin, bivalirudin, or dabigatran).
[0074] In some embodiments, the fibrinolytic agent is selected from
the group consisting of ancrod, E-aminocaproic acid,
antiplasmin-a.sub.1, prostacyclin, and defibrotide.
[0075] In some embodiments, the anti-inflammatory agent is an
anti-cytokine agent such as an antagonist antibody (or
antigen-binding fragment thereof) or a soluble cytokine receptor,
which binds to an inflammatory cytokine and inhibits the activity
of the cytokine. The anti-cytokine agent can be, e.g., a TNF
inhibitor (e.g., an anti-TNF antibody or soluble TNF receptor
protein) or an anti-CD20 agent.
[0076] Anti-inflammatory agents also include, e.g., steroids (e.g.,
dexamethasone), non-steroidal anti-inflammatory drugs (NSAIDs)
(e.g., indomethacin, naproxen, sulindac, diclofenac, aspirin,
flurbiprofen, oxaprozin, salsalate, difunisal, piroxicam, etodolac,
meclofenamate, ibuprofen, fenoprofen, ketoprofen, nabumetone,
tolmetin, choline magnesium salicylate, COX-2 inhibitors, TNF alpha
antagonists (etanercept, adalimumab, infliximab, golimumab),
disease modifying anti-rheumatic drugs (DMARDS) (e.g.,
sulfasalazine, methotrexate), cyclosporin, retinoids and
corticosteroids.
[0077] In yet another aspect, the disclosure features a method for
determining whether the concentration of one or more
aHUS-associated biomarker proteins are elevated in a patient
having, suspected of having, or at risk for developing, atypical
hemolytic uremic syndrome (aHUS), wherein the method comprises: (i)
measuring in a biological sample obtained from the patient the
concentration of each of at least two aHUS-associated biomarkers
from Table 11 (infra), i.e., selected from the group consisting of:
a proteolytic fragment of factor B, C5a, soluble C5b-9 (sC5b-9),
soluble TNFR1 (sTNFR1), soluble VCAM-1 (sVCAM-1), thrombomodulin,
prothrombin fragments 1 and 2 (F1+2), D-dimer, clusterin, TIMP-1,
FABP-1, beta-2 microglobulin (.beta.2m), and cystatin-C, and (ii)
determining whether the patient has an elevated concentration of
each of at least two of the aHUS-associated biomarkers as compared
to a normal control concentration of the same at least two
biomarkers. In some embodiments, the at least two aHUS-associated
biomarker proteins are measured using an immunoassay, such as, an
enzyme-linked immunosorbent assay (ELISA) or a radioimmunoassay
(RIA). The biological fluid can be, e.g., blood, a blood fraction
(e.g., plasma or serum), or urine. It is understood that any
combination of any two or more (e.g., three, four, five, six,
seven, eight, nine, 10, 11 or 12) of the aforementioned
aHUS-biomarkers can be measured and analyzed in accordance with the
methods described herein.
[0078] In another aspect, the disclosure features a method for
diagnosing a patient as having atypical hemolytic uremic syndrome
(aHUS) (or confirming a diagnosis of aHUS, e.g., where the patient
has met two or more of the inclusion criteria discussed under
Example 1), wherein the method comprises: (i) measuring in a
biological sample obtained from a patient suspected of having aHUS
or at risk of developing aHUS the concentration of each of at least
two aHUS-associated biomarkers selected from the group consisting
of: a proteolytic fragment of factor B, C5a, soluble C5b-9
(sC5b-9), soluble TNFR1 (sTNFR1), soluble VCAM-1 (sVCAM-1),
thrombomodulin, prothrombin fragments 1 and 2 (F1+2), D-dimer,
clusterin, TIMP-1, FABP-1, beta-2 microglobulin (.beta.2m), and
cystatin-C, and (ii) diagnosing a patient as having aHUS (or
confirming a diagnosis of aHUS) if the concentration of each of at
least two of the aHUS-associated biomarkers are elevated as
compared to a normal control concentration of the same at least two
biomarkers. In some embodiments, the at least two aHUS-associated
biomarker proteins are measured using an immunoassay, such as, an
enzyme-linked immunosorbent assay (ELISA) or a radioimmunoassay
(RIA). The biological fluid can be, e.g., blood, a blood fraction
(e.g., plasma or serum), or urine. It is understood that any
combination of any two or more (e.g., three, four, five, six,
seven, eight, nine, 10, 11 or 12) of the aforementioned
aHUS-biomarkers can be measured and analyzed in accordance with the
methods described herein.
[0079] A normal control concentration, as used in any of the
methods described herein, can be (or can be based on), e.g., the
concentration of a given aHUS-associated biomarker protein in a
biological sample or biological samples obtained from one or more
(e.g., two, three, four, five, six, seven, eight, nine, 10, 15, 20,
25, 30, 35, or 40 or more) healthy individuals. In some
embodiments, a normal control concentration of a biomarker can be
(or can be based on), e.g., the concentration of the biomarker in a
pooled sample obtained from two or more (e.g., two, three, four,
five, six, seven, eight, nine, 10, 15, 20, 25, 30, 35, or 40 or
more) healthy individuals. In some embodiments of any of the
methods described herein, the pooled samples can be from healthy
individuals or, at least, individuals who do not have or are not
suspected of having (nor at risk for developing) aHUS. For example,
determining whether a subject is one having aHUS can involve
comparing the measured concentration of one or more complement
component proteins (e.g., Table 1 or Table 11) in a biological
sample (or several different types of biological samples) obtained
from the patient and comparing the measured concentration to the
average concentration of the same proteins in the pooled healthy
samples. Such healthy human control concentrations can be, in some
embodiments, a range of values, or a median or mean value obtained
from the range.
[0080] In some embodiments, the concentration of at least one
aHUS-associated biomarker is measured in two or more types of
biological fluid. In some embodiments, the concentration of the
first of the at least two aHUS biomarker proteins is measured in
one type of biological fluid and the concentration of the second of
the at least two aHUS biomarker proteins is measured in a second
type of fluid.
[0081] In some embodiments of any of the methods described herein,
the concentration of the proteolytic fragment of factor B is
measured. The fragment can be, e.g., Ba. The biological sample can
be a plasma sample. As described in Table 11, the normal control
concentration of Ba can be less than 1000 ng/mL. The normal control
concentration of Ba can be less than 600 ng/mL. The normal control
concentration of Ba can be between 300 and 600 ng/mL.
[0082] In some embodiments, the concentration of Ba in the
biological sample is deemed elevated when it is at least two fold
greater than the normal control concentration of Ba. In some
embodiments, the concentration of Ba in the biological sample is
deemed elevated when it is at least five fold greater than the
normal control concentration of Ba. In some embodiments, the
concentration of Ba in the biological sample is deemed elevated
when it is at least 1500 ng/mL. In some embodiments, the
concentration of Ba in the biological sample is deemed elevated
when it is at least 2500 ng/mL.
[0083] In some embodiments of any of the methods described herein,
the concentration of C5a is measured. The biological sample in
which C5a is measured can be a urine sample. And in some
embodiments, the normal control concentration of C5a is less than 2
ng per mg of urinary creatinine. In some embodiments, the normal
control concentration of C5a is less than 1 ng per mg of urinary
creatinine. In some embodiments, the normal control concentration
of C5a is between 0 and 0.7 ng per mg of urinary creatinine.
[0084] In some embodiments of any of the methods described herein,
the concentration of C5a in the biological sample is deemed
elevated when it is at least two fold greater than the normal
control concentration of C5a. In some embodiments, the
concentration of C5a in the biological sample is deemed elevated
when it is at least ten fold greater than the normal control
concentration of C5a. In some embodiments, the concentration of C5a
in the biological sample is deemed elevated when it is at least
forty fold greater than the normal control concentration of C5a. In
some embodiments, the concentration of C5a in the biological sample
is deemed elevated when it is at least 5 ng per mg of urinary
creatinine. In some embodiments, the concentration of C5a in the
biological sample is deemed elevated when it is at least 9 ng per
mg of urinary creatinine.
[0085] In some embodiments of any of the methods described herein,
the concentration of sC5b-9 is measured. The biological sample in
which sC5b-9 is measured can be a urine sample. And in some
embodiments, the normal control concentration of sC5b-9 is less
than 2 ng per mg of urinary creatinine. The normal control
concentration of sC5b-9 can be less than 1 ng per mg of urinary
creatinine. In some embodiments, the normal control concentration
of sC5b-9 is between 0 and 0.6 ng per mg of urinary creatinine.
[0086] In some embodiments of any of the methods described herein,
the concentration of sC5b-9 in the biological sample is deemed
elevated when it is at least ten fold greater than the normal
control concentration of sC5b-9. In some embodiments, the
concentration of sC5b-9 in the biological sample is deemed elevated
when it is at least fifty fold greater than the normal control
concentration of sC5b-9. In some embodiments, the concentration of
sC5b-9 in the biological sample is deemed elevated when it is at
least one hundred fold greater than the normal control
concentration of sC5b-9. In some embodiments, the concentration of
sC5b-9 in the biological sample is deemed elevated when it is at
least 20 ng per mg of urinary creatinine. In some embodiments, the
concentration of sC5b-9 in the biological sample is deemed elevated
when it is at least 30 ng per mg of urinary creatinine.
[0087] In some embodiments of any of the methods described herein,
the concentration of sTNFR1 is measured. The biological sample in
which sTNFR1 is measured can be a serum sample. And in some
embodiments, the normal control concentration of sTNFR1 is less
than 2000 pg/mL. In some embodiments, the normal control
concentration of sTNFR1 is less than 1500 pg/mL. In some
embodiments, the normal control concentration of sTNFR1 is between
400 and 1500 pg/mL.
[0088] In some embodiments of any of the methods described herein,
the concentration of sTNFR1 in the biological sample is deemed
elevated when it is at least two fold greater than the normal
control concentration of sTNFR1. In some embodiments, the
concentration of sTNFR1 in the biological sample is deemed elevated
when it is at least five fold greater than the normal control
concentration of sTNFR1. In some embodiments, the concentration of
sTNFR1 in the biological sample is deemed elevated when it is at
least fifteen fold greater than the normal control concentration of
sTNFR1. In some embodiments, the concentration of sTNFR1 in the
biological sample is deemed elevated when it is at least 10,000
pg/mL. In some embodiments, the concentration of sTNFR1 in the
biological sample is deemed elevated when it is at least 15,000
pg/mL.
[0089] In some embodiments of any of the methods described herein,
the concentration of sVCAM-1 is measured. The biological sample in
which sVCAM-1 is measured can be a serum sample. And in some
embodiments, the normal control concentration of sVCAM-1 is less
than 500 ng/mL. In some embodiments, the normal control
concentration of sVCAM-1 is less than 300 ng/mL. In some
embodiments, the normal control concentration of sVCAM-1 is between
100 and 500 ng/mL.
[0090] In some embodiments of any of the methods described herein,
the concentration of sVCAM-1 in the biological sample is deemed
elevated when it is at least 10% greater than the normal control
concentration of sVCAM-1. In some embodiments, the concentration of
sVCAM-1 in the biological sample is deemed elevated when it is at
least 30% greater than the normal control concentration of sVCAM-1.
In some embodiments, the concentration of sVCAM-1 in the biological
sample is deemed elevated when it is at least 50% greater than the
normal control concentration of sVCAM-1. In some embodiments, the
concentration of sVCAM-1 in the biological sample is deemed
elevated when it is at least 550 ng/mL. In some embodiments, the
concentration of sVCAM-1 in the biological sample is deemed
elevated when it is at least 650 ng/mL.
[0091] In some embodiments of any of the methods described herein,
the concentration of thrombomodulin is measured. The biological
sample in which thrombomodulin is measured can be a plasma sample.
And in some embodiments, the normal control concentration of
thrombomodulin is less than 5 ng/mL. In some embodiments, the
normal control concentration of thrombomodulin is less than 3
ng/mL. In some embodiments, the normal control concentration of
thrombomodulin is between 2 and 6 ng/mL.
[0092] In some embodiments of any of the methods described herein,
the concentration of thrombomodulin in the biological sample is
deemed elevated when it is at least 10% greater than the normal
control concentration of thrombomodulin. In some embodiments, the
concentration of thrombomodulin in the biological sample is deemed
elevated when it is at least 30% greater than the normal control
concentration of thrombomodulin. In some embodiments, the
concentration of thrombomodulin in the biological sample is deemed
elevated when it is at least 50% greater than the normal control
concentration of thrombomodulin. In some embodiments, the
concentration of thrombomodulin in the biological sample is deemed
elevated when it is at least 8 ng/mL. In some embodiments, the
concentration of thrombomodulin in the biological sample is deemed
elevated when it is at least 10 ng/mL.
[0093] In some embodiments of any of the methods described herein,
the concentration of F1+2 is measured. The biological sample in
which F1+2 is measured can be a plasma sample. And in some
embodiments, the normal control concentration of F1+2 is less than
400 pmol/L. In some embodiments, the normal control concentration
of F1+2 is less than 300 pmol/L. In some embodiments, the normal
control concentration of F1+2 is between 50 and 400 pmol/L.
[0094] In some embodiments of any of the methods described herein,
the concentration of F1+2 in the biological sample is deemed
elevated when it is at least 30% greater than the normal control
concentration of F1+2. In some embodiments, the concentration of
F1+2 in the biological sample is deemed elevated when it is at
least 50% greater than the normal control concentration of F1+2. In
some embodiments, the concentration of F1+2 in the biological
sample is deemed elevated when it is at least 100% greater than the
normal control concentration of F1+2. In some embodiments, the
concentration of F1+2 in the biological sample is deemed elevated
when it is at least 900 pmol/L. In some embodiments, the
concentration of F1+2 in the biological sample is deemed elevated
when it is at least 1000 pmol/L.
[0095] In some embodiments of any of the methods described herein,
the concentration of D-dimer is measured. The biological sample in
which D-dimer is measured can be a plasma sample. And in some
embodiments, the normal control concentration of D-dimer is less
than 500 .mu.g/L. In some embodiments, the normal control
concentration of D-dimer is less than 400 .mu.g/L. In some
embodiments, the normal control concentration of D-dimer is between
100 and 500 .mu.g/L.
[0096] In some embodiments of any of the methods described herein,
the concentration of D-dimer in the biological sample is deemed
elevated when it is at least two-fold greater than the normal
control concentration of D-dimer. In some embodiments, the
concentration of D-dimer in the biological sample is deemed
elevated when it is at least five-fold greater than the normal
control concentration of D-dimer. In some embodiments, the
concentration of D-dimer in the biological sample is deemed
elevated when it is at least ten-fold greater than the normal
control concentration of D-dimer. In some embodiments, the
concentration of D-dimer in the biological sample is deemed
elevated when it is at least 1500 .mu.g/L. In some embodiments, the
concentration of D-dimer in the biological sample is deemed
elevated when it is at least 2500 .mu.g/L.
[0097] In some embodiments of any of the methods described herein,
the concentration of clusterin is measured. The biological sample
in which clusterin is measured can be a urine sample. And in some
embodiments, the normal control concentration of clusterin is less
than 500 ng per mg of urinary creatinine. The normal control
concentration of clusterin can be, e.g., less than 400 ng per mg of
urinary creatinine. In some embodiments, the normal control
concentration of clusterin is between 0 and 500 ng per mg of
urinary creatinine.
[0098] In some embodiments of any of the methods described herein,
the concentration of clusterin in the biological sample is deemed
elevated when it is at least two-fold greater than the normal
control concentration of clusterin. In some embodiments, the
concentration of clusterin in the biological sample is deemed
elevated when it is at least five-fold greater than the normal
control concentration of clusterin. In some embodiments, the
concentration of clusterin in the biological sample is deemed
elevated when it is at least ten-fold greater than the normal
control concentration of clusterin. In some embodiments, the
concentration of clusterin in the biological sample is deemed
elevated when it is at least 900 ng per mg of urinary creatinine.
In some embodiments, the concentration of clusterin in the
biological sample is deemed elevated when it is at least 1200 ng
per mg of urinary creatinine.
[0099] In some embodiments of any of the methods described herein,
the concentration of TIMP-1 is measured. The biological sample in
which TIMP-1 is measured can be a urine sample. And in some
embodiments, the normal control concentration of TIMP-1 is less
than 10 ng per mg of urinary creatinine. In some embodiments, the
normal control concentration of TIMP-1 is less than 5 ng per mg of
urinary creatinine. In some embodiments, the normal control
concentration of TIMP-1 is between 0 and 10 ng per mg of urinary
creatinine.
[0100] In some embodiments of any of the methods described herein,
the concentration of TIMP-1 in the biological sample is deemed
elevated when it is at least two-fold greater than the normal
control concentration of TIMP-1. In some embodiments, the
concentration of TIMP-1 in the biological sample is deemed elevated
when it is at least ten-fold greater than the normal control
concentration of TIMP-1. In some embodiments, the concentration of
TIMP-1 in the biological sample is deemed elevated when it is at
least twenty-fold greater than the normal control concentration of
TIMP-1. In some embodiments, the concentration of TIMP-1 in the
biological sample is deemed elevated when it is at least 15 ng per
mg of urinary creatinine. In some embodiments, the concentration of
TIMP-1 in the biological sample is deemed elevated when it is at
least 20 ng per mg of urinary creatinine.
[0101] In some embodiments of any of the methods described herein,
the concentration of FABP-1 (also referred to herein as L-FABP-1)
is measured. The biological sample in which C5a is measured can be
a urine sample. And in some embodiments, the normal control
concentration of FABP-1 is less than 20 ng per mg of urinary
creatinine. In some embodiments, the normal control concentration
of FABP-1 is less than 15 ng per mg of urinary creatinine. In some
embodiments, the normal control concentration of FABP-1 is between
0 and 20 ng per mg of urinary creatinine.
[0102] In some embodiments of any of the methods described herein,
the concentration of FABP-1 in the biological sample is deemed
elevated when it is at least two-fold greater than the normal
control concentration of FABP-1. In some embodiments, the
concentration of FABP-1 in the biological sample is deemed elevated
when it is at least ten-fold greater than the normal control
concentration of FABP-1. In some embodiments, the concentration of
FABP-1 in the biological sample is deemed elevated when it is at
least twenty-fold greater than the normal control concentration of
FABP-1. In some embodiments, the concentration of FABP-1 in the
biological sample is deemed elevated when it is at least 40 ng per
mg of urinary creatinine. In some embodiments, the concentration of
FABP-1 in the biological sample is deemed elevated when it is at
least 50 ng per mg of urinary creatinine.
[0103] In some embodiments of any of the methods described herein,
the concentration of .beta.2m is measured. The biological sample in
which .beta.2m is measured can be a urine sample. And in some
embodiments, the normal control concentration of .beta.2m is less
than 5 .mu.g per mg of urinary creatinine. In some embodiments, the
normal control concentration of .beta.2m is less than 3 .mu.g per
mg of urinary creatinine. In some embodiments, the normal control
concentration of .beta.2m is between 0 and 5 .mu.g per mg of
urinary creatinine.
[0104] In some embodiments of any of the methods described herein,
the concentration of .beta.2m in the biological sample is deemed
elevated when it is at least two-fold greater than the normal
control concentration of .beta.2m. In some embodiments of any of
the methods described herein, the concentration of .beta.2m in the
biological sample is deemed elevated when it is at least ten-fold
greater than the normal control concentration of .beta.2m. In some
embodiments, the concentration of .beta.2m in the biological sample
is deemed elevated when it is at least twenty-fold greater than the
normal control concentration of .beta.2m. In some embodiments, the
concentration of .beta.2m in the biological sample is deemed
elevated when it is at least 15 .mu.g per mg of urinary creatinine.
In some embodiments, the concentration of .beta.2m in the
biological sample is deemed elevated when it is at least 20 .mu.g
per mg of urinary creatinine.
[0105] In some embodiments of any of the methods described herein,
the concentration of cystatin-C is measured. The biological sample
in which cystatin-C is measured can be a urine sample. And in some
embodiments, the normal control concentration of cystatin-C is less
than 400 ng per mg of urinary creatinine. In some embodiments, the
normal control concentration of cystatin-C is less than 300 ng per
mg of urinary creatinine. In some embodiments, the normal control
concentration of cystatin-C is between 0 and 400 ng per mg of
urinary creatinine.
[0106] In some embodiments of any of the methods described herein,
the concentration of cystatin-C in the biological sample is deemed
elevated when it is at least two-fold greater than the normal
control concentration of cystatin-C. In some embodiments, the
concentration of cystatin-C in the biological sample is deemed
elevated when it is at least ten-fold greater than the normal
control concentration of cystatin-C. In some embodiments, the
concentration of cystatin-C in the biological sample is deemed
elevated when it is at least twenty-fold greater than the normal
control concentration of cystatin-C. In some embodiments, the
concentration of cystatin-C in the biological sample is deemed
elevated when it is at least 900 ng per mg of urinary creatinine.
In some embodiments, the concentration of cystatin-C in the
biological sample is deemed elevated when it is at least 1200 ng
per mg of urinary creatinine.
[0107] In some embodiments of any of the methods described herein,
the concentrations of two or more of proteolytic fragments of
factor B, C5a, and sC5b-9 are measured. In some embodiments of any
of the methods described herein, the concentrations of C5a and
sC5b-9 are measured. In some embodiments of any of the methods
described herein, the concentrations of sVCAM-1 and thrombomodulin
are measured. In some embodiments of any of the methods described
herein, the concentrations of F1+2 and D-dimer are measured. In
some embodiments of any of the methods described herein, the
concentrations of two or more of clusterin, TIMP-1, .beta.2m,
FABP-1, and cystatin-C are measured.
[0108] In yet another aspect, the disclosure features a method for
assessing the level of alternative pathway activation in a patient
having aHUS, suspected of having aHUS, or at risk for developing
aHUS, before, during, or after treatment with a complement
inhibitor, such as, an anti-C5 antibody. The method comprises:
measuring the concentration of a proteolytic fragment of factor B
(e.g., Ba or Bb) in a biological sample obtained from a patient
treated with an inhibitor of complement (e.g., an inhibitor of
human complement component C5, such as, an anti-C5 antibody).
[0109] In yet another aspect, the disclosure features a method for
determining whether a patient has responded to therapy with a
complement inhibitor (e.g., had a reduction in risk of developing
thrombosis or had a reduction in the number, frequency, or
occurrence of thrombotic microangiopathy), the method comprising
measuring the concentration of one or more biomarkers of thrombosis
or coagulation set forth in Table 1 or 11, e.g., F1+2, D-dimer,
vWF, or thrombomodulin, in a biological sample obtained from a
patient at elevated risk of, suffering from, or suspected of
having, thrombotic microangiopathy (TMA) and treated with a
complement inhibitor; and determining that the patient has
responded to the therapy if the concentration of the one or more
biomarkers in the biological sample is reduced, as compared to the
concentration of the one or more biomarkers in a biological sample
of the same type obtained from the patient prior to treatment with
the complement inhibitor or determining that the patient has not
responded to the therapy if the concentration of the one or more
biomarkers in the biological sample is not reduced, as compared to
the concentration of the one or more biomarkers in a biological
sample of the same type obtained from the patient prior to
treatment with the complement inhibitor. In some embodiments, the
patient has, is suspected of having, or is at risk for developing,
aHUS.
[0110] In another aspect, the disclosure features a method for
determining whether an aHUS patient has responded to therapy with a
complement inhibitor, the method comprising measuring the
concentration of one or more biomarkers of terminal complement
activation set forth in Table 1 or 11, e.g., C5a and/or sC5b-9, in
a biological sample obtained from a patient having, suspected of
having, or at risk for developing, aHUS and treated with a
complement inhibitor (e.g., an anti-C5 antibody); and determining
that the patient has responded to the therapy if the concentration
of the one or more biomarkers in the biological sample is reduced,
as compared to the concentration of the one or more biomarkers in a
biological sample of the same type obtained from the patient prior
to treatment with the complement inhibitor or determining that the
patient has not responded to the therapy if the concentration of
the one or more biomarkers in the biological sample is not reduced,
as compared to the concentration of the one or more biomarkers in a
biological sample of the same type obtained from the patient prior
to treatment with the complement inhibitor. Thus, the method can be
used to assess or monitor terminal complement blockade in an aHUS
patient treated with a complement inhibitor. In embodiments in
which the patient is non-responsive, or less responsive to therapy,
the method can also include changing the dose amount or dose
frequency of the complement inhibitor or electing a different
complement inhibitor (e.g., an inhibitor of C3 activation) for use
in treating the patient.
