U.S. patent application number 14/930581 was filed with the patent office on 2016-02-18 for biomarker for diagnosis, prediction and/or prognosis of acute heart failure and uses thereof.
The applicant listed for this patent is MyCartis NV. Invention is credited to Koen Kas.
Application Number | 20160047822 14/930581 |
Document ID | / |
Family ID | 43899854 |
Filed Date | 2016-02-18 |
United States Patent
Application |
20160047822 |
Kind Code |
A1 |
Kas; Koen |
February 18, 2016 |
BIOMARKER FOR DIAGNOSIS, PREDICTION AND/OR PROGNOSIS OF ACUTE HEART
FAILURE AND USES THEREOF
Abstract
The application discloses MCAM as a new biomarker for acute
heart failure; methods for predicting, diagnosing, prognosticating
and/or monitoring acute heart failure based on measuring said
biomarker; and kits and devices for measuring said biomarker and/or
performing said methods.
Inventors: |
Kas; Koen; (Schilde,
BE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MyCartis NV |
Gent |
|
BE |
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|
Family ID: |
43899854 |
Appl. No.: |
14/930581 |
Filed: |
November 2, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13502846 |
Apr 19, 2012 |
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PCT/EP2010/065841 |
Oct 21, 2010 |
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14930581 |
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61253658 |
Oct 21, 2009 |
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61314789 |
Mar 17, 2010 |
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61254537 |
Oct 23, 2009 |
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Current U.S.
Class: |
514/26 |
Current CPC
Class: |
A61P 3/12 20180101; G01N
2333/70596 20130101; G01N 33/6872 20130101; G01N 33/574 20130101;
G01N 2333/70503 20130101; G01N 33/68 20130101; G01N 2800/12
20130101; G01N 2800/325 20130101; G01N 2800/52 20130101 |
International
Class: |
G01N 33/68 20060101
G01N033/68 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 21, 2009 |
EP |
09173601.7 |
Mar 17, 2010 |
EP |
10156705.5 |
Claims
1-48. (canceled)
49. A method for treating acute heart failure (AHF) which involves
systolic dysfunction, the method comprising the steps of: a)
obtaining a plasma sample from a subject presenting with rapid
onset of symptoms, wherein the symptoms comprise dyspnea; b) having
an assay conducted for measurement of melanoma cell adhesion
molecule (MCAM), the assay comprising detecting the quantity of
MCAM, thereby measuring the quantity of circulating MCAM in the
sample from the subject; (c) comparing the quantity of circulating
MCAM measured in (b) with a reference value of the quantity of
circulating MCAM, said reference value representing a known
diagnosis, and/or prognosis of AHF which involves systolic
dysfunction; (d) finding a deviation or no deviation of the
quantity of circulating MCAM measured in (b) from the reference
value; (e) attributing said finding of deviation or no deviation to
a particular diagnosis, and/or prognosis of AHF in the subject,
wherein elevated quantities of circulating MCAM in the sample from
the subject compared to a reference value representing a diagnosis
of no AHF or representing a good prognosis for AHF indicates that
the subject has or is at risk of having AHF which involves systolic
dysfunction or indicates a poor prognosis for AHF which involves
systolic dysfunction in the subject; (f) identifying subjects
having AHF with systolic dysfunction in subjects presenting with
rapid onset of symptoms comprising dyspnea; (g) treating the
subject having AHF with systolic dysfunction with one or more
treatments selected from the group consisting of diuretics, beta
blockers, ACE inhibitors, and inotropic drugs and avoiding
treatment with calcium channel blockers.
50. The method according to claim 49, wherein the subject has a
medical history of heart failure.
51. The method according to claim 49, wherein the subject is
diagnosed with AHF with systolic dysfunction and wherein steps (b)
to (f) of claim 49 are repeated at a time point where a diagnosis
of recovery of AHF has to be made.
52. The method according to claim 49 wherein the subject is
diagnosed with AHF with systolic dysfunction and wherein steps (b)
to (f) of claim 49 are repeated for monitoring a change in the
diagnosis, and/or prognosis of AHF in a subject, comprising: (i)
applying the method of steps (b) to (f) of claim 49 to the subject
at one or more additional successive time points, whereby the
diagnosis, and/or prognosis of AHF in the subject is determined at
said successive time points; (ii) comparing the diagnosis, and/or
prognosis of AHF in the subject at said successive time points as
determined in (i); and (iii) finding the presence or absence of a
change between the diagnosis, and/or prognosis of AHF in the
subject at said successive time points as determined in (i),
wherein the AHF in the subject involves systolic dysfunction.
53. The method according to claim 52, wherein said change in the
diagnosis, and/or prognosis of AHF in the subject is monitored in
the course of a medical treatment of said subject.
54. The method according to claim 49 wherein, the presence or
absence and/or quantity of one or more other biomarkers in the
sample from the subject is measured and wherein said one or more
other biomarker useful for diagnosing, and/or prognosticating AHF
is selected from the group consisting of, B-type-natriuretic
peptide (BNP), pro-B-type natriuretic peptide (proBNP), and amino
terminal pro-B-type natriuretic peptide (NTproBNP).
55. The method according to claim 49, wherein said systolic
dysfunction is characterized by a decreased left ventricular
ejection fraction (LVEF), preferably wherein said LVEF is less than
55% or less than 50% or less than 45%, and/or by increased cardiac
filling pressure.
56. The method according to claim 49, wherein the quantity of
circulating MCAM and/or the presence or absence and/or quantity of
the one or more other biomarkers is measured using, respectively, a
binding agent capable of specifically binding to circulating MCAM
and/or to fragments thereof, and a binding agent capable of
specifically binding to said one or more other biomarkers.
57. The method according to claim 49, wherein the quantity of
circulating MCAM and/or the presence or absence and/or quantity of
the one or more other biomarkers is measured using an immunoassay
technology, such as direct ELISA, indirect ELISA, sandwich ELISA,
competitive ELISA, multiplex ELISA, radioimmunoassay (RIA) or
ELISPOT technologies, or using a mass spectrometry analysis method
or using a chromatography method, or using a combination of said
methods.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 13/502,846, filed Apr. 19, 2012 which is the U.S. National
Phase under 35 U.S.C. .sctn.371 of International Application
PCT/EP2010/065841, filed Oct. 21, 2010, which claims priority to EP
09173601.7, filed Oct. 21, 2009, U.S. 61/253,658, filed Oct. 21,
2009, U.S. 61/254,537, filed Oct. 23, 2009, EP 10156705.5, filed
Mar. 17, 2010 and U.S. 61/314,789, filed Mar. 17, 2010.
FIELD OF THE INVENTION
[0002] The invention relates to protein- and/or peptide-based
biomarkers and to agents specifically binding thereto, for use in
predicting, diagnosing, prognosticating and/or monitoring diseases
or conditions in subjects. More particularly, the application
discloses certain proteins and/or peptides as new biomarkers for
acute heart failure; methods for predicting, diagnosing and/or
prognosticating acute heart failure based on measuring said
biomarker proteins and/or peptides; and kits and devices for
measuring said proteins and/or peptides and/or performing said
methods.
BACKGROUND OF THE INVENTION
[0003] In many diseases and conditions, a favourable outcome of
prophylactic and/or therapeutic treatments is strongly correlated
with early and/or accurate prediction, diagnosis and/or prognosis
of the disease or condition. Therefore, there exists a continuous
need for additional and preferably improved manners for early
and/or accurate prediction, diagnosis and/or prognosis of diseases
and conditions to guide the treatment choices.
[0004] Heart failure is a major public health issue in developed
countries and is the cause of considerable morbidity and mortality
among older adults. It is usually a chronic disease characterised
by frequent recurrent decompensation leading to worsening breathing
problems. Moreover, 5 years after diagnosis 50% of heart failure
patients will have died from the disease.
[0005] Acute heart failure (AHF) is a sudden inability of the heart
to pump efficiently and where it can no longer foresee the bodily
demands for oxygen. AHF is the cause of over two million
hospitalisations annually in US and Europe, and displays a
mortality rate of about 20-40% within one year of hospital
discharge in many populations. About 90% of AHF admissions are
typically from patients with chronic heart disease, the remaining
about 10% are de novo patients. The clinical signs of heart disease
and AHF are often non-specific which can make unambiguous diagnosis
demanding.
[0006] A common symptom of AHF is the shortness of breath (dyspnea
or dyspnoea). However, usually only a fraction of subjects
presenting with dyspnea upon admission to a physician or clinic
suffer from AHF. Therefore, a rapid, proper and effective treatment
of AHF requires to adequately distinguish AHF patients from
patients having dyspnea due to other causes.
[0007] Currently, diagnosis of AHF is mainly done on the basis of
clinical signs, such as, ECG, chest X-ray, etc. One biomarker often
used to complement these diagnostic criteria of AHF such as in
emergency setting is B-type natriuretic peptide (BNP). Typically,
BNP lower than 100 pg/mL is regarded as a "rule-out" criterion for
heart failure, whereas BNP higher than 400 pg/mL is seen as a
"rule-in" criterion for AHF. Although BNP is sensitive, its
specificity is relatively low, and is especially problematic due to
the "grey zone" between 100-400 pg/mL. For example, Chung et al.
2006 (Am Heart J 152(5): 949-55) have determined that the BNP cut
point of 100 pg/mL has 100% sensitivity but only 41% specificity
for diagnosing AHF, whereas the cut point of 400 pg/mL has 87%
sensitivity and 76% specificity.
[0008] Also, BNP levels vary with age, sex, weight and other
medical conditions, thereby confounding the diagnosis. Notably, BNP
levels tend to be elevated in patients with medical history of
heart failure and renal failure. For example, Chung et al. 2006
(supra) have shown that BNP performance for diagnosing AHF in
patients presenting with dyspnea is significantly reduced in
patients with a history of heart failure. In particular, about 40%
of patients presenting with dyspnea not caused by AHF, who had a
history of heart failure, displayed BNP values over 400 pg/mL, the
AHF cut-off point used currently in the clinic. Consequently, the
European Society of Cardiology (ESC) Guidelines 2008 also
characterise BNP as a biomarker of heart failure in general rather
than of acute heart failure.
[0009] In view of this, there exists a persistent need for
additional and preferably specific biomarkers for AHF. Such novel
AHF biomarkers may be comparable to or improved over previously
existing markers, such as over BNP, in one or more of their
characteristics, such as, for example, in their sensitivity and/or
specificity, in their reliability in patients presenting with a
symptom potentially indicative of AHF such as with dyspnea, in
their reliability in patients with history of heart failure and
other frequent co-morbidities of heart failure such renal failure,
obesity, coronary artery disease etc.
[0010] In addition, several causes underlie (acute) heart failure.
Specifically systolic dysfunction and diastolic dysfunction lead to
cardiac remodelling and altered cardiac function, resulting in a
decreased cardiac output. Both dysfunctions are characterized by
defects in the pumping function of the heart. Systolic dysfunction
results from a loss of intrinsic inotropy (contractility), most
likely due to alterations in signal transduction mechanisms
responsible for regulating inotropy, and is characterized by
defects in emptying the heart, in particular the ventricle, of
blood during contraction (i.e. the systole). Diastolic dysfunction
occurs when the ventricle becomes less compliant (i.e., "stiffer"),
which impairs ventricular filling and as such is characterized by
defects in filling the heart, in particular the ventricle, with
blood during relaxation (i.e. the diastole).
[0011] As such, the pathophysiology of systolic and diastolic
dysfunction differs, as intrinsic compensatory mechanisms to cope
with both dysfunctions differ. Although systolic and diastolic
dysfunction share some common symptoms, the nature of treatment at
least partially differs. Whereas both beta blockers and ACE
inhibitors are indicated for the treatment of both systolic and
diastolic dysfunction, possibly in combination with diuretics,
inotropic drugs for instance, such as digoxin, are specifically
indicated for the treatment of systolic dysfunction (and
contra-indicated for the treatment of diastolic dysfunction) and
for instance calcium channel blockers are specifically indicated
for the treatment of diastolic dysfunction (and contra-indicated
for the treatment of systolic dysfunction).
[0012] It may be clear that accurate and reliable diagnosis,
prediction, prognosis and/or monitoring of systolic and/or
diastolic dysfunction as well as the differentiation between both
dysfunctions, is needed for adequate treatment. The present
invention addresses the above needs in the art by identifying
biomarkers for AHF and more preferably systolic dysfunction and
parameters associated therewith, and providing uses therefore.
SUMMARY OF THE INVENTION
[0013] Having conducted extensive experiments and tests, the
inventors have revealed that melanoma cell adhesion molecule (MCAM,
also known as CD146 or MUC18), represents a new biomarker
particularly advantageous for predicting, diagnosing and/or
prognosticating acute heart failure (AHF).
[0014] In particular, in a 3-centre study involving prospective
collection of samples from subjects presenting with dyspnea upon
emergency admission, the inventors have first identified and
subsequently validated MCAM as a biomarker displaying a
significantly altered level in dyspneic patients having AHF, when
compared to dyspneic patients not having AHF. In addition, the
inventors have also realised that MCAM may be a useful biomarker
for monitoring the progression of AHF and/or can be used to predict
an acute event, since the amount of MCAM significantly differed
between dyspneic AHF patients upon admission (i.e., before
treatment) and upon discharge (i.e., following treatment).
[0015] Current data indicates that the performance of the MCAM
marker is at least equivalent to that of BNP.
[0016] Furthermore, for discriminating between the dyspneic
patients with and without AHF, the AUC value (area under the ROC
curve; "ROC" stands for receiver operating characteristic) is
slightly higher for MCAM (0.91) than for each one of BNP (0.88) and
NT-proBNP (0.85). The AUC value is a combined measure of
sensitivity and specificity and a higher AUC value (i.e.,
approaching 1) in general indicates an improved performance of the
test.
[0017] In addition, as mentioned above, the BNP marker diagnosis
has a troublesome "grey zone" between values of 100-400 pg/ml, in
which no exact diagnosis of AHF can be established. Using the MCAM
marker level in said samples of the BNP "grey zone" resulted in a
clear distinction between AHF and non-AHF-dyspnea patients.
[0018] This overall diagnostic performance of MCAM is, depending on
the data set used, better or at least equivalent to BNP and
NT-proBNP, the current gold standard biomarkers for diagnosing AHF
in an acute dyspnea population. At a single ratio or concentration
cut-off MCAM reaches a diagnostic accuracy of 84% while BNP at its
rule-out cut-off (100 pg/mL) has only an accuracy of 71%.
[0019] Taken together, the inventors have identified and validated
MCAM (CD146, or MUC-18) as a further and improved biomarker for
predicting, diagnosing and/or prognosticating AHF, in particular in
patients with a history of heart failure, or suffering from other
non-AHF-disorders causing dyspnea.
[0020] Consequently, in an aspect the invention provides a method
for predicting, diagnosing and/or prognosticating acute heart
failure (AHF) in a subject, characterised in that the examination
phase of the method comprises measuring the quantity of MCAM in a
sample from the subject. One understands that methods of
prediction, diagnosis and/or prognosis of diseases or conditions
generally comprise an examination phase in which data is collected
from and/or about the subject.
[0021] Hence, provided is a method for predicting, diagnosing
and/or prognosticating AHF in a subject that may comprise the
steps: [0022] (i) measuring the quantity of MCAM in a sample from
the subject; [0023] (ii) comparing the quantity of MCAM measured in
(i) with a reference value of the quantity of MCAM, said reference
value representing a known prediction, diagnosis and/or prognosis
of AHF; [0024] (iii) finding a deviation or no deviation of the
quantity of MCAM measured in (i) from the reference value; [0025]
(iv) attributing said finding of deviation or no deviation to a
particular prediction, diagnosis and/or prognosis of AHF in the
subject.
[0026] MCAM provides an improved or even substantially complete
discrimination of AHF from non-AHF dyspnea phenotypes. Therefore,
the inventors contemplate that MCAM can also be beneficial for
population screening setups to select subjects having or being at
risk of having AHF. The use of BNP for such population screening is
complicated especially by the confounding effect of heart history
(e.g., CHF pathology) on the BNP readout, hence BNP fails for
screening due to lack of specificity. Thus, in an embodiment, the
present methods for predicting, diagnosing and/or prognosticating
AHF in a subject may be employed for population screening (such as,
e.g., screening in a general population or in a population
stratified based on one or more criteria, e.g., age, gender,
ancestry, occupation, presence or absence of risk factors of AHF,
etc.).
[0027] As demonstrated in the experimental section, the inventors
have shown that prediction or diagnosis of AHF or a poor prognosis
of AHF can in particular be associated with an elevated level of
MCAM. Hence, in an embodiment of the prediction, diagnosis and/or
prognosis methods as taught herein, an elevated quantity of MCAM in
the sample from the subject compared to a reference value
representing the prediction or diagnosis of no AHF or representing
a good prognosis for AHF respectively indicates that the subject
has or is at risk of having AHF or indicates a poor prognosis for
AHF in the subject.
[0028] In addition, the inventors have tested patients diagnosed
with acute heart failure both at admission to the Emergency
Department (ED) and at discharge from the hospital, i.e. when
patients were deemed to have recovered and to be stable. Most
patients showed a significant decrease of MCAM upon discharge when
compared to levels at the admission stage. A very similar picture
is obtained when BNP levels at admission versus discharge are
compared. This data supports the idea that MCAM levels are a
reflection of disease status and thus could be used to monitor
and/or predict an acute event. The inventors have also observed and
verified that methods using MCAM as a biomarker, and particularly
but without limitation the methods for discriminating between the
dyspneic patients with and without AHF, can achieve a sensitivity
of 80% or more and/or a specificity of 80% or more. Hence, in an
embodiment of the prediction, diagnosis and/or prognosis methods as
taught herein, the sensitivity and/or specificity (and preferably,
the sensitivity and specificity) of the methods is at least 50%, at
least 60%, at least 70% or at least 80%, e.g., .gtoreq.81%,
.gtoreq.82%, .gtoreq.83%, .gtoreq.84%, .gtoreq.85%, .gtoreq.86%, or
.gtoreq.87%, or .gtoreq.90% or .gtoreq.95% (symbol ".gtoreq." is
synonymous with expressions "at least" or "equal to or more"),
e.g., between 80% and 100%, or between 81% and 95%, or between 83%
and 90%, or between 84% and 89%, or between 85% and 88%.
[0029] In another embodiment of the prediction, diagnosis and/or
prognosis methods as taught herein, the subject may present itself
with one or more symptoms and/or signs potentially indicative of
AHF. For example, in an embodiment the subject may present itself
with dyspnea. Hence, in an embodiment the methods may be for
discriminating between subjects presenting with dyspnea due to AHF
and subjects presenting themselves with dyspnea due to causes other
than or unrelated to AHF (such as, e.g., due to COPD, or
pneumonia).
[0030] In a further embodiment of the prediction, diagnosis and/or
prognosis methods as taught herein, the subject may display one or
more risk factors for AHF, such as, for example, a genetic
predisposition or one or more developmental, environmental or
behavioural risk factors, such as, e.g., insulin resistance
(impaired blood glucose), truncal obesity, high serum low density
lipoprotein (LDL) cholesterol levels, low serum high density
lipoprotein (HDL) cholesterol levels, high serum triglyceride
levels, and high blood pressure (hypertension), prior myocardial
infarctus, and/or one or more co-morbidities, such as diabetes,
coronary artery disease, asthma, COPD and/or chronic renal
disease.
[0031] Hence, in various embodiments, the present methods for
predicting, diagnosing and/or prognosticating AHF may be used in
individuals who have not yet been diagnosed as having AHF (for
example, preventative screening), or who have been diagnosed as
having AHF or CHF, or who are suspected of having AHF or CHF (for
example, display one or more symptoms characteristic of AHF or
CHF), or who are at risk of developing AHF or CHF (for example,
genetic predisposition; presence of one or more developmental,
environmental or behavioural risk factors). The methods may also be
used to detect various stages of progression or severity of AHF.
The methods may also be used to detect response of AHF to
prophylactic or therapeutic treatments or other interventions. The
methods can furthermore be used to help the medical practitioner in
deciding upon worsening, status-quo, partial recovery, or complete
recovery of the patient from the acute (AHF) event, resulting in
either further treatment or observation or in discharge of the
patient from the ED. The methods of the present invention enable
the medical practitioner to monitor the disease progress by
measuring the level of MCAM in a sample of the patient, wherein a
decrease in MCAM level as compared to a prior MCAM level (e.g. at
the time of the admission to the ED) indicates the condition of the
subject is improving or has improved, while an increase of the MCAM
level as compared to the level of MCAM as measured upon admission
to the ED indicates the condition of the subject has worsened or is
worsening and could possibly result in a new acute heart failure
event. The invention further provides a method for monitoring a
change in the prediction, diagnosis and/or prognosis of AHF in a
subject, comprising: [0032] (i) applying the prediction, diagnosis
and/or prognosis method as taught here above to the subject at one
or more successive time points, whereby the prediction, diagnosis
and/or prognosis of AHF in the subject is determined at said
successive time points; [0033] (ii) comparing the prediction,
diagnosis and/or prognosis of AHF in the subject at said successive
time points as determined in (i); and [0034] (iii) finding the
presence or absence of a change between the prediction, diagnosis
and/or prognosis of AHF in the subject at said successive time
points as determined in (i).
[0035] This aspect allows to monitor the subject's condition over
time. This can inter alia allow to predict the occurrence of an AHF
event, or to monitor in said subject the disease progression,
disease aggravation or alleviation, disease recurrence, response to
treatment, response to other external or internal factors,
conditions, or stressors, etc. Advantageously, the change in the
prediction, diagnosis and/or prognosis of AHF in the subject may be
monitored in the course of a medical treatment of said subject,
preferably a medical treatment aimed at treating AHF. Such
monitoring may be comprised, e.g., in decision making whether a
patient (e.g., a dyspneic or AHF patient) may be discharged or
needs further hospitalisation.
[0036] Typically, this is done by measuring the MCAM level in a
subject at different time points during the stay in the ED, wherein
a decrease in MCAM level in function of time indicates the
condition of the subject is improving or has improved, while an
increase of the MCAM level in function of time indicates the
condition of the subject has worsened or is worsening and could
possibly result in a new acute heart failure event.
[0037] It shall be appreciated that in the present prediction,
diagnosis and/or prognosis methods the measurement of MCAM may also
be combined with the assessment of one or more further biomarkers
relevant for AHF.
[0038] Consequently, also disclosed herein are methods, wherein the
examination phase of the methods further comprises measuring the
presence or absence and/or quantity of one or more other biomarkers
useful for predicting, diagnosing and/or prognosticating AHF in the
sample from the subject. In this respect, any known or yet unknown
suitable AHF marker could be used. In a preferred embodiment, said
additional AHF marker is selected from the group consisting of:
B-type natriuretic peptide (BNP), pro-B-type natriuretic peptide
(proBNP), amino terminal pro-B-type natriuretic peptide (NTproBNP),
and fragments of any one thereof.
[0039] Hence, disclosed is a method for predicting, diagnosing
and/or prognosticating AHF in a subject comprising the steps:
[0040] (i) measuring the quantity of MCAM and the presence or
absence and/or quantity of said one or more other biomarkers in the
sample from the subject; [0041] (ii) using the measurements of (i)
to establish a subject profile of the quantity of MCAM and the
presence or absence and/or quantity of said one or more other
biomarkers; [0042] (iii) comparing said subject profile of (ii) to
a reference profile of the quantity of MCAM and the presence or
absence and/or quantity of said one or more other biomarkers, said
reference profile representing a known prediction, diagnosis and/or
prognosis of AHF; [0043] (iv) finding a deviation or no deviation
of the subject profile of (ii) from the reference profile; [0044]
(v) attributing said finding of deviation or no deviation to a
particular prediction, diagnosis and/or prognosis of AHF in the
subject.
[0045] In an embodiment, said other biomarker useful for
predicting, diagnosing and/or prognosticating AHF is chosen from
the group consisting of B-type natriuretic peptide (BNP),
pro-B-type natriuretic peptide (proBNP), amino terminal pro-B-type
natriuretic peptide (NTproBNP), and fragments of any one
thereof.
[0046] In preferred embodiments of the methods of the present
invention, the MCAM protein detection is done in a plasma sample
(i.e. a non-blood-cell containing blood sample fraction), implying
that the circulating MCAM protein is detected, regardless of
whether or not this circulating form corresponds to the
MMP-processed soluble form or to a degradation product of the
full-length or of said soluble form of MCAM. In a preferred
embodiment, the MCAM protein detected is not membrane or
cell-bound, regardless of how release of MCAM into plasma or serum
is achieved in vivo.
[0047] As indicated above, the present methods may employ reference
values for the quantity of MCAM, which may be established according
to known procedures previously employed for other biomarkers. Such
reference values may be established either within (i.e.,
constituting a step of) or external to (i.e., not constituting a
step of) the methods of the present invention as defined
herein.
[0048] Accordingly, any one of the methods taught herein may
comprise a step of establishing a reference value for the quantity
of MCAM, said reference value representing either (a) a prediction
or diagnosis of no AHF or a good prognosis for AHF, or (b) a
prediction or diagnosis of AHF or a poor prognosis for AHF.
[0049] A further aspect provides a method for establishing a
reference value for the quantity of MCAM, said reference value
representing: [0050] (a) a prediction or diagnosis of no AHF or a
good prognosis for AHF, or [0051] (b) a prediction or diagnosis of
AHF or a poor prognosis for AHF, comprising: [0052] (i) measuring
the quantity of MCAM in: [0053] (i a) one or more samples from one
or more subjects not having AHF or not being at risk of having AHF
or having a good prognosis for AHF, or [0054] (i b) one or more
samples from one or more subjects having AHF or being at risk of
having AHF or having a poor prognosis for AHF, and [0055] (ii)
storing the quantity of MCAM [0056] (ii a) as measured in (i a) as
the reference value representing the prediction or diagnosis of no
AHF or representing the good prognosis for AHF, or [0057] (ii b) as
measured in (i b) as the reference value representing the
prediction or diagnosis of AHF or representing the poor prognosis
for AHF.
[0058] The present methods may otherwise employ reference profiles
for the quantity of MCAM and the presence or absence and/or
quantity of one or more other biomarkers useful for predicting,
diagnosing and/or prognosticating AHF, which may be established
according to known procedures previously employed for other
biomarkers. Such reference profiles may be established either
within (i.e., constituting a step of) or external to (i.e., not
constituting a step of) the present methods. Accordingly, the
methods taught herein may comprise a step of establishing a
reference profile for the quantity of MCAM and the presence or
absence and/or quantity of said one or more other biomarkers, said
reference profile representing either (a) a prediction or diagnosis
of no AHF or a good prognosis for AHF, or (b) a prediction or
diagnosis of AHF or a poor prognosis for AHF.
[0059] A further aspect provides a method for establishing a
reference profile for the quantity of MCAM and the presence or
absence and/or quantity of one or more other biomarkers useful for
predicting, diagnosing and/or prognosticating AHF, said reference
profile representing: [0060] (a) a prediction or diagnosis of no
AHF or a good prognosis for AHF, or [0061] (b) a prediction or
diagnosis of AHF or a poor prognosis for AHF, comprising: [0062]
(i) measuring the quantity of MCAM and the presence or absence
and/or quantity of said one or more other biomarkers in: [0063] (i
a) one or more samples from one or more subjects not having AHF or
not being at risk of having AHF or having a good prognosis for AHF;
or [0064] (i b) one or more samples from one or more subjects
having AHF or being at risk of having AHF or having a poor
prognosis for AHF; [0065] (ii) [0066] (ii a) using the measurements
of (i a) to create a profile of the quantity of MCAM and the
presence or absence and/or quantity of said one or more other
biomarkers; or [0067] (ii b) using the measurements of (i b) to
create a profile of the quantity of MCAM and the presence or
absence and/or quantity of said one or more other biomarkers;
[0068] (iii) [0069] (iii a) storing the profile of (ii a) as the
reference profile representing the prediction or diagnosis of no
AHF or representing the good prognosis for AHF; or [0070] (iii b)
storing the profile of (ii b) as the reference profile representing
the prediction or diagnosis of AHF or representing the poor
prognosis for AHF.
[0071] In an embodiment, said other biomarker useful for
predicting, diagnosing and/or prognosticating AHF may be chosen
from the group consisting of B-type natriuretic peptide (BNP),
pro-B-type natriuretic peptide (proBNP), amino terminal pro-B-type
natriuretic peptide (NTproBNP), and fragments of any one
thereof.
[0072] The invention further provides a method for establishing a
MCAM base-line or reference value in a subject, comprising: [0073]
(i) measuring the quantity of MCAM in the sample from the subject
at different time points wherein the subject is not suffering from
AHF, and [0074] (ii) calculating the range or mean value of the
subject, which is the MCAM base-line or reference value for said
subject.
[0075] In preferred embodiments of the methods of the present
invention, the MCAM protein detection is done in a plasma sample,
implying that the circulating MCAM protein is detected, regardless
of whether or not this circulating form corresponds to the soluble
form or to a degradation product of the full-length or soluble
form. In a preferred embodiment, the MCAM protein detected in the
methods according to the present invention is not membrane or
cell-bound, but rather is the plasma circulating form of MCAM.
[0076] In preferred embodiments of any one of above methods the
subject may be human. In further preferred embodiments, the subject
is suffering from AHF involving systolic dysfunction. In even more
preferred embodiments, said systolic dysfunction is characterized
by a decreased left ventricular ejection fraction (LVEF),
preferably wherein said LVEF is less than 55% or less than 50% or
less than 45%, and/or by increased cardiac filling pressure.
[0077] The inventors have further found that MCAM levels correlate
with left ventricular ejection fraction (LVEF). Subjects with a
reduced LVEF have been shown to have altered (esp. increased) MCAM
levels, compared to subjects with normal LVEF. As reduced LVEF is a
hallmark for systolic dysfunction, MCAM levels can be used to
predict, diagnose, prognosticate and/or monitor systolic
dysfunction.
[0078] In particular, in a 3-centre study involving prospective
collection of samples from subjects presenting with dyspnea upon
emergency admission, MCAM was significantly increased in dyspneic
patients (esp. AHF patients) showing reduced LVEF indicative of
systolic dysfunction, compared to dyspneic patients with preserved
LVEF and systolic function. Systolic dysfunction may preferably
denote systolic dysfunction of the left ventricle.