[0111] In another aspect, the disclosure features a method for
determining whether an aHUS patient has responded to therapy with a
complement inhibitor, the method comprising measuring the
concentration of one or more biomarkers of vascular inflammation or
endothelial activation set forth in Table 1 or 11, e.g., sTNFR1,
sVCAM-1, or thrombomodulin, in a biological sample obtained from a
patient having, suspected of having, or at risk for developing,
aHUS; and determining that the patient has responded to the therapy
if the concentration of the one or more biomarkers in the
biological sample is reduced, as compared to the concentration of
the one or more biomarkers in a biological sample of the same type
obtained from the patient prior to treatment with the complement
inhibitor or determining that the patient has not responded to the
therapy if the concentration of the one or more biomarkers in the
biological sample is not reduced, as compared to the concentration
of the one or more biomarkers in a biological sample of the same
type obtained from the patient prior to treatment with the
complement inhibitor. Thus, the method can be used to assess or
monitor vascular inflammation in an aHUS patient treated with a
complement inhibitor. In embodiments in which the patient is
non-responsive, or less responsive to therapy, the method can also
include changing the dose amount or dose frequency of the
complement inhibitor or electing a different complement inhibitor
(e.g., an inhibitor of C3 activation) for use in treating the
patient.
[0112] In another aspect, the disclosure features a method for
determining whether an aHUS patient has responded to therapy with a
complement inhibitor, the method comprising measuring the
concentration of one or more biomarkers of renal injury set forth
in Table 1 or 11, e.g., clusterin, TIMP-1, FABP-1, .beta.2m, and/or
cystatin-C, in a biological sample obtained from a patient having,
suspected of having, or at risk for developing, aHUS; and
determining that the patient has responded to the therapy if the
concentration of the one or more biomarkers in the biological
sample is reduced, as compared to the concentration of the one or
more biomarkers in a biological sample of the same type obtained
from the patient prior to treatment with the complement inhibitor
or determining that the patient has not responded to the therapy if
the concentration of the one or more biomarkers in the biological
sample is not reduced, as compared to the concentration of the one
or more biomarkers in a biological sample of the same type obtained
from the patient prior to treatment with the complement inhibitor.
Thus, the method can be used to assess or monitor renal injury in
an aHUS patient treated with a complement inhibitor. In embodiments
in which the patient is non-responsive, or less responsive to
therapy, the method can also include changing the dose amount or
dose frequency of the complement inhibitor or electing a different
complement inhibitor (e.g., an inhibitor of C3 activation) for use
in treating the patient.
[0113] The inventors have also discovered that, in aHUS patients,
the relative elevation of terminal complement activation markers
C5a and sC5b-9 concentrations (e.g., urinary concentrations) are
much higher than the relative elevation of levels of complement
alternative pathway activation markers (e.g., Ba) in these
patients. That is, the median concentration of C5a and sC5b-9 in
aHUS patients was 45 and 305 fold higher, respectively, than the
median concentration of these markers in normal healthy humans,
whereas the median concentration of Ba was only approximately
5-fold higher than the median concentration of Ba in normal healthy
humans. While not being bound by any particular theory or mechanism
of action, the inventors believe that the ratio of terminal
complement activation over alternative pathway activation is a
useful diagnostic tool for aHUS. Thus, in another aspect, the
disclosure features a method of diagnosing aHUS or confirming a
diagnosis of aHUS, which method includes comparing the level of
activation of terminal complement (e.g., sC5b-9 or C5a) to the
level of activation of upstream alternative pathway activation
(e.g., Ba or Bb) (relative to normal healthy humans), wherein a
higher degree of terminal activation relative to the alternative
pathway activation is an indication that the patient has aHUS. For
example, a ratio indicative of aHUS could be, e.g., approximately
45:5 or 305:5, fold-induction of terminal complement activation to
fold-induction alternative pathway activation. Moreover, the
inventors believe that this ratio can be useful for distinguishing
aHUS from other complement-associated diseases, such as, thrombotic
thrombocytopenic purpura (TTP), which may not exhibit such a
difference in terminal complement and upstream alternative pathway
activation levels.
[0114] "Polypeptide," "peptide," and "protein" are used
interchangeably and mean any peptide-linked chain of amino acids,
regardless of length or post-translational modification. The
proteins described herein can contain or be wild-type proteins or
can be variants that have not more than 50 (e.g., not more than
one, two, three, four, five, six, seven, eight, nine, ten, 12, 15,
20, 25, 30, 35, 40, or 50) conservative amino acid substitutions.
Conservative substitutions typically include substitutions within
the following groups: glycine and alanine; valine, isoleucine, and
leucine; aspartic acid and glutamic acid; asparagine, glutamine,
serine and threonine; lysine, histidine and arginine; and
phenylalanine and tyrosine.
[0115] As used herein, percent (%) amino acid sequence identity is
defined as the percentage of amino acids in a candidate sequence
that are identical to the amino acids in a reference sequence,
after aligning the sequences and introducing gaps, if necessary, to
achieve the maximum percent sequence identity. Alignment for
purposes of determining percent sequence identity can be achieved
in various ways that are within the skill in the art, for instance,
using publicly available computer software such as BLAST software.
Appropriate parameters for measuring alignment, including any
algorithms needed to achieve maximal alignment over the full-length
of the sequences being compared can be determined by known
methods.
[0116] As used herein, the term "antibody" includes both whole
antibodies and antigen-binding fragments of the whole antibodies.
Whole antibodies include different antibody isotypes including IgM,
IgG, IgA, IgD, and IgE antibodies. The term "antibody" includes a
polyclonal antibody, a monoclonal antibody, a chimerized or
chimeric antibody, a humanized antibody, a primatized antibody, a
deimmunized antibody, and a fully human antibody. The antibody can
be made in or derived from any of a variety of species, e.g.,
mammals such as humans, non-human primates (e.g., orangutan,
baboons, or chimpanzees), horses, cattle, pigs, sheep, goats, dogs,
cats, rabbits, guinea pigs, gerbils, hamsters, rats, and mice. The
antibody can be a purified or a recombinant antibody.
[0117] As used herein, the term "antibody fragment,"
"antigen-binding fragment," or similar terms refer to a fragment of
an antibody that retains the ability to bind to a target antigen
(e.g., human C5) and inhibit the activity of the target antigen.
Such fragments include, e.g., a single chain antibody, a single
chain Fv fragment (scFv), an Fd fragment, an Fab fragment, an Fab'
fragment, or an F(ab').sub.2 fragment. An scFv fragment is a single
polypeptide chain that includes both the heavy and light chain
variable regions of the antibody from which the scFv is derived. In
addition, intrabodies, minibodies, triabodies, and diabodies are
also included in the definition of antibody and are compatible for
use in the methods described herein. See, e.g., Todorovska et al.
(2001) J Immunol Methods 248(1):47-66; Hudson and Kortt (1999) J
Immunol Methods 231(1):177-189; Poljak (1994) Structure
2(12):1121-1123; and Rondon and Marasco (1997) Annual Review of
Microbiology 51:257-283, the disclosures of each of which are
incorporated herein by reference in their entirety. Bispecific
antibodies (including DVD-Ig antibodies; see below) are also
embraced by the term "antibody." Bispecific antibodies are
monoclonal, preferably human or humanized, antibodies that have
binding specificities for at least two different antigens.
[0118] As used herein, the term "antibody" also includes, e.g.,
single domain antibodies such as camelized single domain
antibodies. See, e.g., Muyldermans et al. (2001) Trends Biochem Sci
26:230-235; Nuttall et al. (2000) Curr Pharm Biotech 1:253-263;
Riechmann et al. (1999) J Immunol Meth 231:25-38; PCT application
publication nos. WO 94/04678 and WO 94/25591; and U.S. Pat. No.
6,005,079, all of which are incorporated herein by reference in
their entireties. In some embodiments, the disclosure provides
single domain antibodies comprising two VH domains with
modifications such that single domain antibodies are formed.
[0119] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this disclosure pertains.
Preferred methods and materials are described below, although
methods and materials similar or equivalent to those described
herein can also be used in the practice or testing of the presently
disclosed methods and compositions. All publications, patent
applications, patents, and other references mentioned herein are
incorporated by reference in their entirety.
[0120] Other features and advantages of the present disclosure,
e.g., methods for treating complement-associated disorders in a
subject, will be apparent from the following description, the
examples, and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0121] FIG. 1A is a dot plot depicting the concentration of C5a (in
ng/mg of urinary creatine) in the urine of aHUS patients both
before treatment with eculizumab (Pre-Tx) and various weeks after
initiating treatment with eculizumab. The concentration of urinary
C5a was also measured in the urine from normal, healthy individuals
(NORM).
[0122] FIG. 1B is a dot plot depicting the concentration of sC5b-9
(in ng/mg of urinary creatine) in the urine of aHUS patients both
before treatment with eculizumab (Pre-Tx) and various weeks after
initiating treatment with eculizumab. The concentration of urinary
C5b9 was also measured in the urine from normal, healthy
individuals (NORM).
[0123] FIG. 1C is a dot plot depicting the concentration of
complement component Ba (in ng/mL) in the plasma of aHUS patients
both before treatment with eculizumab (Pre-Tx) and various weeks
after initiating treatment with eculizumab. The concentration of Ba
was also measured in the plasma from normal, healthy individuals
(normals).
[0124] FIG. 1D is a bar graph depicting the mean percentage (%)
reduction in urinary C5a levels (Y-axis) over time in aHUS patients
(N=26) post initiation of treatment with eculizumab. The x-axis
indicates the week of the aHUS patient visit for evaluation
post-initiation of treatment, e.g., V3 is the patient visit for
evaluation at week 3 post-initiation of treatment.
[0125] FIG. 1E is a bar graph depicting the mean percentage (%)
reduction in urinary sC5b-9 levels (Y-axis) over time in aHUS
patients (N=23) post initiation of treatment with eculizumab. The
x-axis indicates the week of the aHUS patient visit for evaluation
post-initiation of treatment, e.g., V3 is the patient visit for
evaluation at week 3 post-initiation of treatment.
[0126] FIG. 1F is a bar graph depicting the mean percentage (%)
reduction in plasma Ba levels (Y-axis) over time in aHUS patients
(N=35) post initiation of treatment with eculizumab. The x-axis
indicates the week of the aHUS patient visit for evaluation
post-initiation of treatment, e.g., V3 is the patient visit for
evaluation at week 3 post-initiation of treatment.
[0127] FIGS. 2A-2C are bar graphs depicting the percentage of aHUS
patients who achieve normalized concentrations of urinary C5a (FIG.
2A), urinary sC5b9 (FIG. 2B), and plasma Ba (FIG. 2C) at baseline
(pre-treatment with eculizumab) and various weeks following
initiation of treatment with eculizumab.
[0128] FIG. 3A is a dot plot depicting the concentration of
prothrombin fragment 1+2 (in pmol/L) in the plasma of aHUS patients
both before treatment with eculizumab (Pre-Tx) and various weeks
after initiating treatment with eculizumab. The concentration of
plasma F1+2 was also measured in the plasma from normal, healthy
individuals (normals).
[0129] FIG. 3B is a dot plot depicting the concentration of D-dimer
(in .mu.g/L) in the plasma of aHUS patients both before treatment
with eculizumab (Pre-Tx) and various weeks after initiating
treatment with eculizumab. The concentration of plasma D-dimer was
also measured in the plasma from normal, healthy individuals
(normals).
[0130] FIG. 3C is a bar graph depicting the mean percentage (%)
reduction in plasma prothrombin fragment F1+2 levels (Y-axis) over
time in aHUS patients post initiation of treatment with eculizumab.
The x-axis indicates the week of the aHUS patient visit for
evaluation post-initiation of treatment, e.g., V3 is the patient
visit for evaluation at week 3 post-initiation of treatment.
[0131] FIG. 3D is a bar graph depicting the mean percentage (%)
reduction in plasma d-dimer levels (Y-axis) over time in aHUS
patients post initiation of treatment with eculizumab. The x-axis
indicates the week of the aHUS patient visit for evaluation
post-initiation of treatment, e.g., V3 is the patient visit for
evaluation at week 3 post-initiation of treatment.
[0132] FIGS. 4A and 4B are bar graphs depicting the percentage of
aHUS patients who achieve normalized concentrations of plasma
prothrombin fragment 1+2 (FIG. 4A) and plasma D-dimer (FIG. 4B) at
baseline (pre-treatment with eculizumab) and various weeks
following initiation of treatment with eculizumab.
[0133] FIG. 5A is a dot plot depicting the concentration of
thrombomodulin (in ng/mL) in the plasma (EDTA treated plasma) of
aHUS patients both before treatment with eculizumab (Pre-Tx) and
various weeks after initiating treatment with eculizumab. The
concentration of plasma thrombomodulin was also measured in the
plasma from normal, healthy individuals (normals). EOS designates
the results of the analysis of samples obtained at the "end of
study".
[0134] FIG. 5B is a dot plot depicting the concentration of VCAM-1
(in ng/mL) in the serum of aHUS patients both before treatment with
eculizumab (Pre-Tx) and various weeks after initiating treatment
with eculizumab. The concentration of serum VCAM-1 was also
measured in the serum from normal, healthy individuals (normal
pool). EOS designates the results of the analysis of samples
obtained at the "end of study".
[0135] FIG. 5C is a dot plot depicting the activity of vWF (in
mU/mL) in the plasma (EDTA treated plasma) of aHUS patients both
before treatment with eculizumab (Pre-Tx) and various weeks after
initiating treatment with eculizumab. The activity of vWF was also
measured in the plasma from normal, healthy individuals (normals).
EOS designates the results of the analysis of samples obtained at
the "end of study".
[0136] FIGS. 6A and 6B are bar graphs depicting the percentage of
aHUS patients that achieve normalized plasma thrombomodulin
concentrations (FIG. 6A) and plasma vWF activity levels (FIG. 4B)
at baseline (pre-treatment with eculizumab) and various weeks
following initiation of treatment with eculizumab.
[0137] FIG. 6C is a bar graph depicting the mean percentage (%)
reduction in plasma thrombomodulin levels (Y-axis) over time in
aHUS patients (N=33) post initiation of treatment with eculizumab.
The x-axis indicates the week of the aHUS patient visit for
evaluation post-initiation of treatment, e.g., V3 is the patient
visit for evaluation at week 3 post-initiation of treatment.
[0138] FIG. 6D is a bar graph depicting the mean percentage (%)
reduction in serum VCAM-1 levels (Y-axis) over time in aHUS
patients (N=36) post initiation of treatment with eculizumab. The
x-axis indicates the week of the aHUS patient visit for evaluation
post-initiation of treatment, e.g., V3 is the patient visit for
evaluation at week 3 post-initiation of treatment.
[0139] FIG. 7A is a dot plot depicting the concentration of TNFR1
(in pg/mL) in the serum of aHUS patients both before treatment with
eculizumab (Pre-Tx) and various weeks after initiating treatment
with eculizumab. The concentration of serum TNFR1 was also measured
in the serum from normal, healthy individuals (normal pool). EOS
designates the results of the analysis of samples obtained at the
"end of study".
[0140] FIG. 7B is a bar graph depicting the percentage of aHUS
patients that achieve normalized serum TNFR1 concentrations at
baseline (pre-treatment with eculizumab) and various weeks
following initiation of treatment with eculizumab.
[0141] FIG. 8A is a dot plot depicting the concentration of
cystatin C (CysC) (in ng/mg of urinary creatine) in the urine of
aHUS patients both before treatment with eculizumab (Pre-Tx) and
various weeks after initiating treatment with eculizumab. The
concentration of urinary CysC was also measured in the urine from
normal, healthy individuals (NORM).
[0142] FIG. 8B is a dot plot depicting the concentration of
.beta.2M (in .mu.g/mg of urinary creatine) in the urine of aHUS
patients both before treatment with eculizumab (Pre-Tx) and various
weeks after initiating treatment with eculizumab. The concentration
of urinary .beta.2M was also measured in the urine from normal,
healthy individuals (NORM).
[0143] FIG. 8C is a dot plot depicting the concentration of NGAL
(in ng/mg of urinary creatine) in the urine of aHUS patients both
before treatment with eculizumab (Pre-Tx) and various weeks after
initiating treatment with eculizumab. The concentration of urinary
NGAL was also measured in the urine from normal, healthy
individuals (NORM).
[0144] FIGS. 9A-9E are a series of bar graphs depicting the mean
levels of several aHUS biomarker proteins in aHUS patients that
were subjected to dialysis (Dialysis), as compared to those aHUS
patients that were not subjected to dialysis (no Dialysis) prior to
enrollment in the study described herein. FIG. 9A depicts the mean
concentration of serum TNFR1 (in pg/mL); FIG. 9B depicts the mean
concentration of urinary .beta.2M (in .mu.g/mg of urinary
creatine); FIG. 9C depicts the concentration of plasma Ba (in
ng/mL); FIG. 9D depicts the concentration of urinary sC5b9 (in
ng/mg of urinary creatine); and FIG. 9E depicts the concentration
of urinary C5a (in ng/mL).
[0145] FIG. 10A is a dot plot depicting the concentration of serum
TNFR1 (in pg/mL) in aHUS patients exhibiting stable clinical
parameters (clinical remission) (Without TMA) and those aHUS
patients that continue to experience elevated haptoglobin and LDH
levels (and reduced platelet counts) (Others), both at baseline and
at 1 to 2.5 weeks post initiation of treatment with eculizumab.
Also shown are the concentrations of serum TNFR1 from normal,
healthy individuals (Normals).
[0146] FIGS. 10B-10E are a series of bar graphs, each one depicting
the concentration of a given biomarker in patients with normal
hematologic markers LDH and haptoglobin ("normal patients" or
patients deemed to be in clinical remission), patients with
abnormal (elevated) hematologic markers ("abnormal patients" or
patients with active aHUS presentation), and healthy subjects
("normals"). FIG. 10B depicts the levels of plasma Ba (ng/mL) in
these subject populations. FIG. 10C depicts the level of serum
VCAM-1 (ng/mL) in the subject populations. FIG. 10E depicts the
level of plasma prothrombin fragments 1+2 (pmol/L) in the
populations, and FIG. 10D depicts the level of plasma D-dimer (in
.mu.g/L). The P values for the respective group comparisons are
shown in the figures.
[0147] FIGS. 10E-10I are a series of bar graphs, each one depicting
the concentration of a given biomarker in patients with normal
platelet levels ("normal patients"), patients with abnormal
(reduced) platelet levels ("abnormal patients"), and healthy
subjects ("normals"). FIG. 10F depicts the levels of plasma Ba
(ng/mL) in these subject populations. FIG. 10G depicts the level of
serum VCAM-1 (ng/mL) in the subject populations. FIG. 10I depicts
the level of plasma prothrombin fragments 1+2 (pmol/L) in the
populations, and FIG. 10H depicts the level of plasma D-dimer (in
.mu.g/L). The P values for the respective group comparisons are
shown in the figures.
[0148] FIG. 11 is a bar graph depicting the mean percentage change
in serum TNFR1 and urinary clusterin, C5a, and C5b9 levels in those
aHUS patients who achieve a complete TMA response and those
patients who still experience TMA events (incomplete response). (As
noted and elaborated on in the working examples, a complete TMA
response refers to a normalization of hematologic parameters and
preservation of renal function.)
[0149] FIG. 12 is a bar graph depicting the mean percentage change
in plasma Ba levels in eculizumab-treated aHUS patients
experiencing a complete TMA response and those eculizumab-treated
aHUS patients who do not (others).
[0150] FIG. 13 is bar graph depicting the mean change from baseline
(initial visit, prior to treatment with eculizumab) in platelet
count (10.sup.9/L) at weeks 12-17 and week 26 post initiation of
treatment with eculizumab in aHUS patients with normalized levels
of plasma Ba versus persistently elevated plasma Ba levels. The p
values for each observation are also provided in the figure.
[0151] FIGS. 14A-14D are a series of bar graphs depicting the
observation that certain aHUS-associated biomarkers are elevated in
aHUS patients with abnormal TMA markers at baseline. FIG. 14A
depicts the concentration of cystatin C (CysC) (in ng/mg of urinary
creatine) in the urine of aHUS patients with normal platelet counts
(>150,000 per .mu.L of blood) as compared to patients with
reduced platelet counts (<150,000 per .mu.L of blood). FIG. 14B
depicts the concentration of clusterin (in ng/mg of urinary
creatine) in the urine of aHUS patients with normal platelet counts
(>150,000 per .mu.L of blood) as compared to patients with
reduced platelet counts (<150,000 per .mu.L of blood). FIG. 14C
depicts the concentration of VCAM-1 in the serum of aHUS patients
with normal LDH levels as compared to patients with elevated LDH
levels. FIG. 14D depicts the concentration of d-dimer (in .mu.g/L)
in the plasma of aHUS patients with normal LDH levels as compared
to patients with elevated LDH levels. The p values for each
observation are indicated in the figures.
[0152] FIG. 15 is a bar graph depicting the level of cystatin C
(ng/mg of urinary creatine) in the urine of aHUS patients at
baseline having repeated plasma therapy (Repeated PT; N=23), no
plasma therapy (No PT; N=3), or in normal patients (N=9).
[0153] FIG. 16 is a series of bar graphs depicting the mean change
in baseline eGFR (mL/min/1.73 m.sup.2) in aHUS patients who achieve
normalized levels of various biomarkers (plasma Ba, serum VCAM-1,
plasma F1+2, plasma d-dimer, and urinary cystatin C) following
eculizumab treatment as compared to aHUS patients in whom the
concentration of these biomarkers remain elevated.
[0154] FIGS. 17A-E are a series of bar graphs depicting the
observation that certain aHUS-associated biomarkers are elevated in
aHUS patients prior to treatment with a complement inhibitor,
regardless of whether the patients have received plasma exchange
(PE) or plasma infusion (PI) therapy. FIG. 17A depicts the
concentration of Factor B proteolytic fragment Ba (in ng/mL) in the
plasma of normal healthy volunteers (NHV), aHUS patients receiving
PE or PI therapy (PE/PI), or aHUS patients not receiving PE/PI
therapy (no PE/PI). FIG. 17B depicts the concentration of sTNFR1
(in pg/mL) in the serum of normal healthy volunteers (NHV), aHUS
patients receiving PE or PI therapy (PE/PI), or aHUS patients not
receiving PE/PI therapy (no PE/PI). FIG. 17C depicts the
concentration of sVCAM-1 (in ng/mL) in the serum of normal healthy
volunteers (NHV), aHUS patients receiving PE or PI therapy (PE/PI),
or aHUS patients not receiving PE/PI therapy (no PE/PI). FIG. 17D
depicts the concentration of D-dimer (in .mu.g/L) in the plasma of
normal healthy volunteers (NHV), aHUS patients receiving PE or PI
therapy (PE/PI), or aHUS patients not receiving PE/PI therapy (no
PE/PI). FIG. 17E depicts the concentration of cystatin-C (in ng/mg
of urinary creatinine) in the urine of normal healthy volunteers
(NHV), aHUS patients receiving PE or PI therapy (PE/PI), or aHUS
patients not receiving PE/PI therapy (no PE/PI). The p values for
each observation are indicated in the figures.
[0155] FIGS. 18A-E are a series of bar graphs depicting the
observation that certain aHUS-associated biomarkers are elevated in
aHUS patients prior to treatment with a complement inhibitor,
regardless of platelet levels in the patients. FIG. 18A depicts the
concentration of Factor B proteolytic fragment Ba (in ng/mL) in the
plasma of normal healthy volunteers (NHV), aHUS patients having
normal platelet levels (>150.times.10.sup.9), or aHUS patients
having reduced platelet counts (<150.times.10.sup.9). FIG. 18B
depicts the concentration of sTNFR1 (in pg/mL) in the serum of
normal healthy volunteers (NHV), aHUS patients having normal
platelet levels (>150.times.10.sup.9), or aHUS patients having
reduced platelet counts (<150.times.10.sup.9). FIG. 18C depicts
the concentration of sVCAM-1 (in ng/mL) in the serum of normal
healthy volunteers (NHV), aHUS patients having normal platelet
levels (>150.times.10.sup.9), or aHUS patients having reduced
platelet counts (<150.times.10.sup.9). FIG. 18D depicts the
concentration of D-dimer (in .mu.g/L) in the plasma of normal
healthy volunteers (NHV), aHUS patients having normal platelet
levels (>150.times.10.sup.9), or aHUS patients having reduced
platelet counts (<150.times.10.sup.9). FIG. 18E depicts the
concentration of cystatin-C (in ng/mg of urinary creatinine) in the
urine of normal healthy volunteers (NHV), aHUS patients having
normal platelet levels (>150.times.10.sup.9), or aHUS patients
having reduced platelet counts (<150.times.10.sup.9). The p
values for each observation are indicated in the figures.
[0156] FIGS. 19A-E are a series of bar graphs depicting the
observation that certain aHUS-associated biomarkers are elevated in
aHUS patients prior to treatment with a complement inhibitor,
regardless of haptoglobin (Hp) or lactate dehydrogenase (LDH)
levels. FIG. 19A depicts the concentration of Factor B proteolytic
fragment Ba (in ng/mL) in the plasma of normal healthy volunteers
(NHV), aHUS patients having normal Hp and LDH levels, or aHUS
patients having elevated (abnormal) Hp/LDH. FIG. 19B depicts the
concentration of sTNFR1 (in pg/mL) in the serum of normal healthy
volunteers (NHV), aHUS patients having normal Hp and LDH levels, or
aHUS patients having elevated (abnormal) Hp/LDH. FIG. 19C depicts
the concentration of sVCAM-1 (in ng/mL) in the serum of normal
healthy volunteers (NHV), aHUS patients having normal Hp and LDH
levels, or aHUS patients having elevated (abnormal) Hp/LDH. FIG.
19D depicts the concentration of D-dimer (in .mu.g/L) in the plasma
of normal healthy volunteers (NHV), aHUS patients having normal Hp
and LDH levels, or aHUS patients having elevated (abnormal) Hp/LDH.
FIG. 19E depicts the concentration of cystatin-C (in ng/mg of
urinary creatinine) in the urine of normal healthy volunteers
(NHV), aHUS patients having normal Hp and LDH levels, or aHUS
patients having elevated (abnormal) Hp/LDH. The p values for each
observation are indicated in the figures.
[0157] FIGS. 20A-B are Box-Whisker plots depicting the longitudinal
effects of sustained eculizumab treatment on terminal complement
activation in aHUS patients. FIG. 20A depicts the change over time
in the concentration of urinary C5a (ng/mg of urinary creatinine)
of aHUS patients following eculizumab treatment, as compared to the
concentration of urinary C5a in the urine of normal healthy
volunteers (NHV). FIG. 20B depicts the change over time in the
concentration of urinary sC5b-9 (ng/mg of urinary creatinine) of
aHUS patients following eculizumab treatment, as compared to the
concentration of urinary sC5b-9 in the urine of normal healthy
volunteers (NHV). The Box-Whisker plots show median, 25.sup.Th, and
75.sup.th percentiles and range. *First time point at which levels
were significantly reduced vs. baseline (BL); P values versus
baseline at each timepoint were calculated using a restricted
maximum likelihood-based repeated measures approach (Mixed Model).
P values compared with NHV were calculated using the Wilcoxon Rank
Sum test.
[0158] FIGS. 21A-C are Box-Whisker plots depicting the longitudinal
effects of sustained eculizumab treatment on the concentration of
biomarker proteins associated with renal injury in aHUS patients.
FIG. 20A depicts the change over time in the concentration of
urinary FABP-1 (ng/mg of urinary creatinine) of aHUS patients
following eculizumab treatment, as compared to the concentration of
urinary FABP-1 in the urine of normal healthy volunteers (NHV).
FIG. 21B depicts the change over time in the concentration of
urinary cystatin-C (ng/mg of urinary creatinine) of aHUS patients
following eculizumab treatment, as compared to the concentration of
urinary cystatin-C in the urine of normal healthy volunteers (NHV).
FIG. 21C depicts the change over time in the concentration of
urinary clusterin (ng/mg of urinary creatinine) of aHUS patients
following eculizumab treatment, as compared to the concentration of
urinary clusterin in the urine of normal healthy volunteers (NHV).
The Box-Whisker plots show median, 25.sup.Th, and 75.sup.th
percentiles and range. *First time point at which levels were
significantly reduced vs. baseline (BL); P values versus baseline
at each timepoint were calculated using a restricted maximum
likelihood-based repeated measures approach (Mixed Model). P values
compared with NHV were calculated using the Wilcoxon Rank Sum
test.
[0159] FIG. 22 is a Box-Whisker plot depicting the longitudinal
effects of sustained eculizumab treatment on complement alternative
pathway activation in aHUS patients. The change over time in the
concentration of Ba (ng/mL) in the plasma of aHUS patients
following eculizumab treatment is shown along with the
concentration of plasma Ba in normal healthy volunteers (NHV). The
Box-Whisker plot shows median, 25.sup.Th, and 75.sup.th percentiles
and range. *First time point at which levels were significantly
reduced vs. baseline (BL); P values versus baseline at each
timepoint were calculated using a restricted maximum
likelihood-based repeated measures approach (Mixed Model). P values
compared with NHV were calculated using the Wilcoxon Rank Sum
test.
[0160] FIGS. 23A-C are Box-Whisker plots depicting the longitudinal
effects of sustained eculizumab treatment on the concentration of
biomarker proteins associated with inflammation, endothelial cell
activation, and tissue damage in aHUS patients. FIG. 23A depicts
the change over time in the concentration of sTNFR1 (pg/mL) in the
serum of aHUS patients following eculizumab treatment, as compared
to the concentration of sTNFR1 in the serum of normal healthy
volunteers (NHV). FIG. 23B depicts the change over time in the
concentration of sVCAM-1 (ng/mL) in the serum of aHUS patients
following eculizumab treatment, as compared to the concentration of
the analyte in the serum of normal healthy volunteers (NHV). FIG.
23C depicts the change over time in the concentration of
thrombomodulin (ng/mL) in the plasma of aHUS patients following
eculizumab treatment, as compared to the concentration of the
analyte in the plasma of normal healthy volunteers (NHV). The
Box-Whisker plots show median, 25.sup.Th, and 75.sup.th percentiles
and range. *First time point at which levels were significantly
reduced vs. baseline (BL); P values versus baseline at each
timepoint were calculated using a restricted maximum
likelihood-based repeated measures approach (Mixed Model). P values
compared with NHV were calculated using the Wilcoxon Rank Sum
test.
[0161] FIGS. 24A-B are Box-Whisker plots depicting the longitudinal
effects of sustained eculizumab treatment on the concentration of
biomarker proteins associated with thrombosis and coagulation in
aHUS patients. FIG. 24A depicts the change over time in the
concentration of F1+2 (pmol/L) in the plasma of aHUS patients
following eculizumab treatment, as compared to the concentration of
the analyte in the plasma of normal healthy volunteers (NHV). FIG.
24B depicts the change over time in the concentration of D-dimer
(.mu.g/L) in the plasma of aHUS patients following eculizumab
treatment, as compared to the concentration of the analyte in the
plasma of normal healthy volunteers (NHV). The Box-Whisker plots
show median, 25.sup.Th, and 75.sup.th percentiles and range. *First
time point at which levels were significantly reduced vs. baseline
(BL); P values versus baseline at each timepoint were calculated
using a restricted maximum likelihood-based repeated measures
approach (Mixed Model). P values compared with NHV were calculated
using the Wilcoxon Rank Sum test.
OVERVIEW OF THE COMPLEMENT SYSTEM
[0162] 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. A
concise summary of the biologic activities associated with
complement activation is provided, for example, in The Merck
Manual, 16.sup.th Edition.
[0163] The complement cascade progresses via the classical pathway,
the alternative pathway, or the lectin 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.
[0164] The classical pathway (CP) is typically initiated by
antibody recognition of, and binding to, an antigenic site on a
target cell. The alternative pathway (AP) can be antibody
independent, and can be initiated by certain molecules on pathogen
surfaces. Additionally, the lectin pathway is typically initiated
with binding of mannose-binding lectin (MBL) to high mannose
substrates. These pathways converge at the point where complement
component C3 is cleaved by an active protease 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. 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. This opsonic function of C3b
is generally considered to be the most important anti-infective
action of the complement system. C3b also forms a complex with
other components unique to each pathway to form classical or
alternative C5 convertase, which cleaves complement component C5
(hereinafter referred to as "C5") into C5a and C5b.
[0165] Cleavage of C5 releases biologically active species such as
for example C5a, a potent anaphylatoxin and chemotactic factor, and
C5b which through a series of protein interactions leads to the
formation of the lytic terminal complement complex, C5b-9. C5a and
C5b-9 also have pleiotropic cell activating properties, by
amplifying the release of downstream inflammatory factors, such as
hydrolytic enzymes, reactive oxygen species, arachidonic acid
metabolites and various cytokines.
[0166] 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.
[0167] As mentioned above, C3a and C5a are activated complement
components. These can trigger mast cell degranulation, which
releases histamine from basophils and mast cells, and other
mediators of inflammation, resulting in smooth muscle contraction,
increased vascular permeability, 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. C5a receptors are found on the surfaces of
bronchial and alveolar epithelial cells and bronchial smooth muscle
cells. C5a receptors have also been found on eosinophils, mast
cells, monocytes, neutrophils, and activated lymphocytes.
DETAILED DESCRIPTION
[0168] As described herein and exemplified in the working Examples,
the inventors identified biomarkers for aHUS. For example, it has
been discovered that an elevated or, in some cases, reduced
concentration of certain proteins is associated with the presence
of aHUS. Similarly, a reduced or elevated concentration (or
activity) of certain proteins in a biological fluid obtained from
an aHUS patient treated with a complement inhibitor indicates that
the patient has responded to therapy with the inhibitor.
Accordingly, analysis of the concentration and/or activity level of
such proteins can be employed to evaluate, among other things, risk
for aHUS, diagnose aHUS, monitor progression or abatement of aHUS,
and/or monitor treatment response to a complement inhibitor.
aHUS Biomarker Proteins and Applications
[0169] aHUS biomarker proteins (as well as exemplary biological
fluids in which they are found) are set forth in Table 1. The
protein sequence associated with the name of each of the biomarkers
listed in Table 1 in GenBank (National Center for Biotechnology
Information (NCBI)) as available as of the filing date of the
present application are incorporated herein by reference.
TABLE-US-00001 TABLE 1 Tissue Source NCBI Reference Biomarker Abbr.
Serum Plasma Urine Seq no.* Markers of Inflammation/platelet or
endothelial activation Chemokine (C--X--C CXCL9 X NP_002407.1
motif) ligand 9 Chemokine (C--X--C CXCL-10 X NP_001556.2 motif)
ligand 10 Interleukin-1 beta IL-1.beta. X NP_000567.1 Interleukin-6
IL-6 X NP_000591.1 Interleukin-8 IL-8 X NP_000575.1 Interleukin-12
p70 IL-12p70 X NP_000873.2 (p35) NP_002178.2 (p40) Interferon-gamma
IFN-.gamma. X NP_000610.2 platelet-selectin p-selectin X
NP_002996.2 endothelial-selectin e-selectin X NP_000441.2
Intercellular ICAM-1 X NP_000192.2 Adhesion Molecule-1 Vascular
cell VCAM-1 X NP_001069.1 adhesion molecule-1 Monocyte MCP-1 X
NP_002973.1 chemotactic protein-1 Vascular endothelial VEGF X
NP_001020537.2 growth factor Regulated on CCL5 X NP_002976.2
Activation, Normal T cell Expressed and Secreted (CCL5) Soluble
CD40 ligand sCD40L X NP_000065.1** Soluble Tumor sTNFR1 X
NP_001056.1** necrosis factor receptor 1 Interleukin-18 IL-18 X
NP_001553.1 Markers of Inflammation/Renal Injury neutrophil NGAL X
NP_005555.2 gelatinase-associated lipocalin Kidney injury KIM-1 X
NP_001092884.1 molecule-1 Osteopontin OPN X NP_001035147.1 tissue
inhibitor of TIMP-1 X NP_003245.1 metalloproteinases-1
Interleukin-18 IL-18 X Supra Chemokine (C--X--C CXCL9 X Supra
motif) ligand 9 Chemokine (C--X--C CXCL10 X Supra motif) ligand 10
clusterin CLU X NP_001822.3 Cystatin C CyC X NP_000090.1 albumin
ALB X NP_000468.1 Liver-fatty acid L-FABP X NP_001434.1 binding
protein Beta-2- .beta.2M X NP_004039.1 microglobulin Trefoil factor
3 TFF-3 X NP_003217.3 N-acetyl-beta-D- NAG X NP_000511.2
glucosaminidase .pi.-glutathione S- .pi.-GST X NP_000843.1
transferase Alpha-glutathione .alpha.-GST X NP_665683.1
S-transferase Complement Complement Ba Ba X SEQ ID NO: 1; See also
FIG. 2 of Morley and Campbell (1984) EMBO J 3(1): 153-157.
Complement C3a C3a X SEQ ID NO: 2 Complement C5a C5a X X SEQ ID NO:
3 Soluble MAC sC5b9 X X NA CH.sub.50 (hemolysis) CH.sub.50 X NA
Complement C5 C5 X X NP_001726.2 Thrombosis/coagulation D-dimer
D-dimer X P02671*** Prothrombin F1 + 2 F1 + 2 X Activation fragment
1 (SEQ ID NO: 4) corresponds to amino acids 44-198 of SEQ ID NO: 6.
Activation fragment 2 (SEQ ID NO: 5) corresponds to amino acids
199-327 of SEQ ID NO: 4. Von Willebrand vWF X NP_000543.2 factor
Von Willebrand vWF X Id. factor activity activity Thrombomodulin TM
X NP_000352.1 *The NCBI accession number for an exemplary human
sequence is provided for each biomarker protein recited in the
Table. **The soluble form of the receptor is generated by
proteolytic processing of the membrane bound form of the receptor.
***UniProtKB (consortium: European Bioinformatics Institute,
Cambridge, UK; Swiss Institute of Bioinformatics; Geneva,
Switzerland; and Protein Information Resource, Washington, D.C.)
designation for human fibrinogen alpha, which is cleaved by
thrombin to form fibrin. D-dimer is a degradation product of
fibrin. A description of the cleavage-based transition of
fibrinogen to fibrin to D-dimer is set forth in Soheir et al.
(2009) Blood 113(13): 2878-2887, the disclosure of which at least
as it relates to the formation of D-dimer is incorporated herein in
its entirety.
[0170] Biomarkers provided herein can be used alone or in
combination as an indicator to, e.g., evaluate risk for developing
aHUS, diagnosing aHUS, determining whether a subject is
experiencing the first acute presentation of aHUS, monitoring
progression or abatement of aHUS, and/or monitoring response to
treatment with a complement inhibitor or optimizing such treatment.
In some embodiments, an individual aHUS biomarker protein described
herein may be used. In some embodiments, at least two, three, four,
five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 21, 22, 23, 24, or 25 or more) aHUS biomarker proteins
selected from Table 1 may be used in combination as a panel.
[0171] In some embodiments, the aHUS biomarker proteins are
selected from a proteolytic fragment of complement component factor
B (e.g., Ba or Bb), soluble C5b9 (sC5b9), thrombomodulin, VCAM-1,
von Willebrand Factor (vWF), soluble CD40 ligand (sCD40L),
prothrombin fragment F1+2, D-dimer, CXCL10, MCP-1, TNFR1,
IFN-.gamma., ICAM-1, IL-1 beta, IL-12 p70, complement component
C5a, .beta.2 microglobulin (.beta.2M), clusterin, cystatin C, NAG,
TIMP-1, NGAL, fatty acid binding protein 1 (FABP-1), CXCL9, KIM-1,
IL-18, vascular endothelial cell growth factor (VEGF), IL-6,
albumin, IL-8, and CCL5. The concentration and/or activity of one
or more of the biomarkers in Table 1 (or any of the subsets of
biomarkers mentioned herein) can be measured.
[0172] In some embodiments, an elevation in the d-dimer
concentration, relative to the concentration of d-dimer in a normal
control sample, and an elevation in the FABP-1 concentration,
relative to the concentration of FABP-1 in a normal control sample,
indicates that the aHUS patient is experiencing a first acute aHUS
manifestation. In some instances, that elevation of one or both of
these aHUS biomarker proteins is a significant elevation as
compared to the normal control.
[0173] In some embodiments, an elevation in the concentration of
one or more of TNFR1, Ba, C5b-9, F1+2, .beta.2M, clusterin, TIMP-1,
NGAL, CysC, and C5a (see Table 7) in a biological sample obtained
from an aHUS patient, relative to the control concentration of the
analytes obtained, e.g., from a pool of samples from aHUS patients
who have not received repeated dialysis, indicates that the patient
is one who has received repeated dialysis.
[0174] In some embodiments, an elevation in the concentration of
one or both of C5a and FABP-1 (e.g., urinary C5a and FABP-1) in a
biological sample obtained from an aHUS patient, relative to the
control concentration of the analytes obtained, e.g., from a pool
of samples from aHUS patients who have not received a kidney
transplant, indicates that the patient is one who has received a
kidney transplant.
[0175] In some embodiments, an elevation in the concentration of
cystatin C (e.g., urinary cystatin C) in a biological sample
obtained from an aHUS patient, relative to the control
concentration of the analytes obtained, e.g., from a pool of
samples from aHUS patients who have not received repeated plasma
therapy, indicates that the patient is one who has received
repeated plasma therapy.
[0176] In some embodiments, a post-treatment reduction in Ba
concentration (e.g., plasma Ba concentration) of at least 10 (e.g.,
at least 15, 20, 25, 30, 35, 40, 45, or 50) %, relative to the Ba
concentration in a sample of the same type of biological fluid
obtained from the subject prior to treatment, indicates that the
subject has or is likely to achieve a complete
thrombomicroangiopathy (TMA) response (i.e., cessation of TMA
events). In some embodiments, the reduction occurs by week 12
following the first treatment with the complement inhibitor. In
some embodiments, the reduction occurs within weeks 12-17 following
the first treatment with the complement inhibitor. In some
embodiments, the reduction occurs by week 26 following the first
treatment with the complement inhibitor.
[0177] In some embodiments, a post-treatment reduction in one or
both of CCL5 and sCD40L of at least 10 (e.g., at least 15, 20, 25,
30, 35, 40, 45, or 50) %, relative to the respective concentration
in sample(s) of the same type of biological fluid obtained from the
subject prior to treatment, indicates that the subject has or is
likely to achieve increased platelet counts (e.g., platelet
recovery). In some embodiments, a post-treatment reduction in Ba
concentration (e.g., plasma Ba concentration) of at least 10 (e.g.,
at least 15, 20, 25, 30, 35, 40, 45, or 50) % (or normalization of
Ba concentrations), relative to the Ba concentration in a sample of
the same type of biological fluid obtained from the subject prior
to treatment, indicates that the subject has or is likely to have
achieved a complete thrombomicroangiopathy (TMA) response (i.e.,
cessation of TMA events).
[0178] In some embodiments, the status of one or more of the aHUS
biomarkers described herein can be predictive of improvement in the
estimated glomerular filtration rate (eGFR) for an aHUS patient
treated with a complement inhibitor. For example, a reduction in
the concentration of prothrombin F1+2 (e.g., within 4, 5, or 6
weeks post initial treatment in a chronic treatment regimen) and/or
d-dimer (e.g., within 12, 13, 14, 15, 16, or 17 weeks post initial
treatment in a chronic treatment regimen) indicates that an aHUS
patient treated with a complement inhibitor has achieved or is
likely to achieve a clinically meaningful improvement in eGFR.
Achievement or likely achievement of a clinically meaningful
improvement in eGFR is also indicated by a normalization of IL-6
and IFN-.gamma. concentration (e.g., within 4, 5, or 6 weeks post
initial treatment with a complement inhibitor in a chronic
treatment regimen). Achievement or likely achievement of a
clinically meaningful improvement in eGFR is also indicated by a
normalization of Ba, CXCL9, CXCL10, and vWF concentration (e.g.,
within 12, 13, 14, 15, 16, or 17 weeks post initial treatment with
a complement inhibitor in a chronic treatment regimen). In some
embodiments, achievement or likely achievement of a clinically
meaningful improvement in eGFR is also indicated by a normalization
of Ba, CXCL9, CXCL10, 132M (e.g., in urine), CysC (e.g., in urine),
vWF, d-dimer, clusterin (e.g., in urine), and/or FABP-1 (e.g., in
urine) concentration (e.g., within 26 weeks post initial treatment
with a complement inhibitor in a chronic treatment regimen).
[0179] Methods for monitoring or evaluating the status of one or
more atypical hemolytic uremic syndrome (aHUS)-associated biomarker
proteins in a subject (e.g., a mammal, e.g., a human) include:
measuring in a biological fluid obtained from the subject one or
both of (i) the concentration of at least one (e.g., at least two,
three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, or 20) aHUS-associated biomarker protein in the
biological fluid.
[0180] Measuring or determining protein expression levels in a
biological sample may be performed by any suitable method (see,
e.g., Harlow and Lane (1988) "Antibodies: A Laboratory Manual",
Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y.). In
general, protein levels are determined by contacting a biological
sample obtained from a subject with binding agents for one or more
of the aHUS biomarker proteins; detecting, in the sample (e.g., the
biological fluid), the levels of one or more of the aHUS biomarker
proteins that bind to the binding agents; and comparing the levels
of one or more of the aHUS biomarker proteins in the sample with
the levels of the corresponding protein biomarkers in a control
sample (e.g., a normal sample). In certain embodiments, a suitable
binding agent is a ribosome, with or without a peptide component,
an RNA molecule, or a polypeptide (e.g., a polypeptide that
comprises a polypeptide sequence of a protein marker, a peptide
variant thereof, or a non-peptide mimetic of such a sequence).
[0181] Suitable binding agents also include an antibody specific
for an aHUS biomarker protein described herein (e.g., an antibody
specific for any biomarker listed in Table 1). Suitable antibodies
for use in the methods of the present invention include monoclonal
and polyclonal antibodies and antigen-binding fragments (e.g., Fab
fragments or scFvs) of antibodies. Antibodies, including monoclonal
and polyclonal antibodies, fragments and chimeras, may be prepared
using methods known in the art (see, for example, Kohler and
Milstein (1975) Nature 256:495-497; Kozbor et al. (1985) J Immunol
Methods 81:31-42; Cote et al. (1983) Proc Natl Acad Sci USA
80:2026-203; and Zhang et al. (2002) J Biol Chem 277:39379-39387).
Antibodies to be used in the methods of the invention can be
purified by methods well known in the art. Antibodies may also be
obtained from commercial sources.
[0182] In certain embodiments, the binding agent is directly or
indirectly labeled with a detectable moiety. The role of a
detectable agent is to facilitate the detection step of the
diagnostic method by allowing visualization of the complex formed
by binding of the binding agent to the protein marker (or fragment
thereof). The detectable agent can be selected such that it
generates a signal that can be measured and whose intensity is
related (preferably proportional) to the amount of protein marker
present in the sample being analyzed. Methods for labeling
biological molecules such as polypeptides and antibodies are
well-known in the art. Any of a wide variety of detectable agents
can be used in the practice of the present invention. Suitable
detectable agents include, but are not limited to: various ligands,
radionuclides, fluorescent dyes, chemiluminescent agents,
microparticles (such as, for example, quantum dots, nanocrystals,
phosphors and the like), enzymes (such as, e.g., those used in an
ELISA, i.e., horseradish peroxidase, beta-galactosidase,
luciferase, alkaline phosphatase), colorimetric labels, magnetic
labels, and biotin, digoxigenin or other haptens and proteins for
which antisera or monoclonal antibodies are available.
[0183] In certain embodiments, the binding agents (e.g.,
antibodies) may be immobilized on a carrier or support (e.g., a
bead, a magnetic particle, a latex particle, a microtiter plate
well, a cuvette, or other reaction vessel). Examples of suitable
carrier or support materials include agarose, cellulose,
nitrocellulose, dextran, Sephadex.RTM., Sepharose.RTM., liposomes,
carboxymethyl cellulose, polyacrylamides, polystyrene, gabbros,
filter paper, magnetite, ion-exchange resin, plastic film, plastic
tube, glass, polyamine-methyl vinyl-ether-maleic acid copolymer,
amino acid copolymer, ethylene-maleic acid copolymer, nylon, silk,
and the like. Binding agents may be indirectly immobilized using
second binding agents specific for the first binding agents (e.g.,
mouse antibodies specific for the protein markers may be
immobilized using sheep anti-mouse IgG Fc fragment specific
antibody coated on the carrier or support).
[0184] Protein expression levels in a biological sample may be
determined using immunoassays. Examples of such assays are time
resolved fluorescence immunoassays (TR-FIA), radioimmunoassays,
enzyme immunoassays (e.g., ELISA), immunofluorescence
immunoprecipitation, latex agglutination, hemagglutination, Western
blot, and histochemical tests, which are conventional methods
well-known in the art. Methods of detection and quantification of
the signal generated by the complex formed by binding of the
binding agent with the protein marker will depend on the nature of
the assay and of the detectable moiety (e.g., fluorescent
moiety).
[0185] In one example, the presence or amount of protein expression
of a gene (e.g., an aHUS biomarker protein depicted in Table 1) can
be determined using a Western blotting technique. For example, a
lysate can be prepared from a biological sample, or the biological
sample (e.g., biological fluid) itself, can be contacted with
Laemmli buffer and subjected to sodium-dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE-resolved
proteins, separated by size, can then be transferred to a filter
membrane (e.g., nitrocellulose) and subjected to immunoblotting
techniques using a detectably-labeled antibody specific to the
protein of interest. The presence or amount of bound
detectably-labeled antibody indicates the presence or amount of
protein in the biological sample.