[0079] In another aspect, the invention hence relates to a method
for predicting, diagnosing, prognosticating and/or monitoring
systolic dysfunction in a subject, comprising measuring MCAM levels
in a sample from said subject.
[0080] Furthermore, in the above population of AHF patients with a
predominance of heart failure patients with systolic dysfunction,
the AUC value (area under the ROC curve; "ROC" stands for receiver
operating characteristic) for discriminating between the dyspneic
patients with and without AHF, is slightly higher for MCAM (0.91)
than for each one of BNP (0.88) and NT-proBNP (0.85). The AUC value
is a combined measure of sensitivity and specificity and a higher
AUC value (i.e., approaching 1) in general indicates an improved
performance of the test.
[0081] The inventors have further found that MCAM levels correlate
with cardiac filling status. In particular, the inventors have
found that MCAM levels are higher in subjects with an increased
cardiac filling pressure, compared to subjects with normal cardiac
filling pressure.
[0082] Both systolic and diastolic dysfunction can cause fluid
build-up in a subject. Subjects with a systolic dysfunction
however, are more resistant to fluid build-up and hence will
accumulate more volume compared to patients with diastolic
dysfunction before symptoms such as dyspnea occur. The inventors
have found that MCAM levels correlate with fluid build-up, and in
particular the vascular filling status or vascular filling volume
or pressure as a measurement of fluid homeostasis. In particular,
the inventors found that MCAM levels are higher in subjects with an
increased vascular filling volume or pressure and hence MCAM is a
marker for fluid build-up in a subject. As a corollary, MCAM levels
are associated to weight gain due to over-filling or weight loss
due to under-filling or volume contraction of a subject. As such,
the inventors have found that MCAM is a marker for determining
oedema, changes in volume status or dehydration in a subject. In
particular, MCAM levels are correlated with the filling status of a
subject with defects in blood circulation, such as caused by heart
failure, and defects in secretion, such as caused by kidney
dysfunction or kidney failure. Accordingly, in an embodiment, the
invention relates to a method as described herein for diagnosing,
predicting, prognosticating and/or monitoring an impaired fluid
homeostasis in a subject, wherein the subject presents itself with,
is diagnosed with or has a medical history of heart failure, in
particular systolic dysfunction.
[0083] Provided is thus a method for predicting, diagnosing,
prognosticating and/or monitoring dyspnea associated with or caused
by volume overload comprising measuring MCAM levels in a sample
from said subject. Volume overload may be indicative of HF,
preferably HF due to systolic dysfunction, and may be at risk of
decompensation or having decompensated into AHF. The method can
discriminate dyspnea caused by volume overload such as HF or AHF
from other causes of dyspnea (e.g., COPD, pneumonia).
[0084] Disclosed is also a method for predicting, diagnosing,
prognosticating and/or monitoring HF, preferably AHF, associated
with or caused by volume overload in a subject, comprising
measuring MCAM levels in a sample from said subject. The volume
overload may be due to systolic dysfunction.
[0085] Hence, disclosed is also a method for predicting,
diagnosing, prognosticating and/or monitoring HF, preferably AHF,
associated with or caused by systolic dysfunction in a subject,
comprising measuring MCAM levels in a sample from said subject.
[0086] Systolic dysfunction is characterized by a decreased
ejection fraction of the left and/or right ventricle, more
particularly decreased LVEF. The inventors have found that MCAM
levels are correlated with the ventricular ejection fraction.
Disclosed is thus also a method for predicting, diagnosing,
prognosticating and/or monitoring the ventricular ejection fraction
in a subject, comprising measuring MCAM levels in a sample from
said subject.
[0087] A ventricular ejection fraction (e.g., LVEF) in a subject
may be said to be reduced compared to normal, if said ejection
fraction is below normal by any extent, e.g., a reduced ventricular
ejection fraction may mean less than about 45% or less than about
50% or less than about 55%; for example a reduced ventricular
ejection fraction may denote between about 40% and about 70%,
preferably between about 45% and about 65%, or between about 50%
and about 60%, e.g., less than about 55%. In an exemplary but
non-limiting experiment MCAM levels provided particularly
satisfactory discrimination between normal and reduced LVEF when
the threshold between said normal and reduced LVEF was set at 55%.
Hence, in embodiments a threshold for normal vs. reduced
ventricular ejection fraction, in particular LVEF, may be set at a
value between about 50% and about 60%, e.g., at 50%, 51%, 52%, 53%,
54%, 55%, 56%, 57%, 58%, 59% or 60%, and preferably at 55%, wherein
a value above said threshold reflects normal ejection fraction and
a value below said threshold denotes reduced ejection fraction.
[0088] The drop or decrease in cardiac output due to a decreased
ventricular ejection fraction promotes renal salt and water
retention. This appropriate adaptation expands the blood volume,
thereby raising end-diastolic pressure and volume. Thus, systolic
dysfunction is also characterized by an increased cardiac filling
pressure. Hence, provided is also a method for predicting,
diagnosing, prognosticating and/or monitoring the cardiac filling
status in a subject comprising measuring MCAM levels in a sample
from said subject. Cardiac filling status may be represented by the
cardiac filling pressure.
[0089] Hence, provided is a method for predicting, diagnosing
and/or prognosticating systolic dysfunction in a subject that may
comprise the steps: [0090] (i) measuring the quantity of MCAM in a
sample from the subject; [0091] (ii) comparing the quantity of MCAM
measured in (i) with a reference value of the quantity of MCAM,
said reference value representing a known prediction, diagnosis
and/or prognosis of systolic dysfunction; [0092] (iii) finding a
deviation or no deviation of the quantity of MCAM measured in (i)
from the reference value; [0093] (iv) attributing said finding of
deviation or no deviation to a particular prediction, diagnosis
and/or prognosis of systolic dysfunction in the subject.
[0094] The above steps can be applied mutatis mutandis to dyspnea
associated with or caused by volume overload; to HF or AHF
associated with or caused by volume overload; to HF or AHF
associated with or caused by systolic dysfunction; to ventricular
ejection fraction; or to cardiac filling status.
[0095] MCAM provides an improved or even substantially complete
discrimination of dyspnea caused by volume overload such as AHF
from other causes of dyspnea. Therefore, the inventors contemplate
that MCAM can also be beneficial for population screening setups to
select subjects having or being at risk of having an acute
decompensation. Any one of the herein described methods may be
employed for population screening (such as, e.g., screening in a
general population or in a population stratified based on one or
more criteria, e.g., age, gender, ancestry, occupation, presence or
absence of risk factors of AHF, etc.). In any one the above methods
of the present invention, the subject may form part of a patient
population showing signs of dyspnea.
[0096] The inventors have found that MCAM can be used as a specific
biomarker for systolic dysfunction. Hence, in an aspect, the
invention relates to the use of the methods as described herein for
discriminating between systolic and diastolic dysfunction.
[0097] In an embodiment, provided is a method for discriminating
between systolic dysfunction and diastolic dysfunction in a
subject, comprising: [0098] (i) measuring the quantity of MCAM in a
sample from said subject; [0099] (ii) comparing the quantity of
MCAM measured in (i) with a reference value of the quantity of
MCAM, said reference value representing a threshold for the
diagnosis of systolic dysfunction; [0100] (iii) attributing the
diagnosis of systolic dysfunction in said subject if the quantity
of MCAM in said sample of said subject exceeds said threshold.
[0101] As demonstrated in the experimental section, the inventors
have shown that prediction or diagnosis of systolic dysfunction or
a poor prognosis of systolic dysfunction can in particular be
associated with an elevated level of MCAM. Hence, in an embodiment
of the prediction, diagnosis and/or prognosis methods as taught
herein, an elevated quantity of MCAM in the sample from the subject
compared to a reference value representing the prediction or
diagnosis of no systolic dysfunction or representing a good
prognosis for systolic dysfunction respectively indicates that the
subject has or is at risk of having systolic dysfunction or
indicates a poor prognosis for systolic dysfunction in the subject.
Elevated MCAM levels may also be indicative of prediction or
diagnosis or poor prognosis of dyspnea associated with or caused by
volume overload; or of HF or AHF associated with or caused by
volume overload; or of HF or AHF associated with or caused by
systolic dysfunction; or of reduced ventricular ejection fraction;
or of increased cardiac filling pressure.
[0102] In an embodiment, the method for monitoring systolic
dysfunction comprises the steps of: [0103] (i) measuring the
quantity of MCAM in samples from the subject from two or more
successive time points; [0104] (ii) comparing the quantity of MCAM
between the samples as measured in (i); [0105] (iii) finding a
deviation or no deviation of the quantity of MCAM between the
samples as compared in (ii); [0106] (iv) attributing said finding
of deviation or no deviation to a change in systolic dysfunction in
the subject between the two or more successive time points.
[0107] The above steps can be applied mutatis mutandis to dyspnea
associated with or caused by volume overload; to HF or AHF
associated with or caused by volume overload; to HF or AHF
associated with or caused by systolic dysfunction; to ventricular
ejection fraction; or to cardiac filling status.
[0108] The monitoring may be applied in the course of a medical
treatment of the subject.
[0109] In an embodiment of the prediction, diagnosis, prognosis
and/or monitoring methods as taught herein, the sensitivity and/or
specificity (and preferably, the sensitivity and specificity) of
the methods is at least 50%, at least 60%, at least 70% or at least
80%, e.g., .gtoreq.81%, .gtoreq.82%, .gtoreq.83%, .gtoreq.84%,
.gtoreq.85%, .gtoreq.86%, or .gtoreq.87%, or .gtoreq.90% or
.gtoreq.95% (symbol ".gtoreq." is synonymous with expressions "at
least" or "equal to or more"), e.g., between 80% and 100%, or
between 81% and 95%, or between 83% and 90%, or between 84% and
89%, or between 85% and 88%.
[0110] In another embodiment of the prediction, diagnosis,
prognosis and/or monitoring methods as taught herein, the subject
may present itself with one or more symptoms and/or signs
potentially indicative of fluid homeostatic imbalance, acute heart
failure, chronic heart failure, systolic dysfunction or kidney
dysfunction or failure. For example, in an embodiment the subject
may present itself with dyspnea.
[0111] In a further embodiment of the prediction, diagnosis,
prognosis and/or monitoring methods as taught herein, the subject
may display one or more risk factors for the conditions, symptoms
and/or parameter values according to the invention, such as, for
example, a genetic predisposition or one or more developmental,
environmental or behavioural risk factors, such as, e.g., insulin
resistance (impaired blood glucose), truncal obesity, high serum
low density lipoprotein (LDL) cholesterol levels, low serum high
density lipoprotein (HDL) cholesterol levels, high serum
triglyceride levels, and high blood pressure (hypertension), prior
myocardial infarctus, and/or one or more co-morbidities, such as
diabetes, coronary artery disease, asthma, COPD and/or chronic
renal disease.
[0112] For example, in patients suffering from over-filling (volume
overload), a decrease in MCAM level as compared to a prior MCAM
level (e.g. at the time of the admission to the ED) indicates the
condition of the subject is improving or has improved, while an
increase of the MCAM level as compared to a prior MCAM level (e.g.
at the time of the admission to the ED) indicates the condition of
the subject has worsened or is worsening. Such worsening could
possibly result in the recurrence of the conditions, symptoms
and/or parameter values according to the invention, such as in a
new acute heart failure event.
[0113] In another example, in patients suffering from under-filling
(volume contraction), such as for example Intensive Care Unit
patients, an increase in MCAM level as compared to a prior MCAM
level (e.g. at the time of the admission to the ICU) indicates the
condition of the subject is improving or has improved, while a
decrease of the MCAM level as compared to a prior MCAM level (e.g.
at the time of the admission to the ICU) indicates the condition of
the subject has worsened or is worsening.
[0114] Accordingly, further provided are a method for monitoring a
change in the prediction, diagnosis and/or prognosis of the
conditions, symptoms and/or parameter values according to the
invention in a subject, comprising: [0115] (i) applying the
prediction, diagnosis and/or prognosis method as taught here above
to the subject at two or more successive time points, whereby the
prediction, diagnosis and/or prognosis of the conditions, symptoms
and/or parameter values according to the invention in the subject
is determined at said successive time points; [0116] (ii) comparing
the prediction, diagnosis and/or prognosis of the conditions,
symptoms and/or parameter values according to the invention in the
subject at said successive time points as determined in (i); and
[0117] (iii) finding the presence or absence of a change between
the prediction, diagnosis and/or prognosis of the conditions,
symptoms and/or parameter values according to the invention in the
subject at said successive time points as determined in (i).
[0118] This aspect allows to monitor the subject's condition over
time. This can inter alia allow to predict the occurrence the
conditions, symptoms and/or parameter values according to the
invention, or to monitor in said subject the disease progression,
disease aggravation or alleviation, disease recurrence, response to
treatment, response to other external or internal factors,
conditions, or stressors, etc. Advantageously, the change in the
prediction, diagnosis and/or prognosis in the subject may be
monitored in the course of a medical treatment of said subject.
Such monitoring may be comprised, e.g., in decision making whether
a patient may be discharged, needs a change in treatment or needs
further hospitalisation.
[0119] It shall be appreciated that in the present prediction,
diagnosis, prognosis and/or monitoring methods the measurement of
MCAM may also be combined with the assessment of one or more
further biomarkers or clinical parameters relevant for the
conditions, symptoms and/or parameters according to the
invention.
[0120] Consequently, also disclosed herein are methods, wherein the
examination phase of the methods further comprises measuring the
presence or absence and/or quantity of one or more such other
biomarkers in the sample from the subject. In this respect, any
known or yet unknown suitable marker could be used.
[0121] Dyspnea can be caused by AHF, but also is present in other
patients due to causes other than or unrelated to AHF such as, COPD
or pneumonia. The diagnostic methods according to the invention
work particularly well in a patient population showing signs of
dyspnea, enabling the specific diagnosis of AHF based on the MCAM
level. In a preferred embodiment of any one the above methods of
the present invention, the subject thus forms part of a patient
population showing signs of dyspnea.
[0122] In the methods taught herein, the quantity of MCAM and/or
the presence or absence and/or quantity of the one or more other
biomarkers may be measured by any suitable technique such as may be
known in the art.
[0123] In an embodiment, the quantity of MCAM and/or the presence
or absence and/or quantity of the one or more other biomarkers may
be measured using, respectively, a binding agent capable of
specifically binding to MCAM and/or to fragments thereof, and a
binding agent capable of specifically binding to said one or more
other biomarkers.
[0124] In an embodiment, the binding agent may be an antibody,
aptamer, photoaptamer, protein, peptide, peptidomimetic or a small
molecule.
[0125] In a further embodiment, the quantity of MCAM and/or the
presence or absence and/or quantity of the one or more other
biomarkers is measured using an immunoassay technology, such as
direct ELISA, indirect ELISA, sandwich ELISA, competitive ELISA,
multiplex ELISA, radioimmunoassay (RIA) or ELISPOT technologies, or
using a mass spectrometry analysis method or using a chromatography
method, or using a combination of said methods.