[0186] In another example, an immunoassay can be used for detecting
and/or measuring the protein expression of an aHUS biomarker
protein (e.g., one depicted in Table 1). As above, for the purposes
of detection, an immunoassay can be performed with an antibody that
bears a detection moiety (e.g., a fluorescent agent or enzyme).
Proteins from a biological sample can be conjugated directly to a
solid-phase matrix (e.g., a multi-well assay plate, nitrocellulose,
agarose, Sepharose.RTM., encoded particles, or magnetic beads) or
it can be conjugated to a first member of a specific binding pair
(e.g., biotin or streptavidin) that attaches to a solid-phase
matrix upon binding to a second member of the specific binding pair
(e.g., streptavidin or biotin). Such attachment to a solid-phase
matrix allows the proteins to be purified away from other
interfering or irrelevant components of the biological sample prior
to contact with the detection antibody and also allows for
subsequent washing of unbound antibody. Here, as above, the
presence or amount of bound detectably-labeled antibody indicates
the presence or amount of protein in the biological sample.
[0187] Alternatively, the protein expression levels may be
determined using mass spectrometry based methods or image-based
methods known in the art for the detection of proteins. Other
suitable methods include 2D-gel electrophoresis, proteomics-based
methods such as the identification of individual proteins recovered
from the gel (e.g., by mass spectrometry or N-terminal sequencing)
and/or bioinformatics.
[0188] Methods for detecting or measuring protein expression can,
optionally, be performed in formats that allow for rapid
preparation, processing, and analysis of multiple samples. This can
be, for example, in multi-well assay plates (e.g., 96 wells or 386
wells) or arrays (e.g., protein chips). Stock solutions for various
reagents can be provided manually or robotically, and subsequent
sample preparation, pipetting, diluting, mixing, distribution,
washing, incubating (e.g., hybridization), sample readout, data
collection (optical data) and/or analysis (computer aided image
analysis) can be done robotically using commercially available
analysis software, robotics, and detection instrumentation capable
of detecting the signal generated from the assay. Examples of such
detectors include, but are not limited to, spectrophotometers,
luminometers, fluorimeters, and devices that measure radioisotope
decay. Exemplary high-throughput cell-based assays (e.g., detecting
the presence or level of a target protein in a cell) can utilize
ArrayScan.RTM. VTI HCS Reader or KineticScan.RTM. HCS Reader
technology (Cellomics Inc., Pittsburg, Pa.).
[0189] Methods for determining the activity of vWF are also known
in the art and described herein (e.g., the working examples). See
also, e.g., Horvath et al. (2004) Exp Clin Cardiol 9(10):31-34.
Commercial kits are also available--Instrumentation Laboratory
(Bedford, Mass.; catalogue number: 0020004700) and Quest
Diagnostics (Madison, N.J.).
[0190] In some embodiments, the protein expression level (or
activity) of at least two aHUS biomarker proteins (e.g., at least
three proteins, at least four proteins, at least five proteins, at
least six proteins, at least seven proteins, at least eight
proteins, at least nine proteins, at least 10 proteins, at least 11
proteins, at least 12 proteins, at least 13 proteins, at least 14
proteins, at least 15 proteins, at least 16 proteins, at least 17
proteins, at least 18 proteins, at least 19 proteins, at least 20
proteins, at least 21 proteins, at least 22 proteins, at least 23
proteins, or at least 24 proteins or more) can be assessed and/or
measured.
[0191] In some embodiments, the biological fluid in which the aHUS
biomarker proteins are measured is blood. In some embodiments, the
biological fluid is a blood fraction, e.g., serum or plasma. In
some embodiments, the biological fluid is urine. In some
embodiments, all of the measurements are performed on one
biological fluid sample (e.g., a serum sample). In some
embodiments, measurements are performed on at least two different
biological fluids obtained from the subject. For example, in some
embodiments, the concentration or activity of one or more aHUS
biomarker proteins is measured in a serum sample obtained from the
patient. In some embodiments, a blood sample and a urine sample are
available so as to allow for testing of different analytes in two
different sample matrices.
[0192] The subject can be, e.g., a human having, suspected of
having, or at risk for developing, aHUS. The subject can be one who
has been (or is being) treated with an inhibitor of complement
(e.g., an inhibitor of complement component C5 such as an anti-C5
antibody). The treatment can have occurred less than one month
(e.g., less than 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20,
19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or
1 day) prior to obtaining the sample from the subject.
[0193] The method can further include the step of determining
whether the subject has or is at risk of developing aHUS. Where the
subject has been treated or is being treated with a complement
inhibitor (e.g., an anti-C5 antibody) under a predetermined dosing
schedule, the method can further include determining whether the
patient is responsive (therapeutically) to the complement inhibitor
therapy.
[0194] In some embodiments of any of the methods described herein,
the method requires recording the measured value(s) of the
concentration of the at least one aHUS biomarker protein. The
recordation can be written or on a computer readable medium. The
method can also include communicating the measured value(s) of the
concentration of the at least one aHUS biomarker protein to the
subject and/or to a medical practitioner in whose care the subject
is placed.
[0195] In some embodiments, any of the methods described herein can
include the step of administering to the subject the complement
inhibitor at a higher dose or with an increased frequency of
dosing, relative to the predetermined dosing schedule, if the
subject is not responsive to treatment with the inhibitor under the
predetermined dosing schedule.
[0196] Some of the methods described herein involve comparing the
measured concentration or activity of an aHUS biomarker protein (as
measured in a biological sample obtained from a subject) to a
control sample. In some embodiments, control sample is obtained
from the subject prior to administering to the subject a complement
inhibitor (e.g., a C5 inhibitor such as eculizumab). In some
embodiments, the control sample can be (or can be based on), e.g.,
a collection of samples obtained from one or more (e.g., two,
three, four, five, six, seven, eight, nine, 10, 15, 20, 25, 30, 35,
or 40 or more) healthy individuals that have not been administered
a complement inhibitor. In some embodiments, the control sample can
be (or can be based on), e.g., a pooled sample obtained from two or
more (e.g., two, three, four, five, six, seven, eight, nine, 10,
15, 20, 25, 30, 35, or 40 or more) individuals. In some embodiments
of any of the methods described herein, the pooled samples can be
from healthy individuals, or at least, individuals who do not have
or are not suspected of having (nor at risk for developing) aHUS.
For example, determining whether a subject is one having aHUS can
involve comparing the measured concentration of one or more serum
biomarkers in the subject and comparing the measured concentration
to the average concentration of the same biomarkers in the pooled
healthy samples. Similarly, determining whether the concentration
or activity of an aHUS associated biomarker has been reduced
following treatment with a complement inhibitor can involve
comparing the concentration or activity of the protein in a
biological fluid obtained from a subject prior to treatment with a
complement inhibitor to the concentration of protein in a sample of
the same biological fluid obtained from the patient after treatment
with the inhibitor (e.g., one day, two days, three days, four days,
five days, six days, 1 week, 2 weeks, 3 weeks, a month, 6 weeks,
two months, or three months after treatment (e.g., the first of a
series of treatment in chronic therapy) with the inhibitor).
[0197] In some embodiments, determining whether a complement
inhibitor has produced a desired effect (e.g., a reduction in the
concentration or activity of an aHUS biomarker protein) in a human
can be performed by querying whether the post-treatment
concentration of the protein falls within a predetermined range
indicative of responsiveness to a complement inhibitor by a human.
In some embodiments, determining whether a complement inhibitor has
produced a desired effect in a human can include querying if the
post-treatment concentration or activity of one or more aHUS
biomarker proteins falls above or below a predetermined cut-off
value. A cut-off value is typically the concentration or activity
of a given protein in a given biological fluid above or below which
is considered indicative of a certain phenotype--e.g.,
responsiveness to therapy with a complement inhibitor.
[0198] In some embodiments of any of the methods described herein,
the same practitioner may administer the complement inhibitor to
the subject prior to determining whether a change in the
concentration or activity of one or more aHUS biomarker proteins
has occurred, whereas in some embodiments, the practitioner who
administers the inhibitor to the subject is different from the
practitioner who determines whether a response has occurred in the
subject. In some embodiments, the practitioner may obtain a
biological sample (e.g., the blood sample) from the subject prior
to administration of the inhibitor. In some embodiments, the
practitioner may obtain a biological sample (e.g., a blood sample)
from the subject following the administration of the inhibitor to
the subject. In some embodiments, the post-treatment sample can be
obtained from the subject less than 48 (e.g., less than 47, 46, 45,
44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28,
27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11,
10, nine, eight, seven, six, five, four, three, two, or even less
than one) hour following administration of the inhibitor to the
subject. In some embodiments, the post-treatment sample can be
obtained from the subject less than 20 (e.g., less than 19, 18, 17,
16, 15, 14, 13, 12, 11, 10, nine, eight, seven, six, five, four,
three, two, or one) day(s) after administering to the subject the
inhibitor. In some embodiments, the biological sample is obtained
from the subject no more than 20 (e.g., no more than 19, 18, 17,
16, 15, 14, 13, 12, 11, 10, nine, eight, seven, six, five, four,
three, two, or one) day(s) after the inhibitor is administered to
the subject.
[0199] In some embodiments, various steps of the methods described
herein can be performed by more than one practitioner. For example,
one practitioner may analyze (e.g., measure the concentration or
activity of one or more aHUS biomarker proteins in) the pre- and
post-treatment samples obtained from the subject. Another
practitioner may receive information regarding the analysis of the
samples by the first practitioner to thereby determine whether,
e.g., the subject has responded to treatment with a complement
inhibitor. In some embodiments, yet another practitioner may obtain
a pre-treatment biological sample from a patient and a fourth
practitioner may obtain a post-treatment biological sample from the
subject. In some embodiments, all steps are carried out by the same
practitioner.
Biological Samples and Sample Collection
[0200] Suitable biological samples for use in the methods described
herein include, e.g., any biological fluid. A biological sample can
be, for example, a specimen obtained from a subject (e.g., a mammal
such as a human) or can be derived from such a subject. A
biological sample can also be a biological fluid such as urine,
whole blood or a fraction thereof (e.g., plasma or serum), saliva,
semen, sputum, cerebrospinal fluid, tears, or mucus. A biological
sample can be further fractionated, if desired, to a fraction
containing particular analytes (e.g., proteins) of interest. For
example, a whole blood sample can be fractionated into serum or
into fractions containing particular types of proteins. If desired,
a biological sample can be a combination of different biological
samples from a subject such as a combination of two different
fluids.
[0201] Biological samples suitable for the invention may be fresh
or frozen samples collected from a subject, or archival samples
with known diagnosis, treatment and/or outcome history. The
biological samples can be obtained from a subject, e.g., a subject
having, suspected of having, or at risk of developing, a
complement-associated disorder such as aHUS. Any suitable methods
for obtaining the biological samples can be employed, although
exemplary methods include, e.g., phlebotomy, swab (e.g., buccal
swab), lavage, or fine needle aspirate biopsy procedure. Biological
samples can also be obtained from bone marrow.
[0202] In some embodiments, a protein extract may be prepared from
a biological sample. In some embodiments, a protein extract
contains the total protein content. Methods of protein extraction
are well known in the art. See, e.g., Roe (2001) "Protein
Purification Techniques: A Practical Approach", 2.sup.nd Edition,
Oxford University Press. Numerous different and versatile kits can
be used to extract proteins from bodily fluids and tissues, and are
commercially available from, for example, BioRad Laboratories
(Hercules, Calif.), BD Biosciences Clontech (Mountain View,
Calif.), Chemicon International, Inc. (Temecula, Calif.),
Calbiochem (San Diego, Calif.), Pierce Biotechnology (Rockford,
Ill.), and Invitrogen Corp. (Carlsbad, Calif.).
[0203] Methods for obtaining and/or storing samples that preserve
the activity or integrity of cells in the biological sample are
well known to those skilled in the art. For example, a biological
sample can be further contacted with one or more additional agents
such as appropriate buffers and/or inhibitors, including protease
inhibitors, the agents meant to preserve or minimize changes (e.g.,
changes in osmolarity or pH) in protein structure. Such inhibitors
include, for example, chelators such as ethylenediamine tetraacetic
acid (EDTA), ethylene glycol tetraacetic acid (EGTA), protease
inhibitors such as phenylmethylsulfonyl fluoride (PMSF), aprotinin,
and leupeptin. Appropriate buffers and conditions for storing or
otherwise manipulating whole cells are described in, e.g., Pollard
and Walker (1997), "Basic Cell Culture Protocols," volume 75 of
Methods in molecular biology, Humana Press; Masters (2000) "Animal
cell culture: a practical approach," volume 232 of Practical
approach series, Oxford University Press; and Jones (1996) "Human
cell culture protocols," volume 2 of Methods in molecular medicine,
Humana Press.
[0204] A sample also can be processed to eliminate or minimize the
presence of interfering substances. For example, a biological
sample can be fractionated or purified to remove one or more
materials (e.g., cells) that are not of interest. Methods of
fractionating or purifying a biological sample include, but are not
limited to, flow cytometry, fluorescence activated cell sorting,
and sedimentation.
Complement Inhibitors
[0205] Any compound which binds to and inhibits, or otherwise
inhibits, the generation and/or activity of any of the human
complement components may be utilized in accordance with the
present disclosure. For example, an inhibitor of complement can be,
e.g., a small molecule, a nucleic acid or nucleic acid analog, a
peptidomimetic, or a macromolecule that is not a nucleic acid or a
protein. These agents include, but are not limited to, small
organic molecules, RNA aptamers, L-RNA aptamers, Spiegelmers,
antisense compounds, double stranded RNA, small interfering RNA,
locked nucleic acid inhibitors, and peptide nucleic acid
inhibitors. In some embodiments, a complement inhibitor may be a
protein or protein fragment.
[0206] In some embodiments, the compositions contain antibodies
specific to a human complement component. Some compounds include
antibodies directed against complement components C1, C2, C3, C4,
C5, C6, C7, C8, C9, Factor D, Factor B, Factor P, MBL, MASP-1,
MASP-2, properdin, or a biologically-active fragment of any of the
foregoing, thus preventing the generation of the anaphylatoxic
activity associated with C5a and/or preventing the assembly of the
membrane attack complex C5b-9.
[0207] The compositions can also contain 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. Other compounds which may be utilized
to bind to or otherwise block the generation and/or activity of any
of the human complement components include, but are not limited to,
proteins, protein fragments, peptides, small molecules, RNA
aptamers including ARC 187 (which is commercially available from
Archemix Corporation, Cambridge, Mass.), L-RNA aptamers,
spiegelmers, antisense compounds, serine protease inhibitors,
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, etc.
[0208] In some embodiments, the complement inhibitor inhibits the
activation of complement. For example, the complement inhibitor can
bind to and inhibit the complement activation activity of C1 (e.g.,
C1q, C1r, or C1s) or the complement inhibitor can bind to and
inhibit (e.g., inhibit cleavage of) C2, C3, or C4. In some
embodiments, the inhibitor inhibits formation or assembly of the C3
convertase and/or C5 convertase of the alternative and/or classical
pathways of complement. In some embodiments, the complement
inhibitor inhibits terminal complement formation, e.g., formation
of the C5b-9 membrane attack complex. For example, an antibody
complement inhibitor may include an anti-C5 antibody. 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.
[0209] In some embodiments, the compositions described herein can
contain an inhibitor of human complement component C5 (e.g., an
antibody, or antigen-binding fragment thereof, that binds to a
human complement component C5 protein or a biologically-active
fragment thereof such as C5a or C5b). As used herein, an "inhibitor
of complement component C5" is any agent that inhibits: (i) the
expression, or proper intracellular trafficking or secretion by a
cell, of a complement component C5 protein; (ii) the activity of C5
cleavage fragments C5a or C5b (e.g., the binding of C5a to its
cognate cellular receptors or the binding of C5b to C6 and/or other
components of the terminal complement complex; see above); (iii)
the cleavage of a human C5 protein to form C5a and C5b; (iv) the
proper intracellular trafficking of, or secretion by a cell, of a
complement component C5 protein; or (v) the stability of C5 protein
or the mRNA encoding C5 protein. Inhibition of complement component
C5 protein expression includes: inhibition of transcription of a
gene encoding a human C5 protein; increased degradation of an mRNA
encoding a human C5 protein; inhibition of translation of an mRNA
encoding a human C5 protein; increased degradation of a human C5
protein; inhibition of proper processing of a pre-pro human C5
protein; or inhibition of proper trafficking or secretion by a cell
of a human C5 protein. Methods for determining whether a candidate
agent is an inhibitor of human complement component C5 are known in
the art and described herein.
[0210] An inhibitor of human complement component C5 can be, e.g.,
a small molecule, a polypeptide, a polypeptide analog, a nucleic
acid, or a nucleic acid analog.
[0211] "Small molecule" as used herein, is meant to refer to an
agent, which has a molecular weight preferably of less than about 6
kDa and most preferably less than about 2.5 kDa. Many
pharmaceutical companies have extensive libraries of chemical
and/or biological mixtures comprising arrays of small molecules,
often fungal, bacterial, or algal extracts, which can be screened
with any of the assays of the application. This application
contemplates using, among other things, small chemical libraries,
peptide libraries, or collections of natural products. Tan et al.
described a library with over two million synthetic compounds that
is compatible with miniaturized cell-based assays (J Am Chem Soc
(1998) 120:8565-8566). It is within the scope of this application
that such a library may be used to screen for agents that bind to a
target antigen of interest (e.g., complement component C5). There
are numerous commercially available compound libraries, such as the
Chembridge DIVERSet. Libraries are also available from academic
investigators, such as the Diversity set from the NCI developmental
therapeutics program. Rational drug design may also be employed.
For example, rational drug design can employ the use of crystal or
solution structural information on the human complement component
C5 protein. See, e.g., the structures described in Hagemann et al.
(2008) J Biol Chem 283(12):7763-75 and Zuiderweg et al. (1989)
Biochemistry 28(1):172-85. Rational drug design can also be
achieved based on known compounds, e.g., a known inhibitor of C5
(e.g., an antibody, or antigen-binding fragment thereof, that binds
to a human complement component C5 protein).
[0212] Peptidomimetics can be compounds in which at least a portion
of a subject polypeptide is modified, and the three dimensional
structure of the peptidomimetic remains substantially the same as
that of the subject polypeptide. Peptidomimetics may be analogues
of a subject polypeptide of the disclosure that are, themselves,
polypeptides containing one or more substitutions or other
modifications within the subject polypeptide sequence.
Alternatively, at least a portion of the subject polypeptide
sequence may be replaced with a nonpeptide structure, such that the
three-dimensional structure of the subject polypeptide is
substantially retained. In other words, one, two or three amino
acid residues within the subject polypeptide sequence may be
replaced by a non-peptide structure. In addition, other peptide
portions of the subject polypeptide may, but need not, be replaced
with a non-peptide structure. Peptidomimetics (both peptide and
non-peptidyl analogues) may have improved properties (e.g.,
decreased proteolysis, increased retention or increased
bioavailability). Peptidomimetics generally have improved oral
availability, which makes them especially suited to treatment of
disorders in a human or animal. It should be noted that
peptidomimetics may or may not have similar two-dimensional
chemical structures, but share common three-dimensional structural
features and geometry. Each peptidomimetic may further have one or
more unique additional binding elements.
[0213] Nucleic acid inhibitors can be used to bind to and inhibit a
target antigen of interest. The nucleic acid antagonist can be,
e.g., an aptamer. Aptamers are short oligonucleotide sequences that
can be used to recognize and specifically bind almost any molecule,
including cell surface proteins. The systematic evolution of
ligands by exponential enrichment (SELEX) process is powerful and
can be used to readily identify such aptamers. Aptamers can be made
for a wide range of proteins of importance for therapy and
diagnostics, such as growth factors and cell surface antigens.
These oligonucleotides bind their targets with similar affinities
and specificities as antibodies do (see, e.g., Ulrich (2006) Handb
Exp Pharmacol. 173:305-326).
[0214] In some embodiments, the complement inhibitor is a
non-antibody scaffold protein. These proteins are, generally,
obtained through combinatorial chemistry-based adaptation of
pre-existing antigen-binding proteins. For example, the binding
site of human transferrin for human transferrin receptor can be
modified using combinatorial chemistry to create a diverse library
of transferrin variants, some of which have acquired affinity for
different antigens. Ali et al. (1999) J Biol Chem 274:24066-24073.
The portion of human transferrin not involved with binding the
receptor remains unchanged and serves as a scaffold, like framework
regions of antibodies, to present the variant binding sites. The
libraries are then screened, as an antibody library is, against a
target antigen of interest to identify those variants having
optimal selectivity and affinity for the target antigen.
Non-antibody scaffold proteins, while similar in function to
antibodies, are touted as having a number of advantages as compared
to antibodies, which advantages include, among other things,
enhanced solubility and tissue penetration, less costly
manufacture, and ease of conjugation to other molecules of
interest. Hey et al. (2005) TRENDS Biotechnol 23(10):514-522.
[0215] One of skill in the art would appreciate that the scaffold
portion of the non-antibody scaffold protein can include, e.g., all
or part of: the Z domain of S. aureus protein A, human transferrin,
human tenth fibronectin type III domain, kunitz domain of a human
trypsin inhibitor, human CTLA-4, an ankyrin repeat protein, a human
lipocalin, human crystallin, human ubiquitin, or a trypsin
inhibitor from E. elaterium. Id.
[0216] In some embodiments, the complement inhibitor is an
antibody, or antigen-binding fragment thereof, which binds to a
human complement component C5 protein. (Hereinafter, the antibody
may sometimes be referred to as an "anti-C5 antibody.")
[0217] In some embodiments, the anti-C5 antibody can bind to an
epitope in the alpha chain of the human complement component C5
protein. Antibodies that bind to the alpha chain of C5 are
described in, for example, PCT application publication no. WO
2010/015608 and U.S. Pat. No. 6,355,245. In some embodiments, the
anti-C5 antibody can bind to an epitope in the beta chain of the
human complement component C5 protein. Antibodies that bind to the
C5 beta chain are described in, e.g., Moongkarndi et al. (1982)
Immunobiol 162:397; Moongkarndi et al. (1983) Immunobiol 165:323;
and Mollnes et al. (1988) Scand J Immunol 28:307-312.
[0218] Additional exemplary antigenic fragments of human complement
component C5 are disclosed in, e.g., U.S. Pat. No. 6,355,245, the
disclosure of which is incorporated herein by reference.
[0219] Additional anti-C5 antibodies, and antigen-binding fragments
thereof, suitable for use in the fusion proteins described herein
are described in, e.g., PCT application publication no. WO
2010/015608, the disclosure of which is incorporated herein by
reference in its entirety.
[0220] In some embodiments, the anti-C5 antibody specifically binds
to a human complement component C5 protein (e.g., the human C5
protein having the amino acid sequence depicted in SEQ ID NO:1).
The terms "specific binding" or "specifically binds" refer to two
molecules forming a complex (e.g., a complex between an antibody
and a complement component C5 protein) that is relatively stable
under physiologic conditions. Typically, binding is considered
specific when the association constant (K.sub.a) is higher than
10.sup.6 M.sup.-1. Thus, an antibody can specifically bind to a C5
protein with a K.sub.a of at least (or greater than) 10.sup.6
(e.g., at least or greater than 10.sup.7, 10.sup.8, 10.sup.9,
10.sup.10, 10.sup.1110.sup.12, 10.sup.13, 10.sup.14, or 10.sup.15
or higher) M.sup.-1. Examples of antibodies that specifically bind
to a human complement component C5 protein are described in, e.g.,
U.S. Pat. No. 6,355,245, the disclosure of which is incorporated
herein by reference in its entirety.
[0221] The anti-C5 antibodies described herein can have activity in
blocking the generation or activity of the C5a and/or C5b active
fragments of a complement component C5 protein (e.g., a human C5
protein). Through this blocking effect, the anti-C5 antibodies
inhibit, e.g., the proinflammatory effects of C5a and the
generation of the C5b-9 membrane attack complex (MAC) at the
surface of a cell. Anti-C5 antibodies that have the ability to
block the generation of C5a are described in, e.g., Moongkarndi et
al. (1982) Immunobiol 162:397 and Moongkarndi et al. (1983)
Immunobiol 165:323.
[0222] In some embodiments, an anti-C5 antibody, or antigen-binding
fragment thereof, can reduce the ability of a C5 protein to bind to
human complement component C3b (e.g., C3b present in an AP or CP C5
convertase complex) by greater than 50 (e.g., greater than 55, 60,
65, 70, 75, 80, 85, 90, or 95 or more) %. In some embodiments, upon
binding to a C5 protein, the anti-C5 antibody or antigen-binding
fragment thereof can reduce the ability of the C5 protein to bind
to complement component C4b (e.g., C4b present in a CP C5
convertase) by greater than 50 (e.g., greater than 55, 60, 65, 70,
75, 80, 85, 90, or 95 or more) %. Methods for determining whether
an antibody can block the generation or activity of the C5a and/or
C5b active fragments of a complement component C5 protein, or
binding to complement component C4b or C3b, are known in the art
and described in, e.g., U.S. Pat. No. 6,355,245 and Wurzner et al.
(1991) Complement Inflamm 8:328-340.
[0223] In some embodiments, the composition comprises, and/or the
antibody is, eculizumab (Soliris.RTM.; Alexion Pharmaceuticals,
Inc., Cheshire, Conn.). (See, e.g., Kaplan (2002) Curr Opin
Investig Drugs 3(7):1017-23; Hill (2005) Clin Adv Hematol Oncol
3(11):849-50; and Rother et al. (2007) Nature Biotechnology
25(11):1256-1488.) In some embodiments, the composition comprises,
and/or the antibody is, pexelizumab (Alexion Pharmaceuticals, Inc.,
Cheshire, Conn.). (See, e.g., Whiss (2002) Curr Opin Investig Drugs
3(6):870-7; Patel et al. (2005) Drugs Today (Barc) 41(3):165-70;
and Thomas et al. (1996) Mol Immunol 33(17-18):1389-401.)
[0224] In some embodiments, the C5 inhibitor is an antibody that
binds to C5a (sometimes referred to herein as "an anti-C5a
antibody"). In some embodiments, the antibody binds to C5a, but not
to full-length C5. In some embodiments, the binding of an antibody
to C5a can inhibit the biological activity of C5a. Methods for
measuring C5a activity include, e.g., chemotaxis assays, RIAs, or
ELISAs (see, e.g., Ward and Zvaifler (1971) J Clin Invest
50(3):606-16 and Wurzner et al. (1991) Complement Inflamm
8:328-340). In some embodiments, the binding of an antibody to C5a
can inhibit the interaction between C5a and C5aR1. Suitable methods
for detecting and/or measuring the interaction between C5a and
C5aR1 (in the presence and absence of an antibody) are known in the
art and described in, e.g., Mary and Boulay (1993) Eur J Haematol
51(5):282-287; Kaneko et al. (1995) Immunology 86(1):149-154;
Giannini et al. (1995) J Biol Chem 270(32):19166-19172; and U.S.
Patent Application Publication No. 20060160726. For example, the
binding of detectably labeled (e.g., radioactively labeled) C5a to
C5aR1-expressing peripheral blood mononuclear cells can be
evaluated in the presence and absence of an antibody. A decrease in
the amount of detectably-labeled C5a that binds to C5aR1 in the
presence of the antibody, as compared to the amount of binding in
the absence of the antibody, is an indication that the antibody
inhibits the interaction between C5a and C5aR1. In some
embodiments, the binding of an antibody to C5a can inhibit the
interaction between C5a and C5L2 (see below). Methods for detecting
and/or measuring the interaction between C5a and C5L2 are known in
the art and described in, e.g., Ward (2009) J Mol Med 87(4):375-378
and Chen et al. (2007) Nature 446(7132):203-207 (see below).
[0225] In some embodiments, the C5 inhibitor is an antibody that
binds to C5b (sometimes referred to herein as "an anti-C5b
antibody"). In some embodiments, the antibody binds to C5b, but
does not bind to full-length C5. The structure of C5b is described
in, e.g., Muller-Eberhard (1985) Biochem Soc Symp 50:235-246; and
Yamamoto and Gewurz (1978) J Immunol 120(6):2008-2015. As described
above, C5b combines with C6, C7, and C8 to form the C5b-8 complex
at the surface of the target cell. Protein complex intermediates
formed during the series of combinations include C5b-6 (including
C5b and C6), C5b-7 (including C5b, C6, and C7), and C5b-8
(including C5b, C6, C7, and C8). 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.
[0226] In some embodiments, the binding of an antibody to C5b can
inhibit the interaction between C5b and C6. In some embodiments,
the binding of the antibody to C5b can inhibit the assembly or
activity of the C5b-9 MAC-TCC. In some embodiments, the binding of
an antibody to C5b can inhibit complement-dependent cell lysis
(e.g., in vitro and/or in vivo). Suitable methods for evaluating
whether an antibody inhibits complement-dependent lysis include,
e.g., hemolytic assays or other functional assays for detecting the
activity of soluble C5b-9. For example, a reduction in the
cell-lysing ability of complement in the presence of an antibody
can be measured by a hemolysis assay described by Kabat and Mayer
(eds.), "Experimental Immunochemistry, 2.sup.nd Edition," 135-240,
Springfield, Ill., C C Thomas (1961), pages 135-139, or a
conventional variation of that assay such as the chicken
erythrocyte hemolysis method as described in, e.g., Hillmen et al.
(2004) N Engl J Med 350(6):552.
[0227] Antibodies that bind to C5b as well as methods for making
such antibodies are known in the art. Commercially available
anti-C5b antibodies are available from a number of vendors
including, e.g., Hycult Biotechnology (catalogue number: HM2080;
clone 568) and Abcam.TM. (ab46151 or ab46168).
[0228] Methods for determining whether a particular agent is an
inhibitor of human complement component C5 are described herein and
are known in the art. For example, the concentration and/or
physiologic activity of C5a and C5b in a body fluid can be measured
by methods well known in the art. Methods for measuring C5a
concentration or activity include, e.g., chemotaxis assays, RIAs,
or ELISAs (see, e.g., Ward and Zvaifler (1971) J Clin Invest.
50(3):606-16 and Wurzner et al. (1991) Complement Inflamm.
8:328-340). For C5b, hemolytic assays or assays 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 agents capable of inhibiting human complement component
C5 such as an anti-C5 antibody, can be screened in order to, e.g.,
identify compounds that are useful in the methods described herein
and determine the appropriate dosage levels of such compounds.
[0229] Methods for determining whether a candidate compound
inhibits the cleavage of human C5 into forms C5a and C5b are known
in the art and described in, e.g., Moongkarndi et al. (1982)
Immunobiol 162:397; Moongkarndi et al. (1983) Immunobiol 165:323;
Isenman et al. (1980) J Immunol 124(1):326-31; Thomas et al. (1996)
Mol. Immunol 33(17-18):1389-401; and Evans et al. (1995) Mol.
Immunol 32(16):1183-95.
[0230] Inhibition of human complement component C5 can also reduce
the cell-lysing ability of complement in a subject's body fluids.
Such reductions of the cell-lysing ability of complement present
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, 2.sup.nd 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 as described
in, e.g., Hillmen et al. (2004) N Engl J Med 350(6):552.
[0231] Antibodies that bind to C3b and, for example, inhibit the
C3b convertase are also well known in the art. See for example, PCT
application publication nos. WO 2010/136311, WO 2009/056631, and WO
2008/154251, the disclosures of each of which are incorporated
herein by reference in their entirety. Antagonistic anti-C6 and
anti-C7 antibodies have been described in, e.g., Brauer et al.
(1996) Transplantation 61(4):588-594 and U.S. Pat. No.
5,679,345.
[0232] In some embodiments, the antibody is an anti-factor B
antibody (such as the monoclonal antibody 1379 produced by ATCC
Deposit No. PTA-6230). Anti-factor B antibodies are also described
in, e.g., Ueda et al. (1987) J Immunol 138(4):1143-9; Tanhehco et
al. (1999) Transplant Proc 31(5):2168-71; U.S. Pat. Nos. 7,999,082
and 7,964,705; and PCT publication no. WO 09/029,669.
[0233] In some embodiments, the antibody is an anti-factor D
antibody, e.g., an antibody described in Pascual et al. (1990) J
Immunol Methods 127:263-269; Sahu et al. (1993) Mol Immunol
30(7):679-684; Pascual et al. (1993) Eur J Immunol 23:1389-1392;
Niemann et al. (1984) J Immunol 132(2):809-815; U.S. Pat. No.
7,439,331; or U.S. patent application publication no.
20080118506.
[0234] In some embodiments, the antibody is an anti-properdin
antibody. Suitable anti-properdin antibodies are also well-known in
the art and include, e.g., U.S. patent application publication nos.
20110014614 and PCT application publication no. WO2009110918.
Methods for Treatment
[0235] Also provided herein are compositions and methods for
treating or preventing aHUS in a subject (e.g., a human). The
compositions (e.g., complement inhibitors and/or secondary agents)
can be administered to a subject, e.g., a human subject, using a
variety of methods that depend, in part, on the route of
administration. The route can be, e.g., intravenous injection or
infusion (IV), subcutaneous injection (SC), intraperitoneal (IP)
injection, or intramuscular injection.
[0236] Administration can be achieved by, e.g., local infusion,
injection, or by means of an implant. The implant can be of a
porous, non-porous, or gelatinous material, including membranes,
such as sialastic membranes, or fibers. The implant can be
configured for sustained or periodic release of the composition to
the subject. See, e.g., U.S. patent publication no. 20080241223;
U.S. Pat. Nos. 5,501,856; 4,863,457; and 3,710,795; and European
patent nos. EP488401 and EP430539, the disclosures of each of which
are incorporated herein by reference in their entirety. The
composition can be delivered to the subject by way of an
implantable device based on, e.g., diffusive, erodible or
convective systems, e.g., osmotic pumps, biodegradable implants,
electrodiffusion systems, electroosmosis systems, vapor pressure
pumps, electrolytic pumps, effervescent pumps, piezoelectric pumps,
erosion-based systems, or electromechanical systems.
[0237] A suitable dose of a complement inhibitor (e.g., an anti-C5
antibody or fragment thereof), which dose is capable of treating or
preventing aHUS in a subject, can depend on a variety of factors
including, e.g., the age, sex, and weight of a subject to be
treated and the particular inhibitor compound used. For example, a
different dose of an siRNA specific for human C5 may be required to
treat a subject with aHUS as compared to the dose of an anti-C5
antibody required to treat the same patient. Other factors
affecting the dose administered to the subject include, e.g., the
type or severity of the aHUS. For example, a subject having
CFH-associated aHUS may require administration of a different
dosage of the inhibitor than a subject with MCP-associated aHUS.
Other factors can include, e.g., other medical disorders
concurrently or previously affecting the subject, the general
health of the subject, the genetic disposition of the subject,
diet, time of administration, rate of excretion, drug combination,
and any other additional therapeutics that are administered to the
subject. It should also be understood that a specific dosage and
treatment regimen for any particular subject will depend upon the
judgment of the treating medical practitioner (e.g., doctor or
nurse).
[0238] The inhibitor can be administered as a fixed dose, or in a
milligram per kilogram "mg/kg" dose. In some embodiments, the dose
can also be chosen to reduce or avoid production of antibodies or
other host immune responses against one or more active agents in
the composition. While in no way intended to be limiting, exemplary
dosages of an inhibitor, such as an anti-C5 antibody, include,
e.g., 1-100 mg/kg, 0.5-50 mg/kg, 0.1-100 mg/kg, 0.5-25 mg/kg, 1-20
mg/kg, and 1-10 mg/kg of body weight.
[0239] In some embodiments, a human can be intravenously
administered an anti-C5 antibody (e.g., eculizumab) at a dose of
about 900 mg about every 12 (e.g., about every 10, 11, 13, 14, 15,
16, 17, 18, 19, 20, 21, 28, 30, 42, or 49 or more) days. See, e.g.,
Hill et al. (2005) Blood 106(7):2559.
[0240] In some embodiments, a human can be intravenously
administered an anti-C5 antibody (e.g., eculizumab) at a dose of
about 600 (e.g., about 625, 650, 700, 725, 750, 800, 825, 850, 875,
900, 925, 950, or 1,000 or more) mg every week, optionally, for two
or more (e.g., three, four, five, six, seven, or eight or more)
weeks. Following the initial treatment, the human can be
administered the antibody at a dose of about 900 mg about every 14
(e.g., about every 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 28,
30, 42, or 49 or more) days, e.g., as a maintenance dose. See,
e.g., Hillmen et al. (2004) N Engl J Med. 350(6):552-9 and
Dmytrijuk et al. (2008) The Oncologist 13(9):993.
[0241] In some embodiments, a human can be intravenously
administered an anti-C5 antibody (e.g., eculizumab) at a dose of
about 900 (e.g., 925, 950, 975, 1000, 1100, or 1200 or more) mg
every week, optionally, for two or more (e.g., three, four, five,
six, seven, or eight or more) weeks. Following the initial
treatment, the human can be administered the antibody at a dose of
about 1200 mg about every 14 (e.g., about every 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 28, 30, 42, or 49 or more) days, e.g., as a
maintenance dose. See, e.g., International patent application
publication no. WO 2010/054403.
[0242] As used herein, "chronically administered," "chronic
treatment," "treating chronically," or similar grammatical
variations thereof refer to a treatment regimen that is employed to
maintain a certain threshold concentration of a therapeutic agent
in the blood of a patient in order to completely or substantially
suppress systemic complement activity in the patient over a
prolonged period of time. Accordingly, a patient chronically
treated with a complement inhibitor can be treated for a period of
time that is greater than or equal to 2 weeks (e.g., 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,
25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,
42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or 52 weeks; 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, or 12 months; or 1, 1.5, 2, 2.5, 3, 3.5, 4,
4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, or 12 years
or for the remainder of the patient's life) with the inhibitor in
an amount and with a dosing frequency that are sufficient to
maintain a concentration of the inhibitor in the patient's blood
that inhibits or substantially inhibits systemic complement
activity in the patient. In some embodiments, the complement
inhibitor can be chronically administered to a patient in need
thereof in an amount and with a frequency that are effective to
maintain serum hemolytic activity at less than or equal to 20
(e.g., 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5) %.
See, e.g., Hill et al. (2005) Blood 106(7):2559. In some
embodiments, the complement inhibitor can be administered to a
patient in an amount and with a frequency that are effective to
maintain serum lactate dehydrogenase (LDH) levels at within at
least 20 (e.g., 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6,
or 5) % of the normal range for LDH. See Hill et al. (2005) supra.
In some embodiments, the complement inhibitor is administered to
the patient in an amount and with a frequency that are effective to
maintain a serum LDH level less than 550 (e.g., less than 540, 530,
520, 510, 500, 490, 480, 470, 460, 450, 440, 430, 420, 410, 400,
390, 380, 370, 360, 350, 340, 330, 320, 310, 300, 290, 280, or less
than 270) IU/L. To maintain systemic complement inhibition in a
patient, the complement inhibitor can be chronically administered
to the patient, e.g., once a week, once every two weeks, twice a
week, once a day, once a month, or once every three weeks.
[0243] A pharmaceutical composition can include a therapeutically
effective amount of an inhibitor of human complement component C5
(e.g., an anti-C5 antibody or antigen-binding fragment thereof).
Such effective amounts can be readily determined by one of ordinary
skill in the art based, in part, on the effect of the administered
inhibitor, or the combinatorial effect of the antibody and one or
more additional active agents, if more than one agent is used. A
therapeutically effective amount of an inhibitor of human
complement component C5 (e.g., an anti-C5 antibody) can also vary
according to factors such as the disease state, age, sex, and
weight of the individual, and the ability of the antibody (and one
or more additional active agents) to elicit a desired response in
the individual, e.g., amelioration of at least one condition
parameter, e.g., amelioration of at least one symptom of aHUS. For
example, a therapeutically effective amount of an inhibitor of
human complement component C5 (e.g., an anti-C5 antibody) can
inhibit (lessen the severity of or eliminate the occurrence of)
and/or prevent thrombocytopenia, microangiopathic hemolytic anemia,
renal failure, and/or any one of the symptoms of aHUS known in the
art or described herein. A therapeutically effective amount is also
one in which any toxic or detrimental effects of the composition
are outweighed by the therapeutically beneficial effects.
[0244] The terms "therapeutically effective amount" or
"therapeutically effective dose," or similar terms used herein are
intended to mean an amount of an agent (e.g., an inhibitor of human
complement component 5) that will elicit the desired biological or
medical response (e.g., an improvement in one or more symptoms of
aHUS). In some embodiments, a composition described herein contains
a therapeutically effective amount of an inhibitor of human
complement component C5. In some embodiments, a composition
described herein contains a therapeutically effective amount of an
antibody, or antigen-binding fragment thereof, which binds to a
complement component C5 protein. In some embodiments, the
composition contains two or more (e.g., three, four, five, six,
seven, eight, nine, 10, or 11 or more) different inhibitors of
human complement component C5 such that the composition as a whole
is therapeutically effective. For example, a composition can
contain an antibody that binds to a human C5 protein and an siRNA
that binds to, and promotes the degradation of, an mRNA encoding a
human C5 protein, wherein the antibody and siRNA are each at a
concentration that when combined are therapeutically effective. In
some embodiments, the composition contains the inhibitor and one or
more second active agents such that the composition as a whole is
therapeutically effective. For example, the composition can contain
an antibody that binds to a human C5 protein and another agent
useful for treating or preventing aHUS.
[0245] Toxicity and therapeutic efficacy of such compositions can
be determined by known pharmaceutical procedures in cell cultures
or experimental animals (animal models of aHUS). These procedures
can be used, e.g., for determining the LD.sub.50 (the dose lethal
to 50% of the population) and the ED.sub.50 (the dose
therapeutically effective in 50% of the population). The dose ratio
between toxic and therapeutic effects is the therapeutic index and
it can be expressed as the ratio LD.sub.50/ED.sub.50. Compositions,
or inhibitors (e.g., anti-C5 antibodies) of the compositions, that
exhibit high therapeutic indices are preferred. While compositions
that exhibit toxic side effects may be used, care should be taken
to design a delivery system that targets such compounds to the site
of affected tissue and to minimize potential damage to normal cells
and, thereby, reduce side effects.
[0246] The data obtained from the cell culture assays and animal
studies can be used in formulating a range of dosage for use in
humans. Suitable animal models of aHUS are known in the art and are
described in, e.g., Atkinson et al. (2007) Journal of Experimental
Medicine 204(6):1245-1248. The dosage of such inhibitors lies
generally within a range of circulating concentrations of the
inhibitors (e.g., an anti-C5 antibody or antigen-binding fragment
thereof) that include the ED.sub.50 with little or no toxicity. The
dosage may vary within this range depending upon the dosage form
employed and the route of administration utilized. For an inhibitor
of human complement component C5 (e.g., an anti-C5 antibody) used
as described herein (e.g., for treating or preventing aHUS), the
therapeutically effective dose can be estimated initially from cell
culture assays. A dose can be formulated in animal models to
achieve a circulating plasma concentration range that includes the
IC.sub.50 (i.e., the concentration of the test compound which
achieves a half-maximal inhibition of symptoms) as determined in
cell culture. Such information can be used to more accurately
determine useful doses in humans. Levels in plasma may be measured,
for example, by high performance liquid chromatography.
[0247] In some embodiments, the required dose of an inhibitor of
human complement component C5 can be determined based on the
concentration of human C5 protein in the subject's blood. For
example, a subject having a higher concentration of circulating
human C5 protein levels may require a higher dose of a human C5
inhibitor than a subject having lower levels of circulating human
C5. Methods for determining the concentration of human complement
component C5 in a blood-derived fluid sample from a subject are
known in the art and described in, e.g., Rawal et al. (1998) J Biol
Chem 273(27):16828-16835.
[0248] In some embodiments, the methods can be performed in
conjunction with other therapies for aHUS. For example, the
composition can be administered to a subject at the same time,
prior to, or after, nephrectomy (e.g., bilateral nephrectomy),
dialysis, a plasma exchange, or a plasma infusion (see, e.g., Noris
et al. (2005) "Non-shiga toxin-associated hemolytic uremic
syndrome." In: Zipfel P (ed). Complement and Kidney Disease. Basel:
Birkhauser-Verlag, 65-83).
[0249] A "subject," as used herein, can be any mammal. For example,
a subject can be a human, a non-human primate (e.g., monkey,
baboon, or chimpanzee), a horse, a cow, a pig, a sheep, a goat, a
dog, a cat, a rabbit, a guinea pig, a gerbil, a hamster, a rat, or
a mouse. In some embodiments, the subject is an infant (e.g., a
human infant).
[0250] As used herein, a subject "in need of prevention," "in need
of treatment," or "in need thereof," refers to one, who by the
judgment of an appropriate medical practitioner (e.g., a doctor, a
nurse, or a nurse practitioner in the case of humans; a
veterinarian in the case of non-human mammals), would reasonably
benefit from a given treatment (such as treatment with a
composition comprising an inhibitor of human complement component
C5).
[0251] As used herein, a subject "at risk for developing aHUS" is a
subject having one or more (e.g., two, three, four, five, six,
seven, or eight or more) risk factors for developing the disorder.
Risk factors for aHUS are well known in the art of medicine and
include, e.g., a predisposition to develop the condition, i.e., a
family history of the condition or a genetic predisposition to
develop the condition such as, e.g., one or more mutations in
complement Factor H(CFH), membrane cofactor protein (MCP; CD46),
C4b-binding protein, complement factor B (CFB), or complement
factor I (CFI). See, e.g., Warwicker et al. (1998) Kidney Int
53:836-844; Richards et al. (2001) Am J Hum Genet 68:485-490;
Caprioli et al. (2001) Am Soc Nephrol 12:297-307; Neuman et al.
(2003) J Med Genet 40:676-681; Richards et al. (2006) Proc Natl
Acad Sci USA 100:12966-12971; Fremeaux-Bacchi et al. (2005) J Am
Soc Nephrol 17:2017-2025; Esparza-Gordillo et al. (2005) Hum Mol
Genet 14:703-712; Goicoechea de Jorge et al. (2007) Proc Natl Acad
Sci USA 104(1):240-245; Blom et al. (2008) J Immunol
180(9):6385-91; and Fremeaux-Bacchi et al. (2004) J Medical Genet
41:e84). See also Kavanagh et al. (2006), supra. Risk factors also
include, e.g., infection with Streptococcus pneumoniae, pregnancy,
cancer, exposure to anti-cancer agents (e.g., quinine, mitomycin C,
cisplatin, or bleomycin), exposure to immunotherapeutic agents
(e.g., cyclosporine, OKT3, or interferon), exposure to
anti-platelet agents (e.g., ticlopidine or clopidogrel), HIV
infection, transplantation, autoimmune disease, and combined
methylmalonic aciduria and homocystinuria (cblC). See, e.g.,
Constantinescu et al. (2004) Am J Kidney Dis 43:976-982; George
(2003) Curr Opin Hematol 10:339-344; Gottschall et al. (1994) Am J
Hematol 47:283-289; Valavaara et al. (1985) Cancer 55:47-50;
Miralbell et al. (1996) J Clin Oncol 14:579-585; Dragon-Durey et
al. (2005) J Am Soc Nephrol 16:555-63; and Becker et al. (2004)
Clin Infect Dis 39:S267-S275. Thus, a human at risk for developing
aHUS can be, e.g., one who has a family history of aHUS and/or one
who has an HIV infection. From the above it will be clear that
subjects "at risk for developing aHUS" are not all the subjects
within a species of interest.
[0252] A subject "suspected of having aHUS" is one having one or
more symptoms of the condition. Symptoms of this condition are
well-known to those of skill in the art of medicine and include,
e.g., severe hypertension, proteinuria, uremia, lethargy/fatigue,
irritability, thrombocytopenia, microangiopathic hemolytic anemia,
and renal function impairment (e.g., acute renal failure). It will
be clear from the foregoing passage that subjects "suspected of
having aHUS" are not all the subjects within a species of
interest.
[0253] aHUS can be genetic, acquired, or idiopathic. aHUS can be
considered genetic when two or more (e.g., three, four, five, or
six or more) members of the same family are affected by the disease
at least six months apart and exposure to a common triggering agent
has been excluded, or when one or more aHUS-associated gene
mutations (e.g., one or more mutations in CFH, MCP/CD46, CFB, or
CFI) are identified in a subject. For example, a subject can have
CFH-associated aHUS, CFB-associated aHUS, CFI-associated aHUS, or
MCP-associated aHUS. Up to 30% of genetic aHUS is associated with
mutations in CFH, 12% with mutations in MCP, 5-10% with mutations
in CFI, and less than 2% with mutations in CFB. Genetic aHUS can be
multiplex (i.e., familial; two or more affected family members) or
simplex (i.e., a single occurrence in a family). aHUS can be
considered acquired when an underlying environmental factor (e.g.,
a drug, systemic disease, or viral or bacterial agents that do not
result in Shiga-like exotoxins) can be identified. aHUS can be
considered idiopathic when no trigger (genetic or environmental) is
evident.