[0126] Another aspect discloses a kit for predicting, diagnosing
and/or prognosticating AHF in a subject, the kit comprising means
for measuring the quantity of MCAM in a sample from the
subject.
[0127] An embodiment provides the kit for predicting, diagnosing
and/or prognosticating AHF in the subject, the kit comprising:
[0128] (i) means for measuring the quantity of MCAM in the sample
from the subject; and [0129] (ii) a reference value of the quantity
of MCAM or means for establishing said reference value, wherein
said reference value represents a known prediction, diagnosis
and/or prognosis of AHF.
[0130] The kit thus allows one to: measure the quantity of MCAM in
the sample from the subject by means (i); compare the quantity of
MCAM measured by means (i) with the reference value of (ii) or
established by means (ii); find a deviation or no deviation of the
quantity of MCAM measured by means (i) from the reference value of
(ii); and consequently attribute said finding of deviation or no
deviation to a particular prediction, diagnosis and/or prognosis of
AHF in the subject.
[0131] A further embodiment provides a kit for predicting,
diagnosing and/or prognosticating AHF in a subject, the kit
comprising means for measuring the quantity of MCAM in a sample
from the subject and means for measuring the presence or absence
and/or quantity of one or more other biomarkers useful for
predicting, diagnosing and/or prognosticating AHF in the sample
from the subject.
[0132] An embodiment provides the kit for predicting, diagnosing
and/or prognosticating AHF in the subject, the kit comprising:
[0133] (i) means for measuring the quantity of MCAM in the sample
from the subject; [0134] (ii) means for measuring the presence or
absence and/or quantity of the one or more other biomarkers useful
for predicting, diagnosing and/or prognosticating AHF in the sample
from the subject; [0135] (iii) optionally, means for establishing a
subject profile of the quantity of MCAM and the presence or absence
and/or quantity of said one or more other biomarkers; and [0136]
(iv) a reference profile of the quantity of MCAM and the presence
or absence and/or quantity of said one or more other biomarkers, or
means for establishing said reference profile, said reference
profile representing a known prediction, diagnosis and/or prognosis
of AHF.
[0137] Such kit thus allows one to: measure the quantity of MCAM
and the presence or absence and/or quantity of said one or more
other biomarkers in the sample from the subject by respectively
means (i) and (ii); establish (e.g., using means included in the
kit or using suitable external means) a subject profile of the
quantity of MCAM and the presence or absence and/or quantity of
said one or more other biomarkers based on said measurements;
compare the subject profile with the reference profile of (iv) or
established by means (iv); find a deviation or no deviation of said
subject profile from said reference profile; and consequently
attribute said finding of deviation or no deviation to a particular
prediction, diagnosis and/or prognosis of AHF in the subject.
[0138] In an embodiment of the above kits, said other biomarker
useful for predicting, diagnosing and/or prognosticating AHF may be
chosen from the group consisting of B-type natriuretic peptide
(BNP), pro-B-type natriuretic peptide (proBNP), amino terminal
pro-B-type natriuretic peptide (NTproBNP), and fragments of any one
thereof.
[0139] In a further embodiment of the above kits, the means for
measuring the quantity of MCAM and/or the presence or absence
and/or quantity of the one or more other biomarkers may comprise,
respectively, one or more binding agents capable of specifically
binding to MCAM and/or to fragments thereof, and one or more
binding agents capable of specifically binding to said one or more
other biomarkers.
[0140] In an embodiment, any one of said one or more binding agents
may be an antibody, aptamer, photoaptamer, protein, peptide,
peptidomimetic or a small molecule.
[0141] In an embodiment, any one of said one or more binding agents
may be advantageously immobilised on a solid phase or support.
[0142] In a further embodiment of the above kits, the means for
measuring the quantity of MCAM and/or the presence or absence
and/or quantity of the one or more other biomarkers may employ an
immunoassay technology, such as direct ELISA, indirect ELISA,
sandwich ELISA, competitive ELISA, multiplex ELISA,
radioimmunoassay (RIA) or ELISPOT technologies, or may employ a
mass spectrometry analysis technology or may employ a
chromatography technology, or may employ a combination of said
technologies.
[0143] An embodiment thus discloses a kit for predicting,
diagnosing and/or prognosticating AHF comprising: [0144] (a) one or
more binding agents capable of specifically binding to MCAM and/or
to fragments thereof; [0145] (b) preferably, a known quantity or
concentration of MCAM and/or a fragment thereof (e.g., for use as
controls, standards and/or calibrators); [0146] (c) preferably, a
reference value of the quantity of MCAM, or means for establishing
said reference value.
[0147] Said components under (a) and/or (c) may be suitably
labelled as taught elsewhere in this specification.
[0148] Another embodiment discloses a kit for predicting,
diagnosing and/or prognosticating AHF comprising: [0149] (a) one or
more binding agents capable of specifically binding to MCAM and/or
to fragments thereof; [0150] (b) one or more binding agents capable
of specifically binding to one or more other biomarkers useful for
predicting, diagnosing and/or prognosticating AHF, preferably
wherein said other biomarkers are chosen from the group consisting
of BNP, proBNP, NTproBNP and fragments of any one thereof; [0151]
(c) preferably, a known quantity or concentration of MCAM and/or a
fragment thereof and a known quantity or concentration of said one
or more other biomarkers (e.g., for use as controls, standards
and/or calibrators); [0152] (d) preferably, a reference profile of
the quantity of MCAM and the presence or absence and/or quantity of
said one or more other biomarkers, or means for establishing said
reference profiles.
[0153] Said components under (a), (b) and/or (c) may be suitably
labelled as taught elsewhere in this specification.
[0154] Also disclosed are reagents and tools useful for measuring
MCAM and optionally the one or more other AHF-related biomarkers
concerned herein.
[0155] For example, a further aspect relates to a protein,
polypeptide or peptide array or microarray comprising [0156] (a)
MCAM and/or a fragment thereof, preferably a known quantity or
concentration of said MCAM and/or fragment thereof; and [0157] (b)
optionally and preferably, one or more other biomarkers useful for
predicting, diagnosing and/or prognosticating AHF, preferably a
known quantity or concentration of said one or more other
biomarkers, and wherein said other biomarkers are preferably chosen
from the group consisting of: BNP, proBNP, NTproBNP and fragments
of any one thereof.
[0158] Another aspect relates to a binding agent array or
microarray comprising: [0159] (a) one or more binding agents
capable of specifically binding to MCAM and/or to fragments
thereof, preferably a known quantity or concentration of said
binding agents; and [0160] (b) optionally and preferably, one or
more binding agents capable of specifically binding to one or more
other biomarkers useful for predicting, diagnosing and/or
prognosticating AHF, preferably a known quantity or concentration
of said binding agents, and preferably wherein said other
biomarkers are chosen from the group consisting of BNP, proBNP,
NTproBNP and fragments of any one thereof.
[0161] Also disclosed are kits as taught here above configured as
portable devices, such as, for example, bed-side devices, for use
at home or in clinical settings.
[0162] A related aspect thus provides a portable testing device
capable of measuring the quantity of MCAM in a sample from a
subject comprising: [0163] (i) means for obtaining a sample from
the subject, [0164] (ii) means for measuring the quantity of MCAM
in said sample, and [0165] (iii) means for visualising the quantity
of MCAM measured in the sample.
[0166] In an embodiment, the means of parts (ii) and (iii) may be
the same, thus providing a portable testing device capable of
measuring the quantity of MCAM in a sample from a subject
comprising (i) means for obtaining a sample from the subject; and
(ii) means for measuring the quantity of MCAM in said sample and
visualising the quantity of MCAM measured in the sample.
[0167] In an embodiment, said visualising means is capable of
indicating whether the quantity of MCAM in the sample is above or
below a certain threshold level and/or whether the quantity of MCAM
in the sample deviates or not from a reference value of the
quantity of MCAM, said reference value representing a known
prediction, diagnosis and/or prognosis of AHF (as taught elsewhere
in this application). Hence, in an embodiment, the portable testing
device may suitably also comprise said reference value or means for
establishing said reference value.
[0168] In an embodiment, the threshold level is chosen such that
the quantity of MCAM in the sample above said threshold level
indicates that the subject has or is at risk of having AHF or
indicates a poor prognosis for AHF in the subject, and the quantity
of MCAM in the sample below said threshold level indicates that the
subject does not have or is not at risk of having AHF or indicates
a good prognosis for AHF in the subject.
[0169] In an embodiment, the portable testing device comprises a
reference value representing the prediction or diagnosis of no AHF
or representing a good prognosis for AHF, or comprises means for
establishing said reference value, and an elevated quantity of MCAM
in the sample from the subject compared to said reference value
indicates that the subject has or is at risk of having AHF or
indicates a poor prognosis for AHF in the subject.
[0170] In another embodiment, the portable testing device comprises
a reference value representing the prediction or diagnosis of AHF
or representing a poor prognosis for AHF, or comprises means for
establishing said reference value, and a comparable quantity of
MCAM in the sample from the subject compared to said reference
value indicates that the subject has or is at risk of having AHF or
indicates a poor prognosis for AHF in the subject.
[0171] In a further embodiment, the measuring (and optionally
visualisation) means of the portable testing device may comprise a
solid support having a proximal and distal end, comprising: [0172]
a sample application zone in the vicinity of the proximal end;
[0173] a reaction zone distal to the sample application zone; and
[0174] a detection zone distal to the reaction zone; [0175]
optionally control standards comprising MCAM protein or peptide
fragments, whereby said support has a capillary property that
directs a flow of fluid sample applied in the application zone in a
direction from the proximal end to the distal end, and [0176]
optionally comprising a fluid source improving the capillary flow
of a more viscous sample.
[0177] In an embodiment, the reaction zone may comprise one or more
bands of a MCAM-specific binding molecules conjugated to a
detection agent, which MCAM specific binding molecule conjugate is
disposed on the solid support such that it can migrate with the
capillary flow of fluid; and wherein the detection zone comprises
one or more capture bands comprising a population of MCAM specific
molecule immobilised on the solid support.
[0178] In an embodiment, the reaction zone may additionally
comprise one or more bands of capture MCAM-specific binding
molecules in an amount sufficient to prevent a threshold quantity
of MCAM specific binding molecule conjugates to migrate to the
detection zone. In an alternative embodiment, said device
additionally comprises means for comparing the amount of captured
MCAM specific binding molecule conjugate with a threshold value.
[0179] The invention also provides a testing device capable of
measuring the quantity of MCAM in a sample from a subject
comprising: [0180] (i) means for measuring the quantity of MCAM in
said sample, and [0181] (ii) means of storing the reference value
in the device, and [0182] (iii) means of comparing the obtained
quantity with the stored reference value, and [0183] (iv) means for
visualising the quantity of MCAM measured in the sample.
[0184] In preferred embodiments of the kits and devices of the
present invention, the MCAM protein detection is done in a plasma
sample, implying that the circulating MCAM protein is detected,
regardless of whether or not this circulating form corresponds to
the soluble form or to a degradation product of the full-length or
soluble form. In a preferred embodiment, the MCAM protein detected
by said kits or devices is not membrane or cell-bound. Preferably
the means for detecting said MCAM protein or fragment is capable of
detecting both the full-length protein, mature protein or processed
protein or the plasma circulating form thereof. More preferably,
said means for detecting the MCAM protein is specifically
recognising the plasma circulating from of MCAM as defined
herein.
[0185] These and further aspects and preferred embodiments are
described in the following sections and in the appended claims.
BRIEF DESCRIPTION OF FIGURES
[0186] FIG. 1 illustrates the protein sequence of the MCAM
biomarker, taken from NP.sub.--006491 (SEQ ID NO. 1). The protein
is known as melanoma cell adhesion molecule (MCAM), or as MUC18 or
CD146. The signal peptide and transmembrane and cytoplasmic domains
are indicated in small caps. Also indicated is the selected
MASSterclass quantified peptide (pept25--bold, underlined: SEQ ID
NO. 2). This MASSterclass peptide can quantify both the full length
and cleaved soluble form of MCAM.
[0187] FIG. 2 illustrates sequences of preproBNP and peptides
derived there from: preproBNP (SEQ ID NO. 3), proBNP (SEQ ID NO.
4), NT-pro-BNP (SEQ ID NO. 5) and mature BNP (SEQ ID NO. 6).
[0188] FIG. 3 illustrates that MCAM shows comparable performance to
B-type natriuretic peptides in discriminating AHF from dyspneic
non-acute heart failure patients. Receiver operating characteristic
curve of BNP compared to MCAM (A) and NT-proBNP compared to MCAM
(B) respectively for diagnosis of heart failure cause of dyspnea in
the ED. Calculated median area under the curve (AUC) and 95%
confidence intervals (CI) are given in Table 1 below.
[0189] FIG. 4: illustrates the complementary value of MCAM and BNP
and the impact of combining these two protein markers on the
diagnostic accuracy. BNP levels measured by standard ELISA are
shown in the X-axis and MCAM levels as measured by MASSterclass are
depicted in the Y-axis. The calculated best cut-off for MCAM
(horizontal line) and the routinely used cut-offs for BNP (two
vertical lines encompassing the "grey zone") are also shown.
Calculated accuracy for the independent markers and the combination
of both markers are given in Table 2 below.
[0190] FIG. 5 illustrates the levels of MCAM (A) and BNP (B)
measured in AHF patients at admission and in the same patients at
discharge from hospital. The top plot shows the raw values as
measured by MASSterclass or ELISA, while the bottom plot shows
normalized values which are fold changes between admission and
discharge.
[0191] FIG. 6: Plan (A) and side view (B) of a test strip according
to the invention.
[0192] FIG. 7: Plan view of a test cartridge according to the
invention.
[0193] FIG. 8 A-B shows a side view and a top view, respectively,
of a reagent strip according to the invention comprising several
test pads.
[0194] FIG. 9: illustrates in box and whisker plots the correlation
between weight gain and MCAM levels in AHF patients at
admission.
[0195] FIG. 10: illustrates in box and whisker plots the
correlation between LVEF and MCAM levels in AHF patients at
admission.
DETAILED DESCRIPTION OF THE INVENTION
[0196] As used herein, the singular forms "a", "an", and "the"
include both singular and plural referents unless the context
clearly dictates otherwise.
[0197] The terms "comprising", "comprises" and "comprised of" as
used herein are synonymous with "including", "includes" or
"containing", "contains", and are inclusive or open-ended and do
not exclude additional, non-recited members, elements or method
steps.
[0198] The recitation of numerical ranges by endpoints includes all
numbers and fractions subsumed within the respective ranges, as
well as the recited endpoints.
[0199] The term "about" as used herein when referring to a
measurable value such as a parameter, an amount, a temporal
duration, and the like, is meant to encompass variations of and
from the specified value, in particular variations of +/-10% or
less, preferably +/-5% or less, more preferably +/-1% or less, and
still more preferably +/-0.1% or less of and from the specified
value, insofar such variations are appropriate to perform in the
disclosed invention. It is to be understood that the value to which
the modifier "about" refers is itself also specifically, and
preferably, disclosed.
[0200] All documents cited in the present specification are hereby
incorporated by reference in their entirety.