[0254] In some embodiments, the methods can include identifying the
subject as one having, suspected of having, or at risk for
developing aHUS. In addition to use of the aHUS biomarker profiling
described herein, laboratory tests can be performed to determine
whether a human subject has thrombocytopenia, microangiopathic
hemolytic anemia, or acute renal insufficiency. Thrombocytopenia
can be diagnosed by a medical professional as one or more of: (i) a
platelet count that is less than 150,000/mm.sup.3 (e.g., less than
60,000/mm.sup.3); (ii) a reduction in platelet survival time that
is reduced, reflecting enhanced platelet disruption in the
circulation; and (iii) giant platelets observed in a peripheral
smear, which is consistent with secondary activation of
thrombocytopoiesis. Microangiopathic hemolytic anemia can be
diagnosed by a medical professional as one or more of: (i)
hemoglobin concentrations that are less than 10 mg/dL (e.g., less
than 6.5 mg/dL); (ii) increased serum lactate dehydrogenase (LDH)
concentrations (>460 U/L); (iii) hyperbilirubinemia,
reticulocytosis, circulating free hemoglobin, and low or
undetectable haptoglobin concentrations; and (iv) the detection of
fragmented red blood cells (schistocytes) with the typical aspect
of burr or helmet cells in the peripheral smear together with a
negative Coombs test. See, e.g., Kaplan et al. (1992) "Hemolytic
Uremic Syndrome and Thrombotic Thrombocytopenic Purpura," Informa
Health Care (ISBN 0824786637) and Zipfel (2005) "Complement and
Kidney Disease," Springer (ISBN 3764371668).
[0255] Blood concentrations of C3 and C4 can also be used as a
measure of complement activation or dysregulation. In addition, a
subject's condition can be further characterized by identifying the
subject as harboring one or more mutations in a gene associated
with aHUS such as CFI, CFB, CFH, or MCP (supra). Suitable methods
for detecting a mutation in a gene include, e.g., DNA sequencing
and nucleic acid array techniques. See, e.g., Breslin et al. (2006)
Clin Am Soc Nephrol 1:88-99 and Goicoechea de Jorge et al. (2007)
Proc Natl Acad Sci USA 104:240-245.
[0256] In some embodiments, the inhibitor of human complement
component C5 (e.g., an anti-C5 antibody or antigen-binding fragment
thereof) can be administered to a subject as a monotherapy.
Alternatively, as described above, the inhibitor can be
administered to a subject as a combination therapy with another
treatment, e.g., another treatment for aHUS. For example, the
combination therapy can include administering to the subject (e.g.,
a human patient) one or more additional agents (e.g.,
anti-hypertensives) that provide a therapeutic benefit to the
subject who has, or is at risk of developing, aHUS. In some
embodiments, the inhibitor of human complement component C5 and the
one or more additional active agents are administered at the same
time. In other embodiments, the inhibitor is administered first in
time and the one or more additional active agents are administered
second in time. In some embodiments, the one or more additional
active agents are administered first in time and the inhibitor is
administered second in time.
[0257] The inhibitor of human complement component C5 can replace
or augment a previously or currently administered therapy. For
example, upon treating with an anti-C5 antibody or antigen-binding
fragment thereof, administration of the one or more additional
active agents can cease or diminish, e.g., be administered at lower
levels. In some embodiments, administration of the previous therapy
can be maintained. In some embodiments, a previous therapy will be
maintained until the level of inhibitor of human C5 reaches a level
sufficient to provide a therapeutic effect. The two therapies can
be administered in combination.
[0258] Monitoring a subject (e.g., a human patient) for an
improvement in aHUS, as defined herein, means evaluating the
subject for a change in a disease parameter, e.g., an improvement
in one or more symptoms of the disease. Such symptoms include any
of the symptoms of aHUS described herein. In some embodiments, the
evaluation is performed at least 1 hour, e.g., at least 2, 4, 6, 8,
12, 24, or 48 hours, or at least 1 day, 2 days, 4 days, 10 days, 13
days, 20 days or more, or at least 1 week, 2 weeks, 4 weeks, 10
weeks, 13 weeks, 20 weeks or more, after treatment begins. The
subject can be evaluated in one or more of the following periods:
prior to beginning of treatment; during the treatment; or after one
or more elements of the treatment have been administered.
Evaluating can include evaluating the need for further treatment,
e.g., evaluating whether a dosage, frequency of administration, or
duration of treatment should be altered. It can also include
evaluating the need to add or drop a selected therapeutic modality,
e.g., adding or dropping any of the treatments for aHUS described
herein.
Kits
[0259] Also provided are kits comprising various reagents and
materials useful for carrying out the methods described herein. The
procedures for measuring, diagnosing, evaluating, and/or assessing
described herein may be performed by diagnostic laboratories,
experimental laboratories, or individual practitioners. The
invention provides kits which can be used in any or all of these
settings.
[0260] In some embodiments, the kits described herein comprise
materials and reagents for, among other things, characterizing or
processing biological samples (e.g., biological fluids), measuring
biomarker levels (e.g., protein or nucleic acid levels), diagnosing
aHUS in a subject, or monitoring treatment response in a subject
according to the methods provided herein. In certain embodiments,
an inventive kit comprises at least one or more reagents that
specifically detect protein levels of one or more aHUS biomarker
proteins (e.g., those selected from Table 1) and, optionally,
instructions for using the kit. The kit can include, e.g., any of
the arrays described herein.
[0261] In some embodiments, the kits may include suitable control
samples (e.g., biological fluids from normal healthy individuals or
a solution comprising a known, control amount of a particular
analyte of interest). In some embodiments, kits of the invention
may include instructions for using the kit according to one or more
methods described herein and may comprise instructions for
processing the biological sample (e.g., a biological fluid)
obtained from the subject and/or for performing the test or
instructions for interpreting the results.
[0262] The following examples are intended to illustrate, not
limit, the invention.
EXAMPLES
[0263] To better understand the pathology of aHUS, the inventors
have collected samples of biological fluids (whole blood, serum,
plasma, and urine) from patients having aHUS or suspected of having
aHUS both before and, at several points, after initiating treatment
with a complement inhibitor (the anti-C5 antibody eculizumab). One
objective of this study was to define a series of clinically
definable parameters that could be used to monitor responsiveness
of patients to treatment with the complement inhibitor as well as
markers of the disease and progression or abatement thereof. The
inventors identified several proteins whose expression and/or
activity was correlated with either the aHUS disease state and/or
responsiveness of an aHUS patient to treatment with a complement
inhibitor. The proteins were those involved or associated with
complement and/or endothelial cell activation, inflammation, renal
injury, and coagulation (see Table 1).
[0264] For the study, a total of 41 adult subjects (27 females and
12 males) with a confirmed diagnosis of aHUS were recruited as were
normal healthy adult volunteers. All patients had confirmed aHUS at
screening based on one or more of the following characteristics:
platelet count less than 150.times.10.sup.9/L; hemoglobin levels at
less than the lower limit of normal; LDH levels that were greater
than or equal to 1.5 times the upper limit of normal; serum
creatinine levels that were greater than or equal to the upper
limit of normal; and an ADAMTS13 activity level that was greater
than 5%. All patients tested negative for Shiga toxin.
[0265] The mean patient age at inclusion was 40.3 years old. 68% of
the patients were female; 2 (4.8%) were black or African-American;
and 1 patient (2.4%) was of Asian descent. Six patients (14.6%)
reported a family history of aHUS. Twenty (48.7%) had at least one
identified complement regulatory protein mutation or tested
positive for an autoantibody that binds to a complement regulatory
protein. Thirty patients (73.2%) presented with a first clinical
manifestation of aHUS. Six patients (14%) immediately initiated
eculizumab without use of plasma exchange/infusion (PE/PI).
Twenty-four patients (58.5%) were on dialysis at baseline (prior to
eculizumab treatment). Nine patients (22%) had previously undergone
a renal transplant. Twenty-seven (66%) had a platelet count that
was less than 150.times.10.sup.9/L. Thirty-two (78%) patients had a
serum LDH level that was greater than the upper limit of normal.
The mean haptoglobin (Hp) levels for the patients in this cohort
was 0.6.+-.0.4 g/L; whereas the mean serum creatinine levels in
this patient cohort was 411.+-.264.6 .mu.mol/L (N=40).
[0266] Biological fluids were collected at enrollment in the study
(prior to treatment) and then following treatment at each
administration of the drug. Eculizumab was administered to the
subjects under the following schedule: 900 mg once per week for
four weeks; 1200 mg as the fifth dose; and 1200 mg once every two
weeks thereafter for up to 55 weeks as part of a Phase 2 clinical
trial.
Example 1
Materials and Methods
Urine Samples
[0267] Freshly collected urine was immediately mixed with protease
inhibitors. The concentrations of several analytes including NGAL,
cystatin C, clusterin, TIMP-1, .beta.2-microglobulin, C5b9, C5a,
and creatinine in urine collected from the subjects were measured
using commercially-available kits as described briefly below.
[0268] NGAL levels were measured in urine using a commercially
available kit (R&D Systems, Minneapolis, Minn.; catalogue
number: DLCN20). Briefly, urine samples were diluted 1:3 using kit
supplied calibrator diluent RD5-24. 50 .mu.L of each sample or kit
standard control (NS0-expressed recombinant human Lipocalin-2) were
added to wells of an assay plate in duplicate, each well containing
100 .mu.L of kit-supplied Assay Diluent RD1-52. After a two hour
incubation at 4.degree. C. in the refrigerator, wells were washed
four times with 200 .mu.L per well of wash solution. An
enzymatically (horseradish peroxidase)-labeled anti-NGAL conjugate
was added at 200 .mu.L per well and incubated for two hours at
4.degree. C. in the refrigerator. Wells were washed four times with
200 .mu.L per well of wash solution and developed by adding 200
.mu.L per well of kit-supplied TMB Substrate Solution (substrate
for the enzyme of the anti-NGAL conjugate) and incubated at room
temperature in the dark for 30 minutes. TMB is a substrate for
horseradish peroxidase often used in ELISA. Reaction between the
substrate and immobilized horseradish peroxidase (HRP) conjugated
to antibodies in the ELISA wells produces a blue colored solution.
After reaching the desired color intensity, the reaction is
terminated by addition of the stop solution (acidic), which changes
the solution color from blue to yellow. Thus, the reactions were
stopped after the incubation by adding 50 .mu.L per well of
kit-supplied Stop Solution to each well and the absorbance read at
450 nm.
[0269] Cystatin C levels were measured with a commercially
available kit (R&D Systems, Minneapolis, Minn.; catalogue
number: DSCTC0). Briefly, urine samples were diluted 1:3 using kit
supplied calibrator diluent RD5-24. 50 .mu.L of each sample or kit
standard control (recombinant human CysC) were added to wells of an
assay plate in duplicate, each well containing 100 .mu.L of
kit-supplied Assay Diluent RD1-52. After a two hour incubation at
4.degree. C. in the refrigerator, wells were washed four times with
200 .mu.L per well of wash solution. An enzymatically-labeled
anti-CysC conjugate was added at 200 .mu.L per well and incubated
for two hours at 4.degree. C. in the refrigerator. Wells were
washed four times with 200 .mu.L per well of wash solution and
developed by adding 200 .mu.L per well of kit-supplied TMB
Substrate Solution (substrate for the enzyme of the anti-CysC
conjugate) and incubated at room temperature in the dark for 30
minutes. The reactions were stopped after the incubation by adding
50 .mu.L per well of kit-supplied Stop Solution to each well and
the absorbance read at 450 nm.
[0270] Clusterin levels were measured with a commercially available
kit (R&D Systems, Minneapolis, Minn.; catalogue number:
DCLU00). Briefly, urine samples were diluted 1:3 using kit supplied
calibrator diluent RD5T. 50 .mu.L of each sample or kit standard
control (recombinant human clusterin) were added to wells of an
assay plate in duplicate, each well containing 100 .mu.L of
kit-supplied Assay Diluent RD1-19. After a two hour incubation at
room temperature on the orbital shaker set at 500 rpm, wells were
washed four times with 200 .mu.L per well of wash solution. An
enzymatically-labeled anti-clusterin conjugate was added at 200
.mu.L per well and incubated for two hours at room temperature on
the orbital shaker set at 500 rpm. Wells were washed four times
with 200 .mu.L per well of wash solution and developed by adding
200 .mu.L per well of TMB Substrate Solution and incubated at room
temperature in the dark for 30 minutes. The reactions were stopped
after the incubation by adding 50 .mu.L per well of Stop Solution
to each well and the absorbance read at 450 nm.
[0271] TIMP-1 levels were measured with a commercially available
kit (R&D Systems, Minneapolis, Minn.; catalogue number:
DTM100). Briefly, urine samples were diluted 1:2 using kit-supplied
calibrator diluent RD5P. 50 .mu.L of each sample or kit standard
control (recombinant human TIMP-1) were added to wells of an assay
plate in duplicate, each well containing 100 .mu.L of kit-supplied
Assay Diluent RD1X. After a two hour incubation at room temperature
on the orbital shaker set at 500 rpm, wells were washed three times
with 200 .mu.L per well of wash solution. An enzymatically-labeled
anti-TIMP-1 conjugate was added at 200 .mu.L per well and incubated
for two hours at room temperature on the orbital shaker set at 500
rpm. Wells were washed four times with 200 .mu.L per well of wash
solution and developed by adding 200 .mu.L per well of TMB
Substrate Solution and incubated at room temperature in the dark
for 30 minutes. The reactions were stopped after the incubation by
adding 50 .mu.L per well of Stop Solution to each well and the
absorbance read at 450 nm.
[0272] .beta.2M levels were measured with a commercially available
kit (R&D Systems, Minneapolis, Minn.; catalogue number:
DBM200). Briefly, urine samples were diluted 1:10 using kit
supplied Sample Diluent. 20 .mu.L of each sample, kit controls or
kit standards were added to wells in duplicate, containing 100
.mu.L of a solution containing enzymatically-labeled anti-.beta.2M
conjugate. After a one hour incubation at room temperature, wells
were washed six times with 200 .mu.L per well of wash solution.
Wells were developed by adding 100 .mu.L per well of TMB Substrate
solution and incubated at room temperature in the dark for 15
minutes. The reactions were stopped after the incubation by adding
100 .mu.L per well of Stop Solution to each well and the absorbance
read at 450 nm.
[0273] Creatinine levels were measured with a commercially
available kit (R&D Systems, Minneapolis, Minn.; catalogue
number: KGE005). Briefly, urine samples were diluted 1:20 using
water and 50 .mu.L of samples, kit controls or kit standards were
added to wells in duplicate, containing 100 .mu.L of the
kit-supplied Alkaline Picrate Solution. After a 30 minute
incubation at room temperature, the absorbance at 490 nm was
measured.
[0274] C5b-9 levels were measured with a commercially available kit
(BD Biosciences, San Jose, Calif.; catalogue number: 558315) and an
optEIA reagent set B (BD Biosciences, San Jose, Calif.; catalogue
number: 550534). Briefly, an anti-C5b-9 capture antibody was
diluted 1:250 in coating buffer, 100 .mu.L of which was added to
each well of a 96 well maxisorp plate (Nunc; catalogue number:
439454) and incubated overnight at 4.degree. C. in the
refrigerator. Wells were washed three times with 200 .mu.L per well
of wash solution and blocked by adding 200 .mu.L per well of
kit-supplied Assay Diluent for one hour at room temperature. Wells
were washed three times with 200 .mu.L per well of wash solution
and 100 .mu.L of urine samples or kit standards were added to wells
in duplicate. After a two hour incubation at room temperature,
wells were washed three times with 200 .mu.L per well of wash
solution. 100 .mu.L of the kit-supplied C5b-9 Working Detector
Antibody Solution was added to each well and incubated for one hour
at room temperature. Wells were washed seven times with 200 .mu.L
per well of wash solution and developed by adding 100 .mu.L per
well of TMB Substrate Solution and incubated at room temperature in
the dark for 30 minutes. The reactions were stopped after the
incubation by adding 50 .mu.L per well of Stop Solution to each
well and the absorbance read at 450 nm.
[0275] C5a levels were measured with a commercially available kit
(BD Biosciences, San Jose, Calif.; catalogue number: 557965).
Briefly, 100 .mu.L of urine samples or kit standards were added to
wells in duplicate containing 50 .mu.L of kit-supplied ELISA
Diluent. After a two hour incubation at room temperature, wells
were washed five times with 200 .mu.L per well of wash solution.
100 .mu.L of the kit-supplied C5a Working Detector Antibody
Solution was added to each well and incubated for one hour at room
temperature. Wells were washed seven times with 200 .mu.L per well
of wash solution and developed by adding 100 .mu.L per well of TMB
Substrate Solution and incubated at room temperature in the dark
for 30 minutes. The reactions were stopped after the incubation by
adding 50 .mu.L per well of Stop Solution to each well and the
absorbance read at 450 nm.
Plasma
[0276] Plasma samples were prepared as follows. Blood was collected
in a 10 mL BD.TM. P100 tube (Becton Dickinson) containing EDTA.
Whole blood was centrifuged no later than 60 minutes after
collection in a refrigerated centrifuge (set to maintain
4-8.degree. C.) for 10 minutes at 3000 rpm. Plasma was then
obtained from the sample following centrifugation. Hemolyzed
samples were discarded.
[0277] The concentration of several analytes including Ba,
prothrombin fragment 1+2, thrombomodulin, vWF, sC5b-9, and C5a in
plasma fractions of blood collected from the subjects was measured
using commercially-available kits as described briefly below.
[0278] Ba levels were measured with a commercially available kit
(Quidel, San Diego, Calif.; catalogue number: A033). Briefly, wells
of an assay plate were washed three times with wash solution.
Plasma samples were diluted 1:1000 with kit-supplied specimen
diluent and 100 .mu.L of the diluted plasma samples, kit controls
and standards were added to wells in duplicate. After a 60 minute
incubation at room temperature, the wells were washed five times
with 200 .mu.L per well of wash solution. 100 .mu.L of an
enzymatically-labeled anti-Ba antibody conjugate were added to each
well and incubated for sixty minutes at room temperature. After
five washes with wash solution, 100 .mu.L of TMB substrate was
added to each well and incubated for fifteen minutes at room
temperature protected from light. The reaction was stopped with the
addition of 100 .mu.L per well of stop solution and absorbance was
read at 450 nm.
[0279] Prothrombin fragment 1+2 levels in EDTA plasma were measured
with the Enzygnost F1+2 kit (Siemens Healthcare; catalogue number:
OPBD03). Briefly, plasma samples were diluted 1:2 with sample
buffer and 50 .mu.L of the diluted samples, or standard (containing
a known concentration of recombinant human prothrombin fragment
1+2) were added to wells. After a 30 minute incubation at
37.degree. C., the wells were washed three times with 200 .mu.L per
well of wash solution. 100 .mu.L of an enzymatically-labeled
anti-prothrombin fragment 1+2 antibody conjugate were added to each
well and incubated for 15 minutes at 37.degree. C. After three
additional washes, 100 .mu.L of chromagen substrate were added to
each well and incubated 15 minutes at room temperature protected
from light. The reaction was stopped by the addition of 100 .mu.L
of stop solution to each well and absorbance read at 450 nm.
[0280] Levels of thrombomodulin (TM) in EDTA plasma were evaluated
with the TM ELISA kit (American Diagnostica, Stamford, Conn.;
catalogue number: 837). Briefly, plasma samples were diluted 1:4
with sample buffer and 200 .mu.L of diluted samples or standard
(containing a known concentration of recombinant TM) was added to
wells. After a 60 minute incubation at room temperature, wells of
the assay plate were washed four times with 200 .mu.L/well of wash
solution. A solution of an enzymatically-labeled anti-TM antibody
was added (200 .mu.L per well) and incubated for 30 minutes at room
temperature. After 4 washes, 200 .mu.L of substrate were added to
each well and the wells were incubated for 20 minutes at room
temperature protected from light. The reaction was stopped with 100
.mu.L of 0.5 M H.sub.2SO.sub.4 and the absorbance at 450 nm was
measured.
[0281] Levels of von Willebrand Factor (vWF) activity were
determined in EDTA plasma by an ELISA kit utilizing capture
antibody specific for vWF collagen binding sites (American
Diagnostica; catalogue number: 885). Plasma samples and kit
controls were diluted 1:20 with assay diluent and 100 .mu.L of the
diluted samples and controls added to wells in duplicate. After a
60 minute incubation at room temperature, the wells were washed 5
times with wash solution and 100 .mu.L of an enzymatically-labeled
anti-vWF conjugate were added to each well. The wells were
incubated for 15 minutes at room temperature and washed 5 times
with wash solution. 100 .mu.L of TMB substrate (which, upon
cleavage by the enzyme, generates a detectable signal) was added to
each well. The wells were incubated for 15 minutes at room
temperature protected from light followed by the addition of 100
.mu.L of kit-supplied stop solution to each well. Absorbance was
measured at 450 nm within 30 minutes of the addition of stop
solution.
[0282] Circulating levels of sC5b-9 were determined with a human
C5b-9 ELISA set (BD Biosciences, San Diego, Calif.; catalogue
number: 558315) and a BD optEIA reagent set B (BD Biosciences;
catalogue number: 550534). Briefly, an anti-C5b-9 capture antibody
was diluted 1:250 in kit-supplied coating buffer, 100 .mu.L of
which were added to was of a 96 well maxisorp plate (Nunc) and
incubated overnight at 4.degree. C. Following three washes in wash
solution, wells were blocked with 200 .mu.L kit-supplied assay
diluent for one hour at room temperature. Following 3 more washes
with wash solution, 100 .mu.L of the plasma samples (diluted 1:100
in assay diluent) or standards (containing a known concentration of
purified sC5b-9) were added and incubated for two hours at room
temperature. The wells were washed three times with wash solution
and 100 .mu.L of working detector (which contains a biotin-labeled
anti-C5b-9 detection antibody and streptavidin-labeled horseradish
peroxidase diluted 1:250 in assay diluent) added to each well.
Following a one hour incubation at room temperature, the wells were
washed seven times with wash solution and 100 .mu.L of substrate
TMB solutions added to each well. The reaction was allowed to
develop for 30 minutes at room temperature protected from light.
Following the addition of 50 .mu.L of stop solution to each well,
absorbance was determined at 450 nm.
[0283] Circulating levels of C5a were determined with a sandwich
ELISA utilizing the BD optEIA reagent set B (BD Biosciences;
catalogue number: 550534). All incubations were performed in the
presence of further (BD Biosciences; catalogue number: 550236).
Briefly, an anti-C5a capture antibody was diluted to 2 .mu.g/mL in
kit-supplied coating buffer, 100 .mu.L of which were added to wells
of a 96 well maxisorp plate (Nunc) followed by an overnight
incubation at 4.degree. C. Following three washes in wash solution,
wells were blocked with 200 .mu.L assay diluent for one hour at
room temperature. Following 3 more washes with wash solution, 50
.mu.L of plasma samples (diluted 1:5 in assay diluent) or standards
(containing a known concentration of C5a) were added and incubated
for one hour at room temperature. The wells were washed 4 times
with wash solution and 100 .mu.L of working detector added to each
well (which contains a biotin-labeled anti-C5a detection antibody
and streptavidin-labeled horseradish peroxidase diluted 1:250 in
assay diluent). Following an incubation for one hour at room
temperature, wells were washed seven times with wash solution and
100 .mu.L of substrate TMB solutions added to each well. The
reaction was allowed to develop for 30 minutes at room temperature
protected from light. Following the addition of 50 .mu.L of stop
solution to each well, absorbance was measured at 450 nm.
Serum
[0284] Serum samples were processed as follows. Blood was collected
in a 10 mL vacutainer serum separating (SST) tube. The tube was
inverted five times and the blood allowed to clot at room
temperature for at least 30 minutes, but no more than two hours.
The tube was subjected to centrifugation at 1800 rcf with the brake
on. Hemolyzed samples were discarded.
[0285] The quantitative determination of various analytes in serum
was carried out using human Cytometric Bead Array (CBA) Flex Set
Kits (CBA Flex Set; Becton Dickinson Biosciences, San Diego,
Calif.), and acquired by flow cytometer (FACS LSR II, Becton
Dickinson) according to the manufacturer's instructions. A Flex set
capture bead is a single bead population with distinct fluorescent
intensity and is coated with a capture antibody specific for a
soluble protein. Each bead population is given an alphanumeric
position designation, indicating its position relative to other
beads in the BD CBA Flex Set system. Beads with different positions
can be combined in assays to create a multiplex assay. In a Flex
Set assay the capture bead, PE conjugated detection reagent, and
the standard or test samples are incubated together to form
sandwich complexes.