[0201] Unless otherwise specified, all terms used in disclosing the
invention, including technical and scientific terms, have the
meaning as commonly understood by one of ordinary skill in the art
to which this invention belongs. By means of further guidance, term
definitions may be included to better appreciate the teaching of
the present invention.
[0202] The present invention derives from the highly innovative
realisation of the inventors that MCAM is a valuable biomarker for
(acute) heart failure, in particular as a biomarker for
specifically systolic dysfunction as an underlying cause of (acute)
heart failure, including systolic dysfunction associated parameters
such as ejection fraction (EF) and cardiac filling volume and
pressure.
[0203] The term "biomarker" is widespread in the art and may
broadly denote a biological molecule and/or a detectable portion
thereof whose qualitative and/or quantitative evaluation in a
subject is predictive or informative (e.g., predictive, diagnostic
and/or prognostic) with respect to one or more aspects of the
subject's phenotype and/or genotype, such as, for example, with
respect to the status of the subject as to a given disease or
condition.
[0204] The terms "heart failure", "acute heart failure" and
"chronic heart failure" as used herein carry their respective
art-established meanings. By means of further guidance, the term
"heart failure" as used herein broadly refers to pathological
conditions characterised by an impaired diastolic or systolic blood
flow rate and thus insufficient blood flow from the ventricle to
peripheral organs. In preferred embodiments of the invention, the
AHF is linked to systolic dysfunction, preferably characterized by
a decreased left ventricular ejection fraction (LVEF), preferably
wherein said LVEF is less than 55% or less than 50% or less than
45%, and/or by increased cardiac filling pressure.
[0205] "Acute heart failure" or also termed "acute decompensated
heart failure" may be defined as the rapid onset of symptoms and
signs secondary to abnormal cardiac function, resulting in the need
for urgent therapy. AHF can present itself acute de novo (new onset
of acute heart failure in a patient without previously known
cardiac dysfunction) or as acute decompensation of CHF.
[0206] The cardiac dysfunction may be related to systolic or
diastolic dysfunction, to abnormalities in cardiac rhythm, or to
preload and afterload mismatch. It is often life threatening and
requires urgent treatment. According to established classification,
AHF includes several distinct clinical conditions of presenting
patients: (I) acute decompensated congestive heart failure, (II)
AHF with hypertension/hypertensive crisis, (Ill) AHF with pulmonary
oedema, (IVa) cardiogenic shock/low output syndrome, (IVb) severe
cardiogenic shock, (V) high output failure, and (VI) right-sided
acute heart failure. For detailed clinical description,
classification and diagnosis of AHF, and for summary of further AHF
classification systems including the Killip classification, the
Forrester classification and the `clinical severity`
classification, refer inter alia to Nieminen et al. 2005
("Executive summary of the guidelines on the diagnosis and
treatment of acute heart failure: the Task Force on Acute Heart
Failure of the European Society of Cardiology". Eur Heart J 26:
384-416) and references therein. Preferably, said cardiac
dysfunction is systolic dysfunction, more preferably characterized
by a decreased left ventricular ejection fraction (LVEF),
preferably wherein said LVEF is less than 55% or less than 50% or
less than 45%, and/or by increased cardiac filling pressure.
[0207] The term "systolic dysfunction" as used herein carries its
art-established meaning. By means of further guidance, the term
"systolic dysfunction" can be used interchangeably with synonymous
terms known to the skilled person, such as "systolic ventricular
dysfunction or failure" or "systolic heart dysfunction or failure".
Essentially, "systolic dysfunction" refers to a failure of the pump
function of the heart due to a decreased contractility of the
ventricle.
[0208] The term "diastolic dysfunction" as used herein carries its
art-established meaning. By means of further guidance, the term
"diastolic dysfunction" can be used interchangeably with synonymous
terms known to the skilled person, such as "diastolic ventricular
dysfunction or failure" or "diastolic heart dysfunction or
failure". Essentially, "diastolic dysfunction" refers to a failure
of the pump function of the heart due to impaired ventricular
filling.
[0209] As used herein, the term "(left) ventricular ejection
fraction" means the output of the (left) ventricle during systole,
and represents the fraction of blood pumped out of a (left)
ventricle with each heart beat. By definition, the volume of blood
within a ventricle immediately before a contraction is known as the
end-diastolic volume. Similarly, the volume of blood left in a
ventricle at the end of contraction is end-systolic volume. The
difference between end-diastolic and end-systolic volumes is the
stroke volume, the volume of blood ejected with each beat. Ejection
fraction (EF) is the fraction of the end-diastolic volume that is
ejected with each beat; that is, it is stroke volume (SV) divided
by end-diastolic volume (EDV):EF=SV/EDV=(EDV-ESV)/EDV.
[0210] As used herein, the term "cardiac filling pressure" relates
to the pressure with which the ventricle is filled with blood.
Cardiac filling pressures are monitored to estimate cardiac filling
volumes, which, in turn, determine the stroke outputs of the left
and right ventricles. As used herein, cardiac filling pressure is a
representation of left ventricular end-diastolic pressure. Methods
for determining or estimating cardiac filling pressure are known in
the art and include ultrasound (echocardiography) and Doppler
measurements as well as direct measurement through catherization of
the ventricle. Cardiac filling pressure can be indirectly estimated
through measurement of left atrial pressure, central venous
pressure or pulmonary artery or capillary wedge pressure.
[0211] The term "fluid build-up" as used herein means an increase
in body fluid in a subject. As such, fluid build-up is associated
with fluid retention. Fluid build-up can amongst others be caused
for instance by (acute) heart failure, in particular due to
systolic dysfunction, or kidney dysfunction or failure, in
particular a dysfunction that prevents or otherwise interferes with
normal secretion of fluids in a subject, such as nephrotic
syndrome. Characteristics of fluid build-up include an increased
vascular filling volume (or vascular volume expansion) and an
increased vascular filling pressure. As used herein "filling
status" or "fluid load" refers to the fluid content in a subject,
in particular vascular, tissue and interstitial fluid content. As
used herein "vascular filling volume" refers to the amount or
volume of fluids in the vasculature. As used herein "vascular
filling pressure" refers to the pressure which is generated by the
amount or volume of fluids in the vasculature. As used herein the
terms "vascular filling volume" and "vascular filling pressure" may
be used interchangeably. Symptoms of fluid build-up in general and
an increased vascular filling volume and/or pressure include edema.
As used herein, "edema" refers to extravascular fluid build-up or
retention, as caused by an increased vascular filling volume or
pressure. According to the invention, fluid build-up, an increased
vascular filling volume and/or pressure and edema may be caused by
(acute) heart failure, systolic dysfunction, kidney dysfunction or
any pathophysiological mechanism known in the art to cause such
fluid imbalance or abnormal fluid homeostasis.
[0212] The term "chronic heart failure" (CHF) generally refers to a
case of heart failure that progresses so slowly that various
compensatory mechanisms work to bring the disease into equilibrium.
Common clinical symptoms of CHF include inter alia any one or more
of breathlessness, diminishing exercise capacity, fatigue, lethargy
and peripheral oedema. Other less common symptoms include any one
or more of palpitations, memory or sleep disturbance and confusion,
and usually co-occur with one or more of the above recited common
symptoms.
[0213] In studies such as the present one, CHF population may
differ from the AHF population in that CHF patients do not have an
acute decompensation and hence do not represent themselves to the
ED at the time the clinical sample used in such a study or research
is taken. Chronic heart failure patients may, however, easily
decompensate leading to "acute heart failure".
[0214] In studies such as the present one, a population of dyspneic
patients without heart failure may comprise for example patients
who present themselves to the ED with similar symptoms as AHF
population but where the cause of dyspnea is unrelated to acute
decompensated heart failure. Typical examples are COPD or pneumonia
patients. Such patients may or may not have underlying heart
failure history, which may particularly complicate the final
diagnosis using conventional diagnostic means such as BNP or
NT-pro-BNP measurements.
[0215] The terms "predicting" or "prediction", "diagnosing" or
"diagnosis" and "prognosticating" or "prognosis" are commonplace
and well-understood in medical and clinical practice. By means of
further explanation and without limitation, "predicting" or
"prediction" generally refer to an advance declaration, indication
or foretelling of a disease or condition in a subject not (yet)
having said disease or condition. For example, a prediction of a
disease or condition in a subject may indicate a probability,
chance or risk that the subject will develop said disease or
condition, for example within a certain time period or by a certain
age. Said probability, chance or risk may be indicated inter alia
as an absolute value, range or statistics, or may be indicated
relative to a suitable control subject or subject population (such
as, e.g., relative to a general, normal or healthy subject or
subject population). Hence, the probability, chance or risk that a
subject will develop a disease or condition may be advantageously
indicated as increased or decreased, or as fold-increased or
fold-decreased relative to a suitable control subject or subject
population.
[0216] As used herein, the term "prediction of AHF" in a subject
may also particularly mean that the subject has a `positive`
prediction of AHF, i.e., that the subject is at risk of having AHF
(e.g., the risk is significantly increased vis-a-vis a control
subject or subject population). The term "prediction of no AHF" in
a subject may particularly mean that the subject has a `negative`
prediction of AHF, i.e., that the subject's risk of having AHF is
not significantly increased vis-a-vis a control subject or subject
population.
[0217] The terms "diagnosing" or "diagnosis" generally refer to the
process or act of recognising, deciding on or concluding on a
disease or condition in a subject on the basis of symptoms and
signs and/or from results of various diagnostic procedures (such
as, for example, from knowing the presence, absence and/or quantity
of one or more biomarkers characteristic of the diagnosed disease
or condition).
[0218] As used herein, "diagnosis of AHF" in a subject may
particularly mean that the subject has AHF, hence, is diagnosed as
having AHF. "Diagnosis of no AHF" in a subject may particularly
mean that the subject does not have AHF, hence, is diagnosed as not
having AHF. A subject may be diagnosed as taught herein as not
having AHF despite displaying one or more conventional symptoms or
signs reminiscent of AHF.
[0219] The terms "prognosticating" or "prognosis" generally refer
to an anticipation on the progression of a disease or condition and
the prospect (e.g., the probability, duration, and/or extent) of
recovery.
[0220] A good prognosis of AHF may generally encompass anticipation
of a satisfactory partial or complete recovery from AHF, preferably
within an acceptable time period. A good prognosis of AHF may more
commonly encompass anticipation of not further worsening or
aggravating of the heart failure condition, preferably within a
given time period.
[0221] A poor prognosis of AHF may generally encompass anticipation
of a substandard recovery and/or unsatisfactorily slow recovery, or
to substantially no recovery or even further worsening of AHF.
[0222] The various aspects and embodiments taught herein may rely
on measuring the quantity of MCAM, and optionally measuring the
presence or absence and/or quantity of one or more other relevant
biomarkers, such as preferably BNP, proBNP, NTproBNP and/or
fragments of any one thereof, in a sample from a subject.
[0223] The term "subject" or "patient" as used herein typically
denotes humans, but may also encompass reference to non-human
animals, preferably warm-blooded animals, more preferably mammals,
such as, e.g., non-human primates, rodents, canines, felines,
equines, ovines, porcines, and the like.
[0224] The terms "sample" or "biological sample" as used herein
include any biological specimen obtained from a subject. Samples
may include, without limitation, whole blood, plasma, serum, red
blood cells, white blood cells (e.g., peripheral blood mononuclear
cells), saliva, urine, stool (i.e., faeces), tears, sweat, sebum,
nipple aspirate, ductal lavage, tumour exudates, synovial fluid,
cerebrospinal fluid, lymph, fine needle aspirate, amniotic fluid,
any other bodily fluid, cell lysates, cellular secretion products,
inflammation fluid, semen and vaginal secretions. Preferred samples
may include ones comprising MCAM in detectable quantities. In
preferred embodiments, the sample may be whole blood or a
fractional component thereof such as, e.g., plasma, serum, or a
cell pellet. Preferably the sample is readily obtainable by
minimally invasive methods. Samples may also include tissue samples
and biopsies, tissue homogenates and the like. Preferably, the
sample used to detect MCAM levels is blood plasma. The term
"plasma" defines the colorless watery fluid of the blood that
contains no cells, but in which the blood cells (erythrocytes,
leukocytes, thrombocytes, etc.) are suspended, containing
nutrients, sugars, proteins, minerals, enzymes, etc.
[0225] A molecule or analyte such as a protein, polypeptide or
peptide, or a group of two or more molecules or analytes such as
two or more proteins, polypeptides or peptides, is "measured" in a
sample when the presence or absence and/or quantity of said
molecule or analyte or of said group of molecules or analytes is
detected or determined in the sample, preferably substantially to
the exclusion of other molecules and analytes.
[0226] The terms "quantity", "amount" and "level" are synonymous
and generally well-understood in the art. The terms as used herein
may particularly refer to an absolute quantification of a molecule
or an analyte in a sample, or to a relative quantification of a
molecule or analyte in a sample, i.e., relative to another value
such as relative to a reference value as taught herein, or to a
range of values indicating a base-line expression of the biomarker.
These values or ranges can be obtained from a single patient or
from a group of patients.
[0227] An absolute quantity of a molecule or analyte in a sample
may be advantageously expressed as weight or as molar amount, or
more commonly as a concentration, e.g., weight per volume or mol
per volume.
[0228] A relative quantity of a molecule or analyte in a sample may
be advantageously expressed as an increase or decrease or as a
fold-increase or fold-decrease relative to said another value, such
as relative to a reference value as taught herein. Performing a
relative comparison between first and second parameters (e.g.,
first and second quantities) may but need not require to first
determine the absolute values of said first and second parameters.
For example, a measurement method can produce quantifiable readouts
(such as, e.g., signal intensities) for said first and second
parameters, wherein said readouts are a function of the value of
said parameters, and wherein said readouts can be directly compared
to produce a relative value for the first parameter vs. the second
parameter, without the actual need to first convert the readouts to
absolute values of the respective parameters.
[0229] As used herein, the term "MCAM" corresponds to the protein
commonly known as Melanoma Cell Adhesion Molecule (MCAM), MUC18 or
CD146, i.e. the proteins and polypeptides commonly known under
these designations in the art. The terms encompass such proteins
and polypeptides of any organism where found, and particularly of
animals, preferably vertebrates, more preferably mammals, including
humans and non-human mammals, even more preferably of humans. The
terms particularly encompass such proteins and polypeptides with a
native sequence, i.e., ones of which the primary sequence is the
same as that of MCAM found in or derived from nature. A skilled
person understands that native sequences of MCAM may differ between
different species due to genetic divergence between such species.
Moreover, the native sequences of MCAM may differ between or within
different individuals of the same species due to normal genetic
diversity (variation) within a given species. Also, the native
sequences of MCAM may differ between or even within different
individuals of the same species due to post-transcriptional or
post-translational modifications. Accordingly, all MCAM sequences
found in or derived from nature are considered "native". The terms
encompass MCAM proteins and polypeptides when forming a part of a
living organism, organ, tissue or cell, when forming a part of a
biological sample, as well as when at least partly isolated from
such sources. The terms also encompass proteins and polypeptides
when produced by recombinant or synthetic means.
[0230] Exemplary MCAM includes, without limitation, human MCAM
having primary amino acid sequence as annotated under
Uniprot/Swissprot (http://www.expasy.org/) accession number
NP.sub.--006491 as shown in FIG. 1 (SEQ ID NO: 1). A skilled person
can also appreciate that said sequences are of precursor of MCAM
and may include parts which are processed away from mature MCAM.
For example, the MCAM protein can be in a soluble form or can be
attached to the cell membrane. In FIG. 1, the signal peptide and
transmembrane and cytoplasmic domains are indicated in small caps
in the amino acid sequence. Also indicated is the selected
MASSterclass quantified peptide (pept25--bold, underlined: SEQ ID
NO. 2). This MASSterclass peptide can quantify both the full length
and cleaved soluble form of MCAM, although due to the experimental
set-up only the plasma circulating fraction (i.e. the non-cell
bound fraction) is measured.
[0231] The MCAM protein is specific for endothelial cells and
vascular smooth muscle cells and has been used as a tool for
sorting endothelial cells out of a population of blood cells, based
on the membrane bound form of CD146. MCAM belongs to the
immunoglobulin supergene family with five immunoglobulin like
domains (V-V-C2-C2-C2), a transmembrane region and a 63 residue
cytoplasmic tail. It is a membrane glycoprotein that functions as a
Ca2+ independent cell adhesion molecule involved in heterophilic
cell to cell interactions. The protein has a molecular size of 130
kDa in its reduced form (118 kDa unreduced), and N linked
glycosylation accounts for fifty percent of the apparent molecular
weight. Soluble CD146 is released by ectodomain shedding (through
the action of MMPs). Increased plasma levels of soluble CD146 was
observed in patients with chronic renal failure (Healthy serum
levels: .about.270 ng/ml; renal failure patients: .about.500 ng/ml)
as discussed in Saito et al., 2008 (Clin Exp Nephrol. 2008
February; 12(1):58-64. Epub 2008 Jan. 5). On the other hand,
decreased serum levels of sCD146 (soluble CD146) were observed in
patients with Inflammatory Bowel Disease (IBD) such as Crohn's
disease, while the membrane bound CD146 expression is increased in
active IBD (Bardin et al., Inflamm. Bowel Dis. 2006 January;
12(1):16-21 and Reumaux et al., Inflamm. Bowel Dis. 2007 October;
13(10):1315-7). The latter two publications indicate that there is
a clear difference in correlation between the condition of the
patient and the levels of 1) the soluble MCAM and 2) the cell- or
membrane-bound form(s) of MCAM. In a preferred embodiment of the
methods, kits and devices of present invention as defined herein,
the circulating MCAM protein, e.g. the form circulating in the
blood plasma, is detected, as opposed to the membrane- or
cell-bound MCAM protein (e.g. MCAM present on the endothelial cell
surface).
[0232] MCAM has been known as an endothelial cell injury marker,
but has not been shown to be useful to distinguish between AHF and
dyspnea in non-AHF patients. Furthermore, the MCAM marker is often
used as a tool for sorting endothelial cells, implying the membrane
bound (full-length) protein is used (cf. e.g. WO2006/020936).
[0233] The reference herein to MCAM may also encompass fragments of
MCAM. Hence, the reference herein to measuring MCAM, or to
measuring the quantity of MCAM, may encompass measuring the MCAM
protein or polypeptide, such as, e.g., measuring the mature and/or
the MMP-processed soluble form (shortly called "soluble form"
hereinafter) of MCAM and/or measuring one or more fragments
thereof. For example, MCAM and/or one or more fragments thereof may
be measured collectively, such that the measured quantity
corresponds to the sum amounts of the collectively measured
species. In another example, MCAM and/or one or more fragments
thereof may be measured each individually. Preferably, said
fragment of MCAM is a plasma circulating form of MCAM.
[0234] The expression "plasma circulating form of MCAM" or shortly
"circulating form" encompasses all MCAM proteins or fragments
thereof that circulate in the plasma, i.e. are not cell- or
membrane bound. Without wanting to be bound by any theory, such
circulating forms can be derived from the full-length MCAM protein
through natural processing (e.g. MMP-cleavage into its "soluble
form" as indicated above), or can be resulting from known
degradation processes occurring in said sample. In certain
situations, the circulating form can also be the full-length MCAM
protein, which is found to be circulating in the plasma. Said
"circulating form" can thus be any MCAM protein or any processed
soluble form of MCAM or fragments of either one, that is
circulating in the sample, i.e. which is not bound to a cell- or
membrane fraction of said sample.
[0235] As used herein, the terms "pro-B-type natriuretic peptide"
(also abbreviated as "proBNP") and "amino terminal pro-B-type
natriuretic peptide" (also abbreviated as "NTproBNP") and "B-type
natriuretic peptide" (also abbreviated as "BNP") refer to peptides
commonly known under these designations in the art. As further
explanation and without limitation, in vivo proBNP, NTproBNP and
BNP derive from natriuretic peptide precursor B preproprotein
(preproBNP). In particular, proBNP peptide corresponds to the
portion of preproBNP after removal of the N-terminal secretion
signal (leader) sequence from preproBNP. NTproBNP corresponds to
the N-terminal portion and BNP corresponds to the C-terminal
portion of the proBNP peptide subsequent to cleavage of the latter
C-terminally adjacent to amino acid 76 of proBNP. The terms
encompass such peptides from any organism where found, and
particularly from animals, preferably vertebrates, more preferably
mammals, including humans and non-human mammals, even more
preferably from humans.
[0236] The designations proBNP, NTproBNP and BNP as used herein
particularly refer to such peptides with a native sequence, i.e.,
peptides of which the primary sequence is the same as that of
respectively proBNP, NTproBNP or BNP found in or derived from
nature. A skilled person understands that native sequences of
proBNP, NTproBNP or BNP may differ between different species due to
genetic divergence between such species. Moreover, the native
sequences of proBNP, NTproBNP or BNP may differ between or even
within different individuals of the same species due to normal
genetic diversity (variation) within a given species. Also, the
native sequences of proBNP, NTproBNP or BNP may differ between or
even within different individuals of the same species due to
post-transcriptional or post-translational modifications.
Accordingly, all proBNP, NTproBNP or BNP sequences found in or
derived from nature are considered "native".
[0237] The designations proBNP, NTproBNP or BNP as used herein
encompass the respective peptides when forming a part of a living
organism, organ, tissue or cell, when forming a part of a
biological sample, as well as when at least partly isolated from
such sources. The terms also encompass the respective peptides when
produced by recombinant or synthetic means.
[0238] Exemplary human proBNP peptide includes without limitation
the peptide from amino acid position 27 to position 134 of the
natriuretic peptide precursor B preproprotein sequence as annotated
under the NIH Entrez Protein
(http://www.ncbi.nlm.nih.gov/sites/entrez?db=protein) accession
number NP.sub.--002512 (version NP.sub.--002512.1 revised Jan. 25,
2009).
[0239] The sequence of NP.sub.--002512 is shown in FIG. 3A (SEQ ID
NO: 3) and the exemplary sequence of proBNP from NP.sub.--002512 is
shown in FIG. 3B (SEQ ID NO: 4). Exemplary human NTproBNP peptide
includes without limitation the peptide from amino acid position 27
to position 102 of the natriuretic peptide precursor B
preproprotein sequence as annotated under said NIH Entrez Protein
accession number NP.sub.--002512. The exemplary sequence of
NTproBNP from NP.sub.--002512 is shown in FIG. 3C (SEQ ID NO: 5).
Exemplary human BNP peptide includes without limitation the peptide
from amino acid position 103 to position 134 of the natriuretic
peptide precursor B preproprotein sequence as annotated under said
NIH Entrez Protein accession number NP.sub.--002512. The exemplary
sequence of BNP from NP.sub.--002512 is shown in FIG. 3D (SEQ ID
NO: 6). See also Sudoh et al. 1989 (Biochem Biophys Res Commun 159:
1427-1434) for further exemplification of human preproBNP-derived
peptides, including proBNP, NTproBNP and BNP. See also Maisel et
al. 2008 (Eur J Heart Fail 10(9): 824-39) and Miller et al. 2007
(Biomarkers Med 1(4): 503-512) on using natriuretic peptide levels
in clinical practice.
[0240] The reference herein to proBNP, NTproBNP and/or BNP may also
encompass fragments of any one of proBNP, NTproBNP and/or BNP.
Hence, the reference herein to measuring the presence or absence
and/or quantity of proBNP, NTproBNP and/or BNP, may encompass
measuring the proBNP, NTproBNP and/or BNP peptides and/or measuring
one or more fragments of any one of the proBNP, NTproBNP and/or BNP
peptides. For example, the proBNP, NTproBNP and/or BNP peptides
and/or one or more fragments of any one thereof may be measured
collectively, such that the measured quantity corresponds to the
sum amount of the collectively measured species. In another
example, the proBNP, NTproBNP and/or BNP peptides and/or one or
more fragments of any one thereof may be measured each
individually.
[0241] Further, unless otherwise apparent from the context,
reference herein to any protein, polypeptide or peptide (such as,
e.g., MCAM, proBNP, NTproBNP or BNP) and fragments thereof may
generally also encompass modified forms of said protein,
polypeptide or peptide and fragments such as bearing
post-expression modifications including, for example,
phosphorylation, glycosylation, lipidation, methylation,
cysteinylation, sulphonation, glutathionylation, acetylation,
oxidation of methionine to methionine sulphoxide or methionine
sulphone, and the like.
[0242] In an embodiment, MCAM and fragments thereof, or proBNP,
NTproBNP, BNP and fragments thereof may be human, i.e., their
primary sequence may be the same as a corresponding primary
sequence of or present in a naturally occurring human MCAM and
fragments thereof, or proBNP, NTproBNP, BNP and fragments thereof.
Hence, the qualifier "human" in this connection relates to the
primary sequence of the respective proteins, polypeptides, peptides
or fragments, rather than to their origin or source. For example,
such proteins, polypeptides, peptides or fragments may be present
in or isolated from samples of human subjects or may be obtained by
other means (e.g., by recombinant expression, cell-free translation
or non-biological peptide synthesis).
[0243] The term "fragment" of a protein, polypeptide or peptide
generally refers to N-terminally and/or C-terminally deleted or
truncated forms of said protein, polypeptide or peptide. The term
encompasses fragments arising by any mechanism, such as, without
limitation, by alternative translation, exo- and/or
endo-proteolysis and/or degradation of said protein or polypeptide,
such as, for example, in vivo or in vitro, such as, for example, by
physical, chemical and/or enzymatic proteolysis. Without
limitation, a fragment of a protein, polypeptide or peptide may
represent at least about 5%, or at least about 10%, e.g.,
.gtoreq.20%, .gtoreq.30% or .gtoreq.40%, such as .gtoreq.50%, e.g.,
.gtoreq.60%, .gtoreq.70% or .gtoreq.80%, or even .gtoreq.90% or
.gtoreq.95% of the amino acid sequence of said protein, polypeptide
or peptide.
[0244] For example, a fragment of MCAM may include a sequence of
.gtoreq.5 consecutive amino acids, or .gtoreq.10 consecutive amino
acids, or .gtoreq.20 consecutive amino acids, or .gtoreq.30
consecutive amino acids, e.g., .gtoreq.40 consecutive amino acids,
such as for example 50 consecutive amino acids, e.g., .gtoreq.60,
.gtoreq.70, .gtoreq.80, .gtoreq.90, .gtoreq.100, .gtoreq.200,
.gtoreq.300, .gtoreq.400, .gtoreq.500 or .gtoreq.600 consecutive
amino acids of MCAM.
[0245] In an embodiment, a fragment of MCAM may be N-terminally
and/or C-terminally truncated by between 1 and about 20 amino
acids, such as, e.g., by between 1 and about 15 amino acids, or by
between 1 and about 10 amino acids, or by between 1 and about 5
amino acids, compared to mature, full-length MCAM (SEQ ID NO. 1) or
its soluble form (cf. FIG. 1).
[0246] In an embodiment, a fragment of proBNP, NTproBNP or BNP may
be N-terminally and/or C-terminally truncated by between 1 and
about 20 amino acids, such as, e.g., by between 1 and about 15
amino acids, or by between 1 and about 10 amino acids, or by
between 1 and about 5 amino acids, compared to proBNP, NTproBNP or
BNP. By means of example, proBNP, NTproBNP and BNP fragments useful
as biomarkers are disclosed in WO 2004/094460.
[0247] In an embodiment, fragments of a given protein, polypeptide
or peptide may be achieved by in vitro proteolysis of said protein,
polypeptide or peptide to obtain advantageously detectable
peptide(s) from a sample.
[0248] For example, such proteolysis may be effected by suitable
physical, chemical and/or enzymatic agents, e.g., proteinases,
preferably endoproteinases, i.e., protease cleaving internally
within a protein, polypeptide or peptide chain. A non-limiting list
of suitable endoproteinases includes serine proteinases (EC
3.4.21), threonine proteinases (EC 3.4.25), cysteine proteinases
(EC 3.4.22), aspartic acid proteinases (EC 3.4.23),
metalloproteinases (EC 3.4.24) and glutamic acid proteinases.
[0249] Exemplary non-limiting endoproteinases include trypsin,
chymotrypsin, elastase, Lysobacter enzymogenes endoproteinase
Lys-C, Staphylococcus aureus endoproteinase Glu-C (endopeptidase
V8) or Clostridium histolyticum endoproteinase Arg-C(clostripain).
Further known or yet to be identified enzymes may be used; a
skilled person can choose suitable protease(s) on the basis of
their cleavage specificity and frequency to achieve desired peptide
forms.
[0250] Preferably, the proteolysis may be effected by
endopeptidases of the trypsin type (EC 3.4.21.4), preferably
trypsin, such as, without limitation, preparations of trypsin from
bovine pancreas, human pancreas, porcine pancreas, recombinant
trypsin, Lys-acetylated trypsin, trypsin in solution, trypsin
immobilised to a solid support, etc. Trypsin is particularly
useful, inter alia due to high specificity and efficiency of
cleavage. The invention also contemplates the use of any
trypsin-like protease, i.e., with a similar specificity to that of
trypsin.
[0251] Otherwise, chemical reagents may be used for proteolysis.
For example, CNBr can cleave at Met; BNPS-skatole can cleave at
Trp.
[0252] The conditions for treatment, e.g., protein concentration,
enzyme or chemical reagent concentration, pH, buffer, temperature,
time, can be determined by the skilled person depending on the
enzyme or chemical reagent employed.
[0253] Hence, in an aspect the invention also provides an isolated
fragment of MCAM as defined here above. Such fragments may give
useful information about the presence and quantity of MCAM in
biological samples, whereby the detection of said fragments is of
interest. Hence, the herein disclosed fragments of MCAM are useful
biomarkers.
[0254] The term "isolated" with reference to a particular component
(such as for instance, a protein, polypeptide, peptide or fragment
thereof) generally denotes that such component exists in separation
from--for example, has been separated from or prepared in
separation from--one or more other components of its natural
environment. For instance, an isolated human or animal protein,
polypeptide, peptide or fragment exists in separation from a human
or animal body where it occurs naturally.
[0255] The term "isolated" as used herein may preferably also
encompass the qualifier "purified". As used herein, the term
"purified" with reference to protein(s), polypeptide(s), peptide(s)
and/or fragment(s) thereof does not require absolute purity.