[0286] Briefly, standards for each analyte were mixed and a serial
dilution was performed using the assay diluent. Capture beads for
each analyte were prepared and pooled using Capture bead diluent
for serum/plasma. Serum samples were diluted appropriately and
along with the standards were incubated with the mixed capture
beads in a total volume of 100 .mu.L for one hour at room
temperature. Detection phycoerythrin (PE) reagents were mixed for
all analytes and were added to the tubes (50 .mu.L). The samples
were washed with wash buffer after an incubation of two hours at
room temperature in the dark and were acquired by flow cytometer
after reconstitution of the pellet in the wash buffer.
[0287] The following bead sets were incubated with serum samples
diluted 1:4 in kit-supplied assay diluent (wherein the biomarker
protein specified indicates the capture antibody conjugated to the
bead): IFN-.gamma. (Bead E7; catalogue number: 558283); IL-12 p70
(Bead E5; catalogue number: 558283); IL-1.beta. (Bead B4; catalogue
number: 558279); IL-6 (Bead A7; catalogue number: 558276); IL-8
(Bead A9; catalogue number: 558277); CXCL-9 (Bead E8; catalogue
number: 558286); CXCL-10 (Bead B5; catalogue number: 558280); MCP-1
(Bead D8; catalogue number: 558287); VEGF (Bead B8; catalogue
number: 558336); and sCD40L (Bead C7; catalogue number 560305). The
following bead sets were incubated with serum samples diluted 1:50
in kit-supplied diluent: ICAM-1 (Bead A4; catalogue number:
560269); VCAM-1 (Bead D6; catalogue number: 560427); TNFR1 (Bead
C4; catalogue number: 560156); E-selectin (Bead D9; catalogue
number: 560419); P-selectin (Bead D7; catalogue number: 560426);
and CCL5 (Bead D4; catalogue number: 558324).
Example 2
Results
Markers of Ongoing Complement Activation
[0288] As summarized in Table 2 below, relative to the
concentration in a sample of biological fluid from healthy
volunteers, the plasma concentration of complement component Ba and
sC5b9 and the urine concentration of C5a and sC5b-9 were elevated
in the majority of aHUS patients. See also FIG. 1.
TABLE-US-00002 TABLE 2 n/N (%) elevated at aHUS Biomarker Protein
baseline P-value Plasma Ba 35/35 (100.0) <0.0001 Plasma sC5b-9
37/38 (97.4) <0.0001 Urine C5a 26/29 (89.7) 0.0007 Urine sC5b-9
23/27 (85.2) 0.0025 * "N" indicates the total number of patients
evaluated for a given biomarker, and "n" indicates the number of
those "N" patients with elevated levels of the biomarker
protein.
[0289] These results indicate that significant systemic alternative
pathway complement activation is ongoing in aHUS patients.
[0290] Following treatment with eculizumab, the mean levels
(concentrations) of these aHUS biomarkers were reduced (FIGS.
1A-C). The mean levels of urinary C5a and sC5b-9 are reduced
significantly at between 1 to 2.5 weeks following initiation of
treatment and remained so. The mean percentage reduction in urinary
C5a levels was greater than 40% at week 3 post-treatment and over
70% by week 6 (FIG. 1D). Urinary sC5b-9 levels were reduced by over
60% by week 3 (FIG. 1E). These markers eventually normalized.
Plasma Ba levels were also significantly reduced (p=0.0053) at
around four to six weeks following treatment with eculizumab,
suggesting that with time eculizumab reduces the initiation or
amplification of the classical complement pathway (FIG. 1C).
However, the mean percentage reduction in plasma Ba levels was
around 10% at week 6 and over 30% by week 12 (FIG. 1F).
[0291] The percentage of treated aHUS patients who experience
normalized complement biomarker protein levels over time is shown
in FIGS. 2A-C. For example, as shown in FIG. 2B, 50% of treated
aHUS patients exhibit normalized levels of urinary sC5b-9 by 2.5
weeks post-treatment initiation with eculizumab. By 17 weeks
post-initiation of treatment, 79% of treated aHUS patients
exhibited normalized sC5b-9 levels. However, Ba levels do not
normalize in most patients (FIGS. 1C and 2C). These data indicate
that even with eculizumab therapy there may be, in some patients,
low level ongoing complement activation.
Markers of Platelet and Hemostatic Activation
[0292] As summarized in Table 3 below, relative to the
concentration in a sample of biological fluid from healthy
volunteers, the serum concentration of sCD40L and the plasma levels
of prothrombin fragment 1+2 and D-dimer were significantly elevated
in the majority of aHUS patients. See also FIGS. 3A-B.
TABLE-US-00003 TABLE 3 aHUS Biomarker Protein n/N (%) elevated at
baseline P-value sCD40L 36/38 (94.7) <0.0001 Prothrombin
Fragment F1 + 2 36/38 (94.7) <0.0001 D-dimer 34/36 (94.4)
=0.0002 * "N" indicates the total number of patients evaluated for
a given biomarker, and "n" indicates the number of those "N"
patients with elevated levels of the biomarker protein.
[0293] The release of sCD40L is generally associated with platelet
metabolism and activity. Prothrombin fragments F1+2 are generated
during conversion of prothrombin to thrombin, whereas D-dimer is a
fibrin degradation product indicating fibrinolysis.
[0294] Following treatment with eculizumab, the mean levels
(concentrations) of these aHUS biomarkers were reduced. The mean
levels of plasma levels of F1+2 and D-dimer are reduced
significantly at between 1 to 2.5 weeks (p=0.0078 and 0.0083,
respectively) following initiation of treatment and remained so. As
shown in FIG. 3C, the mean percentage reduction in F1+2 was around
15% by week 3 and over 50% by week 12. The mean percentage
reduction in d-dimer levels was around 40% at week 6, but greater
than 60% by week 12. These data indicate that eculizumab therapy
has an immediate effect on the coagulation and fibrinolysis
pathways. As shown in FIGS. 4A-B, 32% of treated aHUS patients
exhibit normalization of F1+2 levels by week 26 post-initiation of
treatment and 46% of the patients have normalized levels of
D-dimer. By contrast, sCD40L levels remained elevated throughout
the study.
Markers of Endothelial Cell Damage and/or Activation
[0295] As summarized in Table 4 below, relative to the
concentration in sample of biological fluid from healthy
volunteers, the plasma concentration of thrombomodulin and vWF and
the serum concentration of VCAM-1 were significantly elevated in
aHUS patients. See also FIGS. 5A-C.
TABLE-US-00004 TABLE 4 aHUS Biomarker Protein n/N (%) elevated at
baseline P value Thrombomodulin 33/34 (97.1) <0.0001 VCAM-1
36/38 (94.7) <0.0001 Von Willebrand Factor Antigen 15/38 (39.5)
<0.02 * "N" indicates the total number of patients evaluated for
a given biomarker, and "n" indicates the number of those "N"
patients with elevated levels of the biomarker protein. n.s.
indicates not significant.
[0296] High concentration of thrombomodulin and VCAM-1 in
biological fluids of aHUS patients indicates significant
endothelial cell activation. Thrombomodulin is released in response
to C3a, which further underscores ongoing complement activation in
aHUS patients. vWF concentration is also significantly elevated.
vWF has a number of physiological roles including platelet adhesion
and coagulation and is also a marker of endothelial damage and
activation.
[0297] Following treatment with eculizumab, the mean levels
(concentrations) of these aHUS biomarkers were reduced (FIGS.
5A-C). The mean levels of thrombomodulin and VCAM-1 were reduced
significantly from baseline by week 17 (p=0.0007 and <0.0001,
respectively) following initiation of treatment (see FIGS. 6C and
6D). By week 26, levels of VCAM-1 and vWF had also been reduced.
However, while vWF normalized in .about.70% of treated aHUS
patients by week 17 post-initiation of treatment (FIG. 6B),
thrombomodulin and VCAM-1 levels remained elevated. Interestingly,
of the 10% of patients who normalized thrombomodulin levels (FIG.
6A), only one patient had both normalized thrombomodulin and vWF
levels. These data indicate that eculizumab therapy has a rapid and
robust positive effect to correct endothelial cell damage and
activation.
Markers of Inflammation
[0298] Table 5 (below) sets forth a series of analytes detected in
plasma and/or serum and indicates the percentage of aHUS patients
in which the respective analytes were elevated prior to treatment
with complement inhibitor therapy.
TABLE-US-00005 TABLE 5 aHUS Biomarker n/N (%) elevated at Protein
baseline P value Serum CXCL10 23/38 (60.5) P < 0.0001 Serum
CXCL9 33/38 (86.8) P < 0.0001 IL-18 19/38 (50.0) P < 0.0001
MCP-1 34/38 (89.5) P < 0.0001 TNFR1 38/38 (100.0) P < 0.0001
VEGF 25/38 (65.8) P < 0.0001 IL-6 21/34 (61.8) P = 0.0019 CCL5
4/38 (10.5) P = 0.0045 IFN-.gamma. 5/34 (14.7) P = 0.0353 IL-8
22/38 (57.9) n.s. (P = 0.0640) ICAM-1 2/34 (5.9) n.s IL-1.beta.
1/38 (2.6) n.s IL-12p70 2/34 (5.9) n.s * "N" indicates the total
number of patients evaluated for a given biomarker, and "n"
indicates the number of those "N" patients with elevated levels of
the biomarker protein. ** The concentrations of the two analytes
marked as "Serum" were measured in serum. The concentrations of all
other analytes in the Table were measured in plasma. n.s. indicates
not significant.
[0299] Prior to therapy with eculizumab, patients with aHUS had
elevated levels of circulating inflammatory cytokines and
chemokines including, e.g., CXCL-10, CXCL-9, IL-18, TNFR1, MCP-1,
VEGF, IL-6, and IL-8. Following initiation of treatment, however,
TNFR1 was the earliest inflammatory marker to be significantly
reduced (by week 6, p=0.0012) (FIG. 7A). Mean concentration of
TNFR1 remained significantly lower than baseline at all subsequent
visits (P<0.0001), but only normalized in 6% of aHUS patients
(FIG. 7B). Similarly, mean levels of CXCL10 were significantly
reduced by week 26 (p=0.0055), but did not normalize in all aHUS
patients (31% of patients did not normalize). By week 26, mean
levels of IFN-.gamma. normalized in approximately 50% of patients;
however, mean levels of serum IL-8 (p=0.01), CXCL-9 (p=0.01), IL-18
(p<0.0001) and VEGF (p<0.0001) remained elevated in most aHUS
patients, as compared to normal controls, and not significantly
different from baseline. Serum IL-6 was significantly reduced
(p=0.04) from baseline at week 26 and remained elevated at week 26
as compared to normal control.
[0300] By contrast, mean levels of CCL-5 were elevated
significantly by week 17 post-initiation of treatment and
thereafter (p=0.0072 and 0.0021 at weeks 12-17 and week 26,
respectively). In response to vascular injury in mice, CCL5 is
upregulated, which promotes selective T cell infiltration as part
of a vascular wound-healing response. See, e.g., Rookmaaker et al.
(2007) Am J Physiol Renal Physiol 293(2):F624-630. These data
indicate that eculizumab therapy has a rapid and robust positive
effect on inflammation in many patients with aHUS, but that low
level inflammation may exist in these patients even during
treatment.
Markers of Renal Tubular and Glomerular Injury
[0301] Table 6A (below) sets forth a series of analytes detected in
urine collected from patients and indicates the percentage of aHUS
patients in which the respective analytes were elevated prior to
treatment with complement inhibitor therapy.
TABLE-US-00006 TABLE 6A Biomarker n/N (%) elevated at baseline P
value Beta-2 Microglobulin 20/28 (71.4) P < 0.0001 (.beta.2M)
Clusterin 24/29 (82.8) P < 0.0001 Cystatin C 18/29 (62.1) P =
0.0002 TIMP-1 22/29 (75.9) P = 0.0003 FABP-1 22/29 (75.9) P =
0.0130 NGAL 5/29 (17.2) P = 0.0151 NAG 3/23 (13.0) P = 0.0413
CXCL10 2/29 (6.9) n.s. CXCL9 2/29 (6.9) n.s. KIM-1 2/29 (6.9) n.s.
* "N" indicates the total number of patients evaluated for a given
biomarker, and "n" indicates the number of those "N" patients with
elevated levels of the biomarker protein. n.s. indicates not
significant.
[0302] Prior to treatment with eculizumab, low molecular weight
molecules that are normally filtered by the kidney were elevated in
the urine of patients with aHUS including .beta.2M, clusterin,
cystatin C, and NAG. Molecules produced by renal tubular epithelial
cells in response to injury were also elevated, such as TIMP-1,
NGAL and L-FABP. However, following treatment with eculizumab, CysC
(p=0.0012) (FIG. 8A), clusterin (p=0.0446), and TIMP-1 (p=0.0353)
are significantly reduced by 1-2.5 weeks post-initiation of
treatment and they remained significantly reduced throughout the
course of the study. NGAL (p=0.0003) (FIG. 8C), L-FABP (p=0.0366),
and NAG (p=0.0369) were significantly reduced from baseline by 4-6
weeks post-initiation of treatment and remained so thereafter.
.beta.2M was significantly reduced at 12-17 weeks (p=0.0008) and
onwards (FIG. 8B). By week 26, mean urinary levels of all analytes
had normalized in treated aHUS patients.
[0303] These data indicate that eculizumab therapy has a rapid,
robust, and durable positive effect redressing renal tubular and
glomerular injury experienced by many patients with aHUS.
SUMMARY
[0304] The following Table provides a summary of exemplary aHUS
biomarkers (though not an exhaustive list), which are elevated in
aHUS patients prior to treatment with eculizumab, but are
significantly reduced following treatment with eculizumab. Also
provided in the Table (Table 6B) is the average time
post-initiation of treatment with eculizumab in which significant
reduction of the aHUS biomarker occurred.
TABLE-US-00007 TABLE 6B Biomarker Week 1-2.5 Week 4-6 Week 12-17
Week 26 U-C5a X U-C5b-9 X F1+2 X D-dimer X U-Cys-C X U-TIMP-1 X
Plasma Ba X TNFR1 X U-CLU X U-NGAL X Thrombomodulin X VCAM X L-FABP
X B2M X CXCL10 X IFN-.gamma. X
Baseline aHUS Marker Levels in aHUS Patients Receiving Dialysis
and/or Receiving Kidney Transplant
[0305] Also assessed were the concentration of plasma and urine
complement, inflammation, and renal injury markers in aHUS patients
who received dialysis prior to therapy with eculizumab. As shown in
Table 7 (below), the mean concentration of serum TNFR1, plasma Ba,
C5b9, prothrombin fragments 1+2, .beta.2M, clusterin, sC5b9,
TIMP-1, NGAL, CysC, and C5a were significantly elevated in aHUS
patients who underwent repeated dialysis (e.g., two or more times
within 6 months prior to treatment) as compared to aHUS patients
that did not undergo repeated dialysis prior to enrollment in the
study (prior to treatment). See also FIGS. 9A-E.
TABLE-US-00008 TABLE 7 Higher with Repeated Analyte Dialysis TNFR1
(serum) p = <0.0001 .beta.2m (urinary) p = 0.0009 Clusterin
(urinary) p = 0.0020 Ba (plasma) p = 0.0021 C5b-9 (urinary) p =
0.0042 TIMP-1 (urinary) p = 0.0070 NGAL (urinary) p = 0.0110 CysC
(urinary) p = 0.020 F1+2 (plasma) p = 0.0191 C5b-9 (plasma) p =
0.0476 C5a (urinary) p = 0.0477 ** The concentration of the one
analyte marked "serum" was measured in serum. The concentration of
analytes designated with "urinary" was measured in urine, whereas
the concentration of analytes labeled with "plasma" was measured in
plasma obtained from the patients.
[0306] In addition, aHUS patients who had received a kidney
transplant prior to treatment with eculizumab had lower urinary
C5b-9 and urinary FABP-1 at baseline as compared to patients who
had not received a kidney transplant.
Baseline aHUS Marker Levels Vis-a-Vis TMA Markers
[0307] Levels of some aHUS-associated biomarkers in some aHUS
patients correlated with abnormal thrombotic microangiopathy (TMA)
markers such as reduced platelet counts, elevated LDH, and
increased haptoglobin levels. For example, aHUS patients with
reduced platelet counts at baseline (<150,000 per .mu.L of
blood), exhibited elevated levels of urinary cystatin C (P=0.0276)
and urinary clusterin (P=0.0401). See FIGS. 14A-B. aHUS patients
having elevated LDH levels exhibited increased levels of VCAM-1
(P=0.0226) (FIG. 14C), d-dimer (P=0.0369) (FIG. 14D), IL-18,
thrombomodulin, and TNFR1 (see below). Elevated haptoglobin levels
were often present in aHUS patients having elevated IL-18
levels.
Baseline aHUS Marker Levels in aHUS Patients Receiving Plasma
Therapy
[0308] aHUS patients with repeated plasma therapy prior to
treatment with eculizumab exhibited higher mean levels of urinary
cystatin C at baseline (see FIG. 15).
Correlations between Biomarker Levels and Clinical Parameters
[0309] Platelets
[0310] An elevated level of CCL5 was positively correlated with
higher platelet counts at baseline (p=<0.0001; cc (correlation
coefficient)=0.8106). An elevated level of sCD40L was also
correlated with higher platelet counts at baseline (p=<0.001;
cc=0.6313).
[0311] Moreover, patients with normalized Ba levels following
eculizumab treatment show significantly higher platelet increases
than patients whose Ba levels remain elevated following treatment.
See FIG. 13.
[0312] Estimated Glomerular Filtration Rate (eGFR), LDH, and
Urinary Complement
[0313] A correlation was also observed between elevated plasma Ba
levels and reduced eGFR (p<0.0001; cc=-0.7219). An elevated
concentration of TNFR1 in serum of aHUS patients prior to treatment
was correlated with lactate dehydrogenase (LDH) levels (p=0.027;
cc=0.3586), but more significantly correlated with lower eGFR
(p<0.0001; cc=-0.6134). In addition, higher levels of urinary
complement components C5a and sC5b-9 and renal injury markers
(.beta.2M, clusterin, cystatin C, NGAL, and TIMP-1) were moderately
correlated with lower eGFR (p=0.0002 to 0.0242; cc=-0.4286 to
-0.6714).
[0314] Elevated levels of urinary sC5b-9, clusterin, and TIMP-1
were modestly correlated with proteinuria (p=0.0086 to 0.0284;
cc=0.40 to 0.4788), whereas elevated levels of plasma Ba (p=0.0017;
cc=0.517), .beta.2M, clusterin, urinary sC5b-9, and cystatin C were
correlated with increased creatinine in the urine of patients prior
to treatment with eculizumab (p=0.0440-0.0018;
cc=0.3982-0.6457).
[0315] First Clinical Presentation of aHUS
[0316] Also observed was a correlation between patients
experiencing their first aHUS manifestation and significantly
elevated plasma D-dimer levels or urinary FABP-1 at baseline (prior
to eculizumab treatment) (see Table 8).
TABLE-US-00009 TABLE 8 Biomarker Elevated (n %) Single aHUS
Multiple Biomarker Manifestation Manifestations p-value Plasma
D-dimer 27 (100.0) 7 (77.8) 0.0571 (.mu.g/L) Urine FABP-1 (ng/mg 19
(90.5) 3 (37.5) 0.0079 normalized to creatinine)
[0317] Smoldering Disease
[0318] Six of the aHUS patients involved in the study presented at
enrollment with normalized hematologic parameters (including
haptoglobin, LDH, and platelet levels). However, these patients
still showed evidence of chronic inflammation and complement
activation despite a stable clinical picture. The patients had
significantly elevated levels of serum TNFR1 (as shown in FIG. 10A)
as well as significantly elevated levels of thrombomodulin, Ba
(FIG. 10B), prothrombin fragments 1+2 (FIG. 10E), VCAM-1 (FIG.
10C), and d-dimer (FIG. 10D). Similarly, patients with normal
(>150.times.10.sup.9 platelets/.mu.L) platelet levels at
baseline still show elevated levels of most biomarkers (e.g., Ba
(FIG. 10F), VCAM-1 (FIG. 10G), D-dimer (FIG. 10H), and F1+2 (FIG.
10I). Taken together, these findings indicate that, even for the
subset of aHUS patients deemed to be in clinical remission
following treatment, there are likely ongoing low levels of
complement activity, coagulopathy, and inflammation.
Correlations Between Biomarker Levels and Clinical Outcomes
[0319] Hematologic Responses
[0320] Patients with complete hematologic responses show more
dramatic reductions in TNFR1, urinary clusterin, and urinary
complement levels (C5a and C5b-9) (FIG. 11). For example, 86% of
patients exhibiting a reduced concentration of these aHUS biomarker
proteins attained a complete hematologic response (normalization of
platelets and LDH) by weeks 12-17 post-initiation of treatment with
eculizumab. Moreover, these patients showed a greater mean
percentage reduction in serum TNFR1, urinary clusterin, urinary
C5a, and urinary C5b-9 than patients who did not attain a complete
hematologic response.
[0321] Also observed was that the rapidity of reduction in TNFR1
(e.g., by week 12 as compared to week 17 or beyond) was correlated
with complete hematologic response (p=0.0008). The rate of
normalization of D-dimer was significantly associated with a
complete hematologic response (p=0.0109; cc=6.26).
[0322] Furthermore, the data show that a significantly greater
increase in platelet counts at weeks 12-17 (p=0.0022) and week 26
(p=0.0110) was achieved in eculizumab-treated aHUS patients having
(at weeks 12-17 and week 26, respectively) normalized plasma Ba
concentrations. Improvement in platelets was also correlated with a
significant reduction in mean F1+2 levels at week 4-6 (P=0.0148;
cc=-0.4087) and week 12-17 (P=0.0073; cc=-0.4396) and more modestly
with a reduction in d-dimer levels at week 12-17 (P=0.0470;
cc=-0.3381). Nevertheless, a subset of patients, despite
demonstrating a greater increase in platelet counts at weeks four
through 26, continued to exhibit significantly elevated levels of
prothrombin fragments 1+2, thrombomodulin, urinary .beta.2M,
clusterin, TIMP-1, and cystatin C, suggesting ongoing underlying
disease activity.
[0323] Analysis of the data collected from the study also revealed
a correlation between the change in other biomarker protein
concentration and platelet recovery. For example, the concentration
of CCL5, MCP-1, and sCD40L were positively correlated with
increased platelet counts in eculizumab-treated patients as shown
in Table 9 below.
TABLE-US-00010 TABLE 9 Week following initiation of treatment with
eculizumab Biomarker P-value Correlation coeff. 1-2.5 CCL-5 p <
0.0001 0.7419 sCD40L P = 0.0141 0.3950 VEGF P = 0.0014 0.5002 4-6
CCL-5 p < 0.0001 0.7743 sCD40L p < 0.0001 0.6818 MCP-1 P =
0.0114 0.4169 12-17 CCL5 P = 0.0003 0.5656 26 CCL-5 p < 0.0001
0.7845 sCD40L P = 0.0012 0.5398
[0324] Thrombomicroangiopathy (TMA)
[0325] Eculizumab-treated aHUS patients having a greater reduction
in plasma Ba levels more frequently achieved a complete TMA
response (e.g., normalization of hematologic parameters (e.g.,
platelet count and LDH levels) and preservation of renal function).
For example, 72.7% of patients attained a complete TMA response by
weeks 12-17, and 85.29% of the patients achieved a complete TMA
response by week 26. As shown in FIG. 12, these patients showed a
greater mean percentage reduction in plasma Ba concentration than
patients who did not attain a complete TMA response (p=0.0018 and
p=0.006, respectively).
[0326] Post-Treatment eGFR
[0327] Also observed was a relationship between the reduction
and/or normalization of certain biomarkers and an improvement in
eGFR. For example, a significantly greater improvement (Table 10)
in eGFR (e.g., eGFR.gtoreq.15 mL/min/1.73 m.sup.2 sustained for at
least two consecutive measurements obtained at least four weeks
apart) was observed among patients with normalized MCP-1, IL-6, and
IFN-.gamma. (at weeks 4-6); normalized VCAM-1, CXCL10, CXCL9, and
Ba (at weeks 12-17), and normalized Ba, urinary .beta.2M, urinary
CysC, vWF, D-dimer, clusterin, CXCL10, CXCL9, urinary FABP-1, and
others (at week 26) (Table 10). See also FIG. 16.
TABLE-US-00011 TABLE 10 Week post- initiation of treatment w/
Normalized eculizumab Biomarker p value 1-2.5 * * 4-6 MCP-1 0.0002
IL-6 0.0251 VCAM-1 0.0166 12-17 VCAM-1 0.0003 CXCL-10 0.0071 Ba
0.0299 CXCL-9 0.0441 26 VCAM-1 <0.0001 Cystatin C <0.0001 Ba
0.0002 U-.beta.2m 0.0013 CXCL9 0.0027 CXCL10 0.0172 vWF 0.0052
D-dimer 0.0224 L-FABP 0.0230 Clusterin 0.0300 F1+2 0.0460
Example 3
Baseline Levels of Selected aHUS Biomarker Proteins in aHUS
Patients
[0328] At baseline, prior to eculizumab treatment, substantial
evidence of significant complement activation, vascular
inflammation/damage, and organ injury was observed in aHUS patients
regardless of use of plasma exchange/plasma infusion or normal
laboratory values for platelet count, Hp or LDH. As evidenced by
the data set forth in Table 11, the concentrations of aHUS
biomarkers of complement activity, vascular inflammation,
endothelial activation and damage, coagulation, and renal injury
were significantly elevated in aHUS patients compared to healthy
subjects.