Instead, it denotes that such protein(s), polypeptide(s),
peptide(s) and/or fragment(s) is (are) in a discrete environment in
which their abundance (conveniently expressed in terms of mass or
weight or concentration) relative to other proteins is greater than
in a biological sample. A discrete environment denotes a single
medium, such as for example a single solution, gel, precipitate,
lyophilisate, etc. Purified peptides, polypeptides or fragments may
be obtained by known methods including, for example, laboratory or
recombinant synthesis, chromatography, preparative electrophoresis,
centrifugation, precipitation, affinity purification, etc.
[0256] Purified protein(s), polypeptide(s), peptide(s) and/or
fragment(s) may preferably constitute by weight 10%, more
preferably 50%, such as 60%, yet more preferably 70%, such as 80%,
and still more preferably 90%, such as 95%, 96%, 97%, 98%, 99% or
even 100%, of the protein content of the discrete environment.
Protein content may be determined, e.g., by the Lowry method (Lowry
et al. 1951. J Biol Chem 193: 265), optionally as described by
Hartree 1972 (Anal Biochem 48: 422-427). Also, purity of peptides
or polypeptides may be determined by SDS-PAGE under reducing or
non-reducing conditions using Coomassie blue or, preferably, silver
stain.
[0257] A further embodiment provides isolated MCAM or fragments of
MCAM as taught herein comprising a detectable label. This
facilitates ready detection of such fragments. The term "label" as
used throughout this specification refers to any atom, molecule,
moiety or biomolecule that can be used to provide a detectable and
preferably quantifiable read-out or property, and that can be
attached to or made part of an entity of interest, such as a
peptide or polypeptide or a specific-binding agent. Labels may be
suitably detectable by mass spectrometric, spectroscopic, optical,
colorimetric, magnetic, photochemical, biochemical, immunochemical
or chemical means. Labels include without limitation dyes;
radiolabels such as .sup.32P, .sup.33P, .sup.35S, .sup.125I,
.sup.131I; electron-dense reagents; enzymes (e.g., horse-radish
peroxidase or alkaline phosphatase as commonly used in
immunoassays); binding moieties such as biotin-streptavidin;
haptens such as digoxigenin; luminogenic, phosphorescent or
fluorogenic moieties; mass tags; and fluorescent dyes alone or in
combination with moieties that can suppress or shift emission
spectra by fluorescence resonance energy transfer (FRET).
[0258] In an embodiment, the isolated MCAM or fragments of MCAM as
taught herein may be labelled by a mass-altering label. Preferably,
a mass-altering label may involve the presence of a distinct stable
isotope in one or more amino acids of the peptide vis-a-vis its
corresponding non-labelled peptide. Mass-labelled peptides are
particularly useful as positive controls, standards and calibrators
in mass spectrometry applications. In particular, peptides
including one or more distinct isotopes are chemically alike,
separate chromatographically and electrophoretically in the same
manner and also ionise and fragment in the same way. However, in a
suitable mass analyser such peptides and optionally select
fragmentation ions thereof will display distinguishable m/z ratios
and can thus be discriminated. Examples of pairs of distinguishable
stable isotopes include H and D, .sup.12C and .sup.13C, .sup.14N
and .sup.15N or .sup.16O and .sup.18O. Usually, peptides and
proteins of biological samples analysed in the present invention
may substantially only contain common isotopes having high
prevalence in nature, such as for example H, .sup.12C, .sup.14N and
.sup.16O. In such case, the mass-labelled peptide may be labelled
with one or more uncommon isotopes having low prevalence in nature,
such as for instance D, .sup.13C, .sup.15N and/or .sup.18O. It is
also conceivable that in cases where the peptides or proteins of a
biological sample would include one or more uncommon isotopes, the
mass-labelled peptide may comprise the respective common
isotope(s).
[0259] Isotopically-labelled synthetic peptides may be obtained
inter alia by synthesising or recombinantly producing such peptides
using one or more isotopically-labelled amino acid substrates, or
by chemically or enzymatically modifying unlabelled peptides to
introduce thereto one or more distinct isotopes. By means of
example and not limitation, D-labelled peptides may be synthesised
or recombinantly produced in the presence of commercially available
deuterated L-methionine
CH.sub.3--S-CD.sub.2CD.sub.2-CH(NH.sub.2)--COOH or deuterated
arginine
H.sub.2NC(.dbd.NH)--NH--(CD.sub.2).sub.3-CD(NH.sub.2)--COOH. It
shall be appreciated that any amino acid of which deuterated or
.sup.15N- or .sup.13C-containing forms exist may be considered for
synthesis or recombinant production of labelled peptides. In
another non-limiting example, a peptide may be treated with trypsin
in H.sub.2.sup.16O or H.sub.2.sup.18O, leading to incorporation of
two oxygens (.sup.16O or .sup.18O, respectively) at the
COOH-termini of said peptide (e.g., US 2006/105415).
[0260] Accordingly, also contemplated is the use of MCAM and
isolated fragments of MCAM as taught herein, optionally comprising
a detectable label, as (positive) controls, standards or calibators
in qualitative or quantitative detection assays (measurement
methods) of MCAM, and particularly in such methods for predicting,
diagnosing and/or prognosticating AHF in subjects as taught herein.
The proteins, polypeptides or peptides may be supplied in any form,
inter alia as precipitate, vacuum-dried, lyophilisate, in solution
as liquid or frozen, or covalently or non-covalently immobilised on
solid phase, such as for example, on solid chromatographic matrix
or on glass or plastic or other suitable surfaces (e.g., as a part
of peptide arrays and microarrays). The peptides may be readily
prepared, for example, isolated from natural sources, or prepared
recombinantly or synthetically.
[0261] Also provided are binding agents capable of specifically
binding to any one or more of the isolated fragments of MCAM as
taught herein. Further provided are binding agents capable of
specifically binding to only one of the isolated fragments of MCAM
as taught herein. Such binding agents may include inter alia an
antibody, aptamer, photoaptamer, protein, peptide, peptidomimetic
or a small molecule.
[0262] In a preferred embodiment, said binding agent is capable of
binding both the membrane-bound and plasma circulating forms of
MCAM. Preferably, said binding agent is capable of specifically
binding or detecting the plasma circulating form of MCAM.
[0263] The term "specifically bind" as used throughout this
specification means that an agent (denoted herein also as
"specific-binding agent") binds to one or more desired molecules or
analytes, such as to one or more proteins, polypeptides or peptides
of interest or fragments thereof substantially to the exclusion of
other molecules which are random or unrelated, and optionally
substantially to the exclusion of other molecules that are
structurally related.
[0264] The term "specifically bind" does not necessarily require
that an agent binds exclusively to its intended target(s). For
example, an agent may be said to specifically bind to protein(s)
polypeptide(s), peptide(s) and/or fragment(s) thereof of interest
if its affinity for such intended target(s) under the conditions of
binding is at least about 2-fold greater, preferably at least about
5-fold greater, more preferably at least about 10-fold greater, yet
more preferably at least about 25-fold greater, still more
preferably at least about 50-fold greater, and even more preferably
at least about 100-fold or more greater, than its affinity for a
non-target molecule.
[0265] Preferably, the agent may bind to its intended target(s)
with affinity constant (K.sub.A) of such binding
K.sub.A.gtoreq.1.times.10.sup.6 M.sup.-1, more preferably
K.sub.A.gtoreq.1.times.10.sup.7 M.sup.-1, yet more preferably
K.sub.A.gtoreq.1.times.10.sup.8 M.sup.-1, even more preferably
K.sub.A.gtoreq.1.times.10.sup.9 M.sup.-1, and still more preferably
K.sub.A.gtoreq.1.times.10.sup.10 M.sup.-1 or
K.sub.A.gtoreq.1.times.10.sup.11 M.sup.-1, wherein
K.sub.A=[SBA_T]/[SBA][T], SBA denotes the specific-binding agent, T
denotes the intended target. Determination of K.sub.A can be
carried out by methods known in the art, such as for example, using
equilibrium dialysis and Scatchard plot analysis.
[0266] Specific binding agents as used throughout this
specification may include inter alia an antibody, aptamer,
photoaptamer, protein, peptide, peptidomimetic or a small
molecule.
[0267] As used herein, the term "antibody" is used in its broadest
sense and generally refers to any immunologic binding agent. The
term specifically encompasses intact monoclonal antibodies,
polyclonal antibodies, multivalent (e.g., 2-, 3- or more-valent)
and/or multi-specific antibodies (e.g., bi- or more-specific
antibodies) formed from at least two intact antibodies, and
antibody fragments insofar they exhibit the desired biological
activity (particularly, ability to specifically bind an antigen of
interest), as well as multivalent and/or multi-specific composites
of such fragments. The term "antibody" is not only inclusive of
antibodies generated by methods comprising immunisation, but also
includes any polypeptide, e.g., a recombinantly expressed
polypeptide, which is made to encompass at least one
complementarity-determining region (CDR) capable of specifically
binding to an epitope on an antigen of interest. Hence, the term
applies to such molecules regardless whether they are produced in
vitro or in vivo.
[0268] In an embodiment, an antibody may be any of IgA, IgD, IgE,
IgG and IgM classes, and preferably IgG class antibody.
[0269] In an embodiment, the antibody may be a polyclonal antibody,
e.g., an antiserum or immunoglobulins purified there from (e.g.,
affinity-purified).
[0270] In another preferred embodiment, the antibody may be a
monoclonal antibody or a mixture of monoclonal antibodies.
Monoclonal antibodies can target a particular antigen or a
particular epitope within an antigen with greater selectivity and
reproducibility.
[0271] By means of example and not limitation, monoclonal
antibodies may be made by the hybridoma method first described by
Kohler et al. 1975 (Nature 256: 495), or may be made by recombinant
DNA methods (e.g., as in U.S. Pat. No. 4,816,567). Monoclonal
antibodies may also be isolated from phage antibody libraries using
techniques as described by Clackson et al. 1991 (Nature 352:
624-628) and Marks et al. 1991 (J Mol Biol 222: 581-597), for
example.
[0272] In further embodiments, the antibody binding agents may be
antibody fragments. "Antibody fragments" comprise a portion of an
intact antibody, comprising the antigen-binding or variable region
thereof. Examples of antibody fragments include Fab, Fab', F(ab')2,
Fv and scFv fragments; diabodies; linear antibodies; single-chain
antibody molecules; and multivalent and/or multispecific antibodies
formed from antibody fragment(s), e.g., dibodies, tribodies, and
multibodies. The above designations Fab, Fab', F(ab')2, Fv, scFv
etc. are intended to have their art-established meaning.
[0273] The term antibody includes antibodies originating from or
comprising one or more portions derived from any animal species,
preferably vertebrate species, including, e.g., birds and
mammals.
[0274] Without limitation, the antibodies may be chicken, turkey,
goose, duck, guinea fowl, quail or pheasant. Also without
limitation, the antibodies may be human, murine (e.g., mouse, rat,
etc.), donkey, rabbit, goat, sheep, guinea pig, camel (e.g.,
Camelus bactrianus and Camelus dromaderius), llama (e.g., Lama
paccos, Lama glama or Lama vicugna) or horse.
[0275] A skilled person will understand that an antibody can
include one or more amino acid deletions, additions and/or
substitutions (e.g., conservative substitutions), insofar such
alterations preserve its binding of the respective antigen. An
antibody may also include one or more native or artificial
modifications of its constituent amino acid residues (e.g.,
glycosylation, etc.).
[0276] Methods of producing polyclonal and monoclonal antibodies as
well as fragments thereof are well known in the art, as are methods
to produce recombinant antibodies or fragments thereof (see for
example, Harlow and Lane, "Antibodies: A Laboratory Manual", Cold
Spring Harbour Laboratory, New York, 1988; Harlow and Lane, "Using
Antibodies: A Laboratory Manual", Cold Spring Harbour Laboratory,
New York, 1999, ISBN 0879695447; "Monoclonal Antibodies: A Manual
of Techniques", by Zola, ed., CRC Press 1987, ISBN 0849364760;
"Monoclonal Antibodies: A Practical Approach", by Dean &
Shepherd, eds., Oxford University Press 2000, ISBN 0199637229;
Methods in Molecular Biology, vol. 248: "Antibody Engineering:
Methods and Protocols", Lo, ed., Humana Press 2004, ISBN
1588290921).
[0277] The term "aptamer" refers to single-stranded or
double-stranded oligo-DNA, oligo-RNA or oligo-DNA/RNA or any
analogue thereof, that can specifically bind to a target molecule
such as a peptide. Advantageously, aptamers can display fairly high
specificity and affinity (e.g., K.sub.A in the order
1.times.10.sup.9 M.sup.-1) for their targets. Aptamer production is
described inter alia in U.S. Pat. No. 5,270,163; Ellington &
Szostak 1990 (Nature 346: 818-822); Tuerk & Gold 1990 (Science
249: 505-510); or "The Aptamer Handbook: Functional
Oligonucleotides and Their Applications", by Klussmann, ed.,
Wiley-VCH 2006, ISBN 3527310592, incorporated by reference herein.
The term "photoaptamer" refers to an aptamer that contains one or
more photoreactive functional groups that can covalently bind to or
crosslink with a target molecule. The term "peptidomimetic" refers
to a non-peptide agent that is a topological analogue of a
corresponding peptide. Methods of rationally designing
peptidomimetics of peptides are known in the art. For example, the
rational design of three peptidomimetics based on the sulphated
8-mer peptide CCK26-33, and of two peptidomimetics based on the
11-mer peptide Substance P, and related peptidomimetic design
principles, are described in Norwell 1995 (Trends Biotechnol 13:
132-134).
[0278] The term "small molecule" refers to compounds, preferably
organic compounds, with a size comparable to those organic
molecules generally used in pharmaceuticals. The term excludes
biological macromolecules (e.g., proteins, nucleic acids, etc.).
Preferred small organic molecules range in size up to about 5000
Da, e.g., up to about 4000, preferably up to 3000 Da, more
preferably up to 2000 Da, even more preferably up to about 1000 Da,
e.g., up to about 900, 800, 700, 600 or up to about 500 Da.
[0279] Also provided are methods for immunising animals, e.g.,
non-human animals such as laboratory or farm, animals using (i.e.,
using as the immunising antigen) the herein taught fragments of
MCAM, optionally attached to a presenting carrier. Immunisation and
preparation of antibody reagents from immune sera is well-known per
se and described in documents referred to elsewhere in this
specification. The animals to be immunised may include any animal
species, preferably warm-blooded species, more preferably
vertebrate species, including, e.g., birds and mammals. Without
limitation, the antibodies may be chicken, turkey, goose, duck,
guinea fowl, quail or pheasant. Also without limitation, the
antibodies may be human, murine (e.g., mouse, rat, etc.), donkey,
rabbit, goat, sheep, guinea pig, camel, llama or horse.
[0280] The term "presenting carrier" or "carrier" generally denotes
an immunogenic molecule which, when bound to a second molecule,
augments immune responses to the latter, usually through the
provision of additional T cell epitopes. The presenting carrier may
be a (poly)peptidic structure or a non-peptidic structure, such as
inter alia glycans, polyethylene glycols, peptide mimetics,
synthetic polymers, etc. Exemplary non-limiting carriers include
human Hepatitis B virus core protein, multiple C3d domains, tetanus
toxin fragment C or yeast Ty particles.