TABLE-US-00012 TABLE 11 Median Level at BL [range] Disease
Biomarker (P Value vs. n/N (%) Elevated Fold increase Process (NHV
range; units) NHV**) at BL over NHV at BL CAP Plasma Ba
(388.0-588.0 ng/mL) 2676.4 [935.0-3668.0] 35/35 (100) 5.53
Activation (<0.0001) Terminal U-C5a (0.0-0.7 ng/mg 9.00
[0.3-76.6] 26/29 (89.7) 45 Complement U-creat) (0.0007) Activation
U-sC5b-9 (0.0-0.6 ng/mg 30.50 [0.2-665.7] 23/27 (85.2) 305 U-creat)
(0.0025) Inflammation sTNFR1 (407.3-1391.3 pg/mL) 17616.85
[4008.5-54158.2] 38/38 (100) 18.71 (<0.0001) Endothelial sVCAM-1
(159.2-444.7 ng/mL) 659.75 [375.4-1865.5] 36/38 (94.7) 1.99
Activation (<0.0001) Endothelial TM (2.0-3.6 ng/mL) 10
[3.4-24.1] 33/34 (97.1) 3.64 Damage (<0.0001) Coagulation F1+2
(82.9-305.5 pmol/L) 1017.55 [217.7-5774.0] 36/38 (94.7) 5.46
(<0.0001) D-dimer (157.0-395.9 .mu.g/L) 2735 [330.0-44100.0]
34/36 (94.4) 9.84 (0.0002) Renal Injury U-clusterin 1232.30
[129.9-6091.2] 24/29 (82.8) 8.62 (5.7-437.1 ng/mg U- (<0.0001)
creat) U-TIMP-1 23.8 [1.4-230.4] 22/29 (75.9) 39.67 (0.0-5.4 ng/mg
U- (0.0003) creat) U-L-FABP-1 58.00 [3.7-1309.8] 22/29 (75.9) 48.33
(0.0-16.9 ng/mg U- (0.0130) creat) U-.beta.2m 18.4 [0.4-127.7]
20/28 (71.4) 46 (0.0-2.7 .mu.g/mg U- (<0.0001) creat)
U-cystatin-C 1256.9 [14.3-7189.6] 18/26 (69.2) 23.85 (0.3-301.3
ng/mg U- (0.0001) creat) NHV means normal human value or
concentration for a given aHUS biomarker protein recited in the
Table. "creat" means creatinine, the concentration of which is used
to normalize certain biomarker concentrations recited in the table.
CAP refers to alternative pathway of complement (see above). "BL"
refers to "baseline", i.e., prior to treatment with eculizumab. "N"
is the total number of patients analyzed for a given disease
process and biomarker. "n" is the number of "N" patients in which a
given biomarker was elevated. "U" indicates that the analyte was
measured in urine. * P values were calculated using a Wilcoxon Rank
Sum test, testing for a difference between groups.
[0329] In addition, the inventors observed that there was no
statistical significance between the baseline elevated levels of
certain aHUS biomarkers observed in patients who had received or
were receiving plasma exchange (PE) or plasma infusion (PI) therapy
as compared to the level of elevation of the aHUS biomarkers in
patients who did not receive PE or PI therapy. For example, the
concentration of Ba, sTNFR1, sVCAM-1, and D-dimer were not reduced
or normalized in patients who had received PE/PI therapy (FIGS.
17A-D). Note that only 3 of 26 patients analyzed in the data
presented in FIGS. 17A-D did not receive PE/PI. The majority of
patients (n=23) had elevated levels of Cystatin C, as compared to
normal healthy volunteers. Cystatin C being a renal injury marker
(glomerular injury), it is possible that the patients who did not
receive PE/PI had less damage to their kidneys and thus had reduced
levels of renal injury-related biomarker proteins in their
urine.
[0330] Similarly, at baseline, prior to eculizumab therapy, the
concentration of protein markers of complement activation (e.g.,
Ba), inflammation (e.g., sTNFR1), endothelial cell activation
(sVCAM-1), coagulation (D-dimer), and renal injury (cystatin-C)
were elevated in patients with aHUS having normal platelet counts.
See FIGS. 18A-E. And patients with normal Hp and LDH levels showed
evidence of ongoing complement activation, inflammation,
endothelial cell activation, coagulation and renal injury (see
FIGS. 19A-E).
[0331] In view of the foregoing, the concentration of biomarkers
reflecting complement activity, vascular inflammation, endothelial
activation and damage, coagulation and renal injury were
chronically elevated in patients with aHUS compared to normal
healthy subjects. Patients with aHUS receiving PE/PI showed strong
evidence of significant ongoing complement activation, vascular
inflammation, endothelial activation, coagulation and renal injury.
While PE/PI may transiently maintain normal platelet count and LDH
in some patients, the above results demonstrate that the underlying
complement dysregulation and TMA processes persist. Despite normal
laboratory values for platelet count, LDH, and Hp, these studies
indicate that significant ongoing complement activation, vascular
inflammation, endothelial activation, coagulation and renal injury
exist in aHUS patients.
Example 4
Effects of Sustained Treatment with Eculizumab on aHUS Biomarker
Concentrations
[0332] The inventors observed that sustained eculizumab treatment
inhibits chronic elevated complement activation and terminal
complement mediated renal injury, and reduces inflammation,
endothelial damage and thrombotic risk in patients with aHUS. For
example, sustained eculizumab treatment rapidly and completely
inhibited terminal complement activation as indicated by the
reduction in the concentration of both C5a and sC5b-9 (e.g.,
urinary C5a and sC5b-9). See FIGS. 20A-B. At baseline, patients
with aHUS showed significant terminal complement activation
compared with NHV, despite use of PE/PI or normal platelet counts
in some patients. In fact, aHUS patients demonstrated 45-fold
higher urinary C5a and 305-fold higher urinary sC5b-9 levels than
NHV. However, during sustained eculizumab treatment, all aHUS
patients demonstrated rapid and potent terminal complement
blockade, with complete normalization of pathogenic terminal
complement activation products and no difference in levels relative
to NHV.
[0333] Furthermore, sustained eculizumab treatment normalized the
concentration of biomarker proteins of renal injury (FIGS. 21A-C).
Prior to initiating eculizumab therapy, the majority of patients
had elevated levels of biomarkers of: tubular interstitial injury
and deterioration of renal function (e.g., L-FABP-1, .about.48 fold
higher than NHV), glomerular filtration (e.g., cystatin C,
.about.24-fold higher than NHV), proximal tubular injury (e.g.,
clusterin, 8.6 fold higher than NHV). However, sustained treatment
with eculizumab dramatically reduced the urinary concentrations of
FABP-1 (by up to 100%), cystatin C (by up to 99%), and clusterin
(by up to 98%). This reduction was significant across all
timepoints (P<0.0001 for all) and the reduced concentration of
all renal injury markers was no different than levels in NHV.
Additional renal injury markers (e.g., TIMP-1 and
.beta.2-microglobulin) also normalized (see above under Example 2).
These results suggest that organ ischemia and damage may be
entirely terminal complement dependent and confirms clinical data
demonstrating that sustained inhibition of complement-mediated TMA
led to clinically meaningful eGFR improvement and discontinuation
of dialysis.
[0334] Sustained treatment with eculizumab also significantly
reduces complement alternative pathway activation (see FIG. 22).
All patients with aHUS showed significant systemic CAP activation
upstream of C5, with 5.5-fold higher levels of Ba compared with
NHV, prior to eculizumab treatment. However, following initiation
of eculizumab therapy, the concentration of upstream biomarkers of
CAP activation (e.g., Ba levels) was reduced by 30% and reduction
after week 4-6 was significant across all timepoints (p<0.005)
as compared with the concentration of the markers in NHV. Yet Ba
levels did not normalize in aHUS patients treated with eculizumab,
suggesting that CAP activation persists, reflecting the underlying
complement dysregulation in patients with aHUS. To be clear,
though, terminal complement blockade with eculizumab protected
patients from the clinical consequences of ongoing CAP
activation.
[0335] In addition, chronic treatment of aHUS patients with
eculizumab resulted in significantly reduced concentrations of
biomarkers associated with inflammation, endothelial activation,
and tissue damage (FIGS. 23A-C). Serum sTNFR1 levels were elevated
(18.7-fold higher than NHV levels) in 100% of patients with aHUS at
baseline. Sustained treatment with eculizumab significantly reduced
sTNFR1 up to 94%. The reduction in the concentration of these
biomarkers at week 4-6 was significant across all timepoints
(P<0.0001). Soluble VCAM-1 and TM levels were elevated in
>95% of aHUS patients at baseline by 2-fold and 3.6-fold,
respectively, as compared to NHV, demonstrating significant
endothelial cell activation and damage prior to eculizumab therapy.
TM and sVCAM-1 concentrations were also significantly reduced
during eculizumab treatment. After week 12-17, reduction in the
concentration of biomarkers of endothelial damage was significant
across all later timepoints (TM; P<0.0001), but still modestly
elevated compared to NHV. Dramatically reduced soluble TM levels
may reflect restoration of membrane bound TM, which is protective
against thrombotic risk.
[0336] Finally, chronic treatment with eculizumab rapidly and
significantly reduced the concentration of biomarkers associated
with thrombotic risk and coagulation (FIGS. 24A-B). The
concentration of coagulation biomarkers F1+2 and D-dimer were
significantly elevated (5.5-fold and 9.8 fold higher than NHV) at
baseline in greater than 94% of patients with aHUS (P<0.0001 and
P=0.0002, respectively). Yet F1+2 and D-dimer were significantly
reduced at 2.5 weeks post initiation of treatment with eculizumab.
The concentration of F1+2 decreased by up to 88% (P<0.05 for all
timepoints) and the D-dimer concentration was reduced by up to 99%
(P<0.0001 for all timepoints) with sustained eculizumab
treatment. However, these two markers remained modestly elevated
over the respective concentrations in normal healthy subjects.
CONCLUSIONS
[0337] In view of the foregoing data, the inventors were able to
draw a number of conclusions. First, at baseline, elevated levels
of all thrombotic microangiopathy (TMA) biomarkers were evident in
patients with aHUS as compared to the levels in samples from normal
healthy volunteers (NHV). In all patient groups--including those
receiving PE/PI or those with normal platelets, Hp or LDH--patients
with aHUS demonstrated significant elevation, over NHVs, in
measures of: terminal complement activation (45-305 fold higher
than NHV levels); vital organ damage; alternative pathway of
complement activation (e.g., as represented by Ba levels; 5.5-fold
higher than NHV levels); vascular inflammation; endothelial
activation and damage; and coagulation.
[0338] Sustained eculizumab treatment of aHUS patients
significantly reduced and normalized highly elevated markers of
terminal complement activation. Inhibition of terminal complement
activation with eculizumab also dramatically reduced and normalized
markers of organ damage. Upstream biomarkers of alternative pathway
activation were also significantly reduced, but did not normalize.
And low levels of alternative pathway activation persisted in
treated patients, reflecting the underlying complement
dysregulation in patients with aHUS. That said, the data clearly
indicate that terminal complement blockade with eculizumab protects
aHUS patients from the clinical consequences of ongoing alternative
pathway activation.
[0339] Moreover, sustained eculizumab treatment also resulted in:
(i) significant and sustained reduction of markers of vascular
inflammation (by up to 94%); (ii) significant inhibition of markers
of endothelial activation (by up to 60%); (iii) significant and
sustained reduction in markers of endothelial damage (by up to 77%)
to near normal levels, demonstrating a clear relationship between
terminal complement activation and endothelial damage; and (iv)
marked reduction (by up to 99%) of the concentration of biomarkers
of thrombotic risk, likely decreasing the potential for clot
formation and thus reducing incidence of TMA in these patients. The
inventors conclude, while not being bound by any theory or
mechanism of action, that inhibition of terminal complement
activation with eculizumab must be sustained, as loss of terminal
complement inhibition in aHUS would lead to a rapid increase in
severely amplified terminal complement activation, subsequently
leading to: increase in underlying subclinical endothelial
activation, significant acceleration of endothelial damage, marked
increase in thrombotic risk, and an early and ongoing risk of
catastrophic vascular ischemia and vital organ damage. Moreover,
these data indicate that the renal injury, vascular inflammation,
and endothelial damage and activation are in whole or in part
dependent on terminal complement activity, which activity is
effectively and safely inhibited using eculizumab.
[0340] While the present disclosure has been described with
reference to the specific embodiments thereof, it should be
understood by those skilled in the art that various changes may be
made and equivalents may be substituted without departing from the
true spirit and scope of the disclosure. In addition, many
modifications may be made to adapt a particular situation,
material, composition of matter, process, or process step or steps,
to the objective, spirit and scope of the present disclosure. All
such modifications are intended to be within the scope of the
disclosure.
Sequence CWU 1
1
61244PRTHomo sapiensmisc_feature(1)..(244)Human Factor B fragment
Ba 1Leu Gly Leu Leu Ser Gly Gly Val Thr Thr Thr Pro Trp Ser Leu Ala
1 5 10 15 Arg Pro Gln Gly Ser Cys Ser Leu Glu Gly Val Glu Ile Lys
Gly Gly 20 25 30 Ser Phe Arg Leu Leu Gln Glu Gly Gln Ala Leu Glu
Tyr Val Cys Pro 35 40 45 Ser Gly Phe Tyr Pro Tyr Pro Val Gln Thr
Arg Thr Cys Arg Ser Thr 50 55 60 Gly Ser Trp Ser Thr Leu Lys Thr
Gln Asp Gln Lys Thr Val Arg Lys 65 70 75 80 Ala Glu Cys Arg Ala Ile
His Cys Pro Arg Pro His Asp Phe Glu Asn 85 90 95 Gly Glu Tyr Trp
Pro Arg Ser Pro Tyr Tyr Asn Val Ser Asp Glu Ile 100 105 110 Ser Phe
His Cys Tyr Asp Gly Tyr Thr Leu Arg Gly Ser Ala Asn Arg 115 120 125
Thr Cys Gln Val Asn Gly Arg Trp Ser Gly Gln Thr Ala Ile Cys Asp 130
135 140 Asn Gly Ala Gly Tyr Cys Ser Asn Pro Gly Ile Pro Ile Gly Thr
Arg 145 150 155 160 Lys Val Gly Ser Gln Tyr Arg Leu Glu Asp Ser Val
Thr Tyr His Cys 165 170 175 Ser Arg Gly Leu Thr Leu Arg Gly Ser Gln
Arg Arg Thr Cys Gln Glu 180 185 190 Gly Gly Ser Trp Ser Gly Thr Glu
Pro Ser Cys Gln Asp Ser Phe Met 195 200 205 Tyr Asp Thr Pro Gln Glu
Val Ala Glu Ala Phe Leu Ser Ser Leu Thr 210 215 220 Glu Thr Ile Glu
Gly Val Asp Ala Glu Asp Gly His Gly Pro Gly Glu 225 230 235 240 Gln
Gln Lys Arg 277PRTHomo sapiensmisc_feature(1)..(77)Human Complement
Component C3a 2Ser Val Gln Leu Thr Glu Lys Arg Met Asp Lys Val Gly
Lys Tyr Pro 1 5 10 15 Lys Glu Leu Arg Lys Cys Cys Glu Asp Gly Met
Arg Glu Asn Pro Met 20 25 30 Arg Phe Ser Cys Gln Arg Arg Thr Arg
Phe Ile Ser Leu Gly Glu Ala 35 40 45 Cys Lys Lys Val Phe Leu Asp
Cys Cys Asn Tyr Ile Thr Glu Leu Arg 50 55 60 Arg Gln His Ala Arg
Ala Ser His Leu Gly Leu Ala Arg 65 70 75 374PRTHomo
sapiensmisc_feature(1)..(74)Human Complement Component C5a 3Thr Leu
Gln Lys Lys Ile Glu Glu Ile Ala Ala Lys Tyr Lys His Ser 1 5 10 15
Val Val Lys Lys Cys Cys Tyr Asp Gly Ala Cys Val Asn Asn Asp Glu 20
25 30 Thr Cys Glu Gln Arg Ala Ala Arg Ile Ser Leu Gly Pro Arg Cys
Ile 35 40 45 Lys Ala Phe Thr Glu Cys Cys Val Val Ala Ser Gln Leu
Arg Ala Asn 50 55 60 Ile Ser His Lys Asp Met Gln Leu Gly Arg 65 70
4155PRTHomo sapiensmisc_feature(1)..(155)Human Prothrombin
Activation Fragment 1 4Ala Asn Thr Phe Leu Glu Glu Val Arg Lys Gly
Asn Leu Glu Arg Glu 1 5 10 15 Cys Val Glu Glu Thr Cys Ser Tyr Glu
Glu Ala Phe Glu Ala Leu Glu 20 25 30 Ser Ser Thr Ala Thr Asp Val
Phe Trp Ala Lys Tyr Thr Ala Cys Glu 35 40 45 Thr Ala Arg Thr Pro
Arg Asp Lys Leu Ala Ala Cys Leu Glu Gly Asn 50 55 60 Cys Ala Glu
Gly Leu Gly Thr Asn Tyr Arg Gly His Val Asn Ile Thr 65 70 75 80 Arg
Ser Gly Ile Glu Cys Gln Leu Trp Arg Ser Arg Tyr Pro His Lys 85 90
95 Pro Glu Ile Asn Ser Thr Thr His Pro Gly Ala Asp Leu Gln Glu Asn
100 105 110 Phe Cys Arg Asn Pro Asp Ser Ser Thr Thr Gly Pro Trp Cys
Tyr Thr 115 120 125 Thr Asp Pro Thr Val Arg Arg Gln Glu Cys Ser Ile
Pro Val Cys Gly 130 135 140 Gln Asp Gln Val Thr Val Ala Met Thr Pro
Arg 145 150 155 5129PRTHomo sapiensmisc_feature(1)..(129)Human
Prothrombin Activation Fragment 2 5Ser Glu Gly Ser Ser Val Asn Leu
Ser Pro Pro Leu Glu Gln Cys Val 1 5 10 15 Pro Asp Arg Gly Gln Gln
Tyr Gln Gly Arg Leu Ala Val Thr Thr His 20 25 30 Gly Leu Pro Cys
Leu Ala Trp Ala Ser Ala Gln Ala Lys Ala Leu Ser 35 40 45 Lys His
Gln Asp Phe Asn Ser Ala Val Gln Leu Val Glu Asn Phe Cys 50 55 60
Arg Asn Pro Asp Gly Asp Glu Glu Gly Val Trp Cys Tyr Val Ala Gly 65
70 75 80 Lys Pro Gly Asp Phe Gly Tyr Cys Asp Leu Asn Tyr Cys Glu
Glu Ala 85 90 95 Val Glu Glu Glu Thr Gly Asp Gly Leu Asp Glu Asp
Ser Asp Arg Ala 100 105 110 Ile Glu Gly Arg Thr Ala Thr Ser Glu Tyr
Gln Thr Phe Phe Asn Pro 115 120 125 Arg 6622PRTHomo
sapiensmisc_feature(1)..(622)Human Prothrombin 6Met Ala His Val Arg
Gly Leu Gln Leu Pro Gly Cys Leu Ala Leu Ala 1 5 10 15 Ala Leu Cys
Ser Leu Val His Ser Gln His Val Phe Leu Ala Pro Gln 20 25 30 Gln
Ala Arg Ser Leu Leu Gln Arg Val Arg Arg Ala Asn Thr Phe Leu 35 40
45 Glu Glu Val Arg Lys Gly Asn Leu Glu Arg Glu Cys Val Glu Glu Thr
50 55 60 Cys Ser Tyr Glu Glu Ala Phe Glu Ala Leu Glu Ser Ser Thr
Ala Thr 65 70 75 80 Asp Val Phe Trp Ala Lys Tyr Thr Ala Cys Glu Thr
Ala Arg Thr Pro 85 90 95 Arg Asp Lys Leu Ala Ala Cys Leu Glu Gly
Asn Cys Ala Glu Gly Leu 100 105 110 Gly Thr Asn Tyr Arg Gly His Val
Asn Ile Thr Arg Ser Gly Ile Glu 115 120 125 Cys Gln Leu Trp Arg Ser
Arg Tyr Pro His Lys Pro Glu Ile Asn Ser 130 135 140 Thr Thr His Pro
Gly Ala Asp Leu Gln Glu Asn Phe Cys Arg Asn Pro 145 150 155 160 Asp
Ser Ser Thr Thr Gly Pro Trp Cys Tyr Thr Thr Asp Pro Thr Val 165 170
175 Arg Arg Gln Glu Cys Ser Ile Pro Val Cys Gly Gln Asp Gln Val Thr
180 185 190 Val Ala Met Thr Pro Arg Ser Glu Gly Ser Ser Val Asn Leu
Ser Pro 195 200 205 Pro Leu Glu Gln Cys Val Pro Asp Arg Gly Gln Gln
Tyr Gln Gly Arg 210 215 220 Leu Ala Val Thr Thr His Gly Leu Pro Cys
Leu Ala Trp Ala Ser Ala 225 230 235 240 Gln Ala Lys Ala Leu Ser Lys
His Gln Asp Phe Asn Ser Ala Val Gln 245 250 255 Leu Val Glu Asn Phe
Cys Arg Asn Pro Asp Gly Asp Glu Glu Gly Val 260 265 270 Trp Cys Tyr
Val Ala Gly Lys Pro Gly Asp Phe Gly Tyr Cys Asp Leu 275 280 285 Asn
Tyr Cys Glu Glu Ala Val Glu Glu Glu Thr Gly Asp Gly Leu Asp 290 295
300 Glu Asp Ser Asp Arg Ala Ile Glu Gly Arg Thr Ala Thr Ser Glu Tyr
305 310 315 320 Gln Thr Phe Phe Asn Pro Arg Thr Phe Gly Ser Gly Glu
Ala Asp Cys 325 330 335 Gly Leu Arg Pro Leu Phe Glu Lys Lys Ser Leu
Glu Asp Lys Thr Glu 340 345 350 Arg Glu Leu Leu Glu Ser Tyr Ile Asp
Gly Arg Ile Val Glu Gly Ser 355 360 365 Asp Ala Glu Ile Gly Met Ser
Pro Trp Gln Val Met Leu Phe Arg Lys 370 375 380 Ser Pro Gln Glu Leu
Leu Cys Gly Ala Ser Leu Ile Ser Asp Arg Trp 385 390 395 400 Val Leu
Thr Ala Ala His Cys Leu Leu Tyr Pro Pro Trp Asp Lys Asn 405 410 415
Phe Thr Glu Asn Asp Leu Leu Val Arg Ile Gly Lys His Ser Arg Thr 420
425 430 Arg Tyr Glu Arg Asn Ile Glu Lys Ile Ser Met Leu Glu Lys Ile
Tyr 435 440 445 Ile His Pro Arg Tyr Asn Trp Arg Glu Asn Leu Asp Arg
Asp Ile Ala 450 455 460 Leu Met Lys Leu Lys Lys Pro Val Ala Phe Ser
Asp Tyr Ile His Pro 465 470 475 480 Val Cys Leu Pro Asp Arg Glu Thr
Ala Ala Ser Leu Leu Gln Ala Gly 485 490 495 Tyr Lys Gly Arg Val Thr
Gly Trp Gly Asn Leu Lys Glu Thr Trp Thr 500 505 510 Ala Asn Val Gly
Lys Gly Gln Pro Ser Val Leu Gln Val Val Asn Leu 515 520 525 Pro Ile
Val Glu Arg Pro Val Cys Lys Asp Ser Thr Arg Ile Arg Ile 530 535 540
Thr Asp Asn Met Phe Cys Ala Gly Tyr Lys Pro Asp Glu Gly Lys Arg 545
550 555 560 Gly Asp Ala Cys Glu Gly Asp Ser Gly Gly Pro Phe Val Met
Lys Ser 565 570 575 Pro Phe Asn Asn Arg Trp Tyr Gln Met Gly Ile Val
Ser Trp Gly Glu 580 585 590 Gly Cys Asp Arg Asp Gly Lys Tyr Gly Phe
Tyr Thr His Val Phe Arg 595 600 605 Leu Lys Lys Trp Ile Gln Lys Val
Ile Asp Gln Phe Gly Glu 610 615 620
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