[0281] Immune sera obtained or obtainable by immunisation as taught
herein may be particularly useful for generating antibody reagents
that specifically bind to one or more of the herein disclosed
fragments of MCAM.
[0282] The invention also teaches a method for selecting
specific-binding agents which bind (a) one or more of the MCAM
fragments taught herein, substantially to the exclusion of (b) MCAM
and/or other fragments thereof. Conveniently, such methods may be
based on subtracting or removing binding agents which cross-react
or cross-bind the non-desired MCAM molecules under (b). Such
subtraction may be readily performed as known in the art by a
variety of affinity separation methods, such as affinity
chromatography, affinity solid phase extraction, affinity magnetic
extraction, etc.
[0283] Any existing, available or conventional separation,
detection and quantification methods can be used herein to measure
the presence or absence (e.g., readout being present vs. absent; or
detectable amount vs. undetectable amount) and/or quantity (e.g.,
readout being an absolute or relative quantity, such as, for
example, absolute or relative concentration) of MCAM and/or
fragments thereof and optionally of the one or more biomarkers
useful for AHF in samples (any molecules or analytes of interest to
be so-measured in samples, including MCAM and fragments thereof,
may be herein below referred to collectively as biomarkers).
[0284] For example, such methods may include immunoassay methods,
mass spectrometry analysis methods, or chromatography methods, or
combinations thereof.
[0285] The term "immunoassay" generally refers to methods known as
such for detecting one or more molecules or analytes of interest in
a sample, wherein specificity of an immunoassay for the molecule(s)
or analyte(s) of interest is conferred by specific binding between
a specific-binding agent, commonly an antibody, and the molecule(s)
or analyte(s) of interest.
[0286] Immunoassay technologies include without limitation direct
ELISA (enzyme-linked immunosorbent assay), indirect ELISA, sandwich
ELISA, competitive ELISA, multiplex ELISA, radioimmunoassay (RIA),
ELISPOT technologies, and other similar techniques known in the
art. Principles of these immunoassay methods are known in the art,
for example John R. Crowther, "The ELISA Guidebook", 1st ed.,
Humana Press 2000, ISBN 0896037282.
[0287] By means of further explanation and not limitation, direct
ELISA employs a labelled primary antibody to bind to and thereby
quantify target antigen in a sample immobilised on a solid support
such as a microwell plate. Indirect ELISA uses a non-labelled
primary antibody which binds to the target antigen and a secondary
labelled antibody that recognises and allows to quantify the
antigen-bound primary antibody. In sandwich ELISA the target
antigen is captured from a sample using an immobilised `capture`
antibody which binds to one antigenic site within the antigen, and
subsequent to removal of non-bound analytes the so-captured antigen
is detected using a `detection` antibody which binds to another
antigenic site within said antigen, where the detection antibody
may be directly labelled or indirectly detectable as above.
Competitive ELISA uses a labelled `competitor` that may either be
the primary antibody or the target antigen. In an example,
non-labelled immobilised primary antibody is incubated with a
sample, this reaction is allowed to reach equilibrium, and then
labelled target antigen is added. The latter will bind to the
primary antibody wherever its binding sites are not yet occupied by
non-labelled target antigen from the sample. Thus, the detected
amount of bound labelled antigen inversely correlates with the
amount of non-labelled antigen in the sample. Multiplex ELISA
allows simultaneous detection of two or more analytes within a
single compartment (e.g., microplate well) usually at a plurality
of array addresses (see, for example, Nielsen & Geierstanger
2004. J Immunol Methods 290: 107-20 and Ling et al. 2007. Expert
Rev Mol Diagn 7: 87-98 for further guidance). As appreciated,
labelling in ELISA technologies is usually by enzyme (such as,
e.g., horse-radish peroxidase) conjugation and the end-point is
typically colorimetric, chemiluminescent or fluorescent.
[0288] Radioimmunoassay (RIA) is a competition-based technique and
involves mixing known quantities of radioactively-labelled (e.g.,
.sup.125I or .sup.131I-labelled) target antigen with antibody to
said antigen, then adding non-labelled or `cold` antigen from a
sample and measuring the amount of labelled antigen displaced (see,
e.g., "An Introduction to Radioimmunoassay and Related Techniques",
by Chard T, ed., Elsevier Science 1995, ISBN 0444821198 for
guidance).
[0289] Further, mass spectrometry methods are suitable for
measuring biomarkers.
[0290] Generally, any mass spectrometric (MS) techniques that can
obtain precise information on the mass of peptides, and preferably
also on fragmentation and/or (partial) amino acid sequence of
selected peptides (e.g., in tandem mass spectrometry, MS/MS; or in
post source decay, TOF MS), are useful herein. Suitable peptide MS
and MS/MS techniques and systems are well-known per se (see, e.g.,
Methods in Molecular Biology, vol. 146: "Mass Spectrometry of
Proteins and Peptides", by Chapman, ed., Humana Press 2000, ISBN
089603609x; Biemann 1990. Methods Enzymol 193: 455-79; or Methods
in Enzymology, vol. 402: "Biological Mass Spectrometry", by
Burlingame, ed., Academic Press 2005, ISBN 9780121828073) and may
be used herein.
[0291] MS arrangements, instruments and systems suitable for
biomarker peptide analysis may include, without limitation,
matrix-assisted laser desorption/ionisation time-of-flight
(MALDI-TOF) MS; MALDI-TOF post-source-decay (PSD); MALDI-TOF/TOF;
surface-enhanced laser desorption/ionization time-of-flight mass
spectrometry (SELDI-TOF) MS; electrospray ionization mass
spectrometry (ESI-MS); ESI-MS/MS; ESI-MS/(MS).sup.n (n is an
integer greater than zero); ESI 3D or linear (2D) ion trap MS; ESI
triple quadrupole MS; ESI quadrupole orthogonal TOF (Q-TOF); ESI
Fourier transform MS systems; desorption/ionization on silicon
(DIOS); secondary ion mass spectrometry (SIMS); atmospheric
pressure chemical ionization mass spectrometry (APCI-MS);
APCI-MS/MS; APCI-(MS).sup.n; atmospheric pressure photoionization
mass spectrometry (APPI-MS); APPI-MS/MS; and APPI-(MS).sup.n.
Peptide ion fragmentation in tandem MS (MS/MS) arrangements may be
achieved using manners established in the art, such as, e.g.,
collision induced dissociation (CID).
[0292] In an embodiment, detection and quantification of biomarkers
by mass spectrometry may involve multiple reaction monitoring
(MRM), such as described among others by Kuhn et al. 2004
(Proteomics 4: 1175-86).
[0293] In an embodiment, MS peptide analysis methods may be
advantageously combined with upstream peptide or protein separation
or fractionation methods, such as for example with the
chromatographic and other methods described herein below.
[0294] Chromatography can also be used for measuring biomarkers. As
used herein, the term "chromatography" encompasses methods for
separating chemical substances, referred to as such and vastly
available in the art. In a preferred approach, chromatography
refers to a process in which a mixture of chemical substances
(analytes) carried by a moving stream of liquid or gas ("mobile
phase") is separated into components as a result of differential
distribution of the analytes, as they flow around or over a
stationary liquid or solid phase ("stationary phase"), between said
mobile phase and said stationary phase. The stationary phase may be
usually a finely divided solid, a sheet of filter material, or a
thin film of a liquid on the surface of a solid, or the like.
Chromatography is also widely applicable for the separation of
chemical compounds of biological origin, such as, e.g., amino
acids, proteins, fragments of proteins or peptides, etc.
[0295] Chromatography as used herein may be preferably columnar
(i.e., wherein the stationary phase is deposited or packed in a
column), preferably liquid chromatography, and yet more preferably
HPLC. While particulars of chromatography are well known in the
art, for further guidance see, e.g., Meyer M., 1998, ISBN:
047198373X, and "Practical HPLC Methodology and Applications",
Bidlingmeyer, B. A., John Wiley & Sons Inc., 1993.
[0296] Exemplary types of chromatography include, without
limitation, high-performance liquid chromatography (HPLC), normal
phase HPLC (NP-HPLC), reversed phase HPLC (RP-HPLC), ion exchange
chromatography (IEC), such as cation or anion exchange
chromatography, hydrophilic interaction chromatography (HILIC),
hydrophobic interaction chromatography (HIC), size exclusion
chromatography (SEC) including gel filtration chromatography or gel
permeation chromatography, chromatofocusing, affinity
chromatography such as immuno-affinity, immobilised metal affinity
chromatography, and the like.
[0297] In an embodiment, chromatography, including single-, two- or
more-dimensional chromatography, may be used as a peptide
fractionation method in conjunction with a further peptide analysis
method, such as for example, with a downstream mass spectrometry
analysis as described elsewhere in this specification.
[0298] Further peptide or polypeptide separation, identification or
quantification methods may be used, optionally in conjunction with
any of the above described analysis methods, for measuring
biomarkers in the present disclosure. Such methods include, without
limitation, chemical extraction partitioning, isoelectric focusing
(IEF) including capillary isoelectric focusing (CIEF), capillary
isotachophoresis (CITP), capillary electrochromatography (CEC), and
the like, one-dimensional polyacrylamide gel electrophoresis
(PAGE), two-dimensional polyacrylamide gel electrophoresis
(2D-PAGE), capillary gel electrophoresis (CGE), capillary zone
electrophoresis (CZE), micellar electrokinetic chromatography
(MEKC), free flow electrophoresis (FFE), etc.
[0299] The various aspects and embodiments taught herein may
further rely on comparing the quantity of MCAM measured in samples
with reference values of the quantity of MCAM, wherein said
reference values represent known predictions, diagnoses and/or
prognoses of AHF.
[0300] For example, distinct reference values may represent the
prediction of a risk (e.g., an abnormally elevated risk) of having
AHF vs. the prediction of no or normal risk of having AHF. In
another example, distinct reference values may represent
predictions of differing degrees of risk of having AHF.
[0301] In a further example, distinct reference values can
represent the diagnosis of AHF vs. the diagnosis of no AHF (such
as, e.g., the diagnosis of healthy, or recovered from AHF, etc.).
In another example, distinct reference values may represent the
diagnosis of AHF of varying severity.
[0302] In yet another example, distinct reference values may
represent a good prognosis for AHF vs. a poor prognosis for AHF. In
a further example, distinct reference values may represent
varyingly favourable or unfavourable prognoses for AHF.
[0303] Such comparison may generally include any means to determine
the presence or absence of at least one difference and optionally
of the size of such different between values or profiles being
compared. A comparison may include a visual inspection, an
arithmetical or statistical comparison of measurements. Such
statistical comparisons include, but are not limited to, applying a
rule. If the values or biomarker profiles comprise at least one
standard, the comparison to determine a difference in said values
or biomarker profiles may also include measurements of these
standards, such that measurements of the biomarker are correlated
to measurements of the internal standards.
[0304] Reference values for the quantity of MCAM may be established
according to known procedures previously employed for other
biomarkers.
[0305] For example, a reference value of the quantity of MCAM for a
particular prediction, diagnosis and/or prognosis of AHF may be
established by determining the quantity of MCAM in sample(s) from
one individual or from a population of individuals characterised by
said particular prediction, diagnosis and/or prognosis of AHF
(i.e., for whom said prediction, diagnosis and/or prognosis of AHF
holds true). Such population may comprise without
limitation.gtoreq.2, .gtoreq.10, .gtoreq.100, or even several
hundreds or more individuals.
[0306] Hence, by means of an illustrative example, reference values
of the quantity of MCAM for the diagnoses of AHF vs. no AHF may be
established by determining the quantity of MCAM in sample(s) from
one individual or from a population of individuals diagnosed (e.g.,
based on other adequately conclusive means, such as, for example,
clinical signs and symptoms, imaging, ECG, etc.) as, respectively,
having or not having AHF.
[0307] In an embodiment, reference value(s) as intended herein may
convey absolute quantities of MCAM. In another embodiment, the
quantity of MCAM in a sample from a tested subject may be
determined directly relative to the reference value (e.g., in terms
of increase or decrease, or fold-increase or fold-decrease).
Advantageously, this may allow to compare the quantity of MCAM in
the sample from the subject with the reference value (in other
words to measure the relative quantity of MCAM in the sample from
the subject vis-a-vis the reference value) without the need to
first determine the respective absolute quantities of MCAM.
[0308] The expression level or presence of a biomarker in a sample
of a patient may sometimes fluctuate, i.e. increase or decrease
significantly without change (appearance of, worsening or improving
of) symptoms. In such an event, the marker change precedes the
change in symptoms and becomes a more sensitive measure than
symptom change. Therapeutic intervention can be initiated earlier
and be more effective than waiting for deteriorating symptoms.
Symptoms can be (but not limited to): shortness of breath, oedema
in lower extremities, heart palpitations, fatigue, etc. Early
intervention at a more benign status may be carried out safely at
home, which is a major improvement from treating seriously
deteriorated patients in the emergency room.
[0309] Measuring the MCAM level of the same patient at different
time points can in such a case thus enable the continuous
monitoring of the status of the patient and can lead to prediction
of worsening or improvement of the patient's condition with regard
to AHF. A home or clinical test kit or device as indicated herein
can be used for this continuous monitoring. One or more reference
values or ranges of MCAM levels linked to a certain disease state
(e.g. AHF or no AHF) for such a test can e.g. be determined
beforehand or during the monitoring process over a certain period
of time in said subject. Alternatively, these reference values or
ranges can be established through data sets of several patients
with highly similar disease phenotypes, e.g. from healthy subjects
or subjects not having AHF. A sudden deviation of the MCAM levels
from said reference value or range can predict the worsening of the
condition of the patient (e.g. at home or in the clinic) before the
(often severe) symptoms actually can be felt or observed.
[0310] The invention therefore also provides a method or algorithm
for determining a significant change in the level of the MCAM
marker in a certain patient, which is indicative for change
(worsening or improving) in clinical status. In addition, the
invention allows establishing the diagnosis that the subject is
recovering or has recovered from the AHF condition.
[0311] In an embodiment the present methods may include a step of
establishing such reference value(s). In an embodiment, the present
kits and devices may include means for establishing a reference
value of the quantity of MCAM for a particular prediction,
diagnosis and/or prognosis of AHF. Such means may for example
comprise one or more samples (e.g., separate or pooled samples)
from one or more individuals characterised by said particular
prediction, diagnosis and/or prognosis of AHF.
[0312] The various aspects and embodiments taught herein may
further entail finding a deviation or no deviation between the
quantity of MCAM measured in a sample from a subject and a given
reference value.
[0313] A "deviation" of a first value from a second value may
generally encompass any direction (e.g., increase: first
value>second value; or decrease: first value<second value)
and any extent of alteration.
[0314] For example, a deviation may encompass a decrease in a first
value by, without limitation, at least about 10% (about 0.9-fold or
less), or by at least about 20% (about 0.8-fold or less), or by at
least about 30% (about 0.7-fold or less), or by at least about 40%
(about 0.6-fold or less), or by at least about 50% (about 0.5-fold
or less), or by at least about 60% (about 0.4-fold or less), or by
at least about 70% (about 0.3-fold or less), or by at least about
80% (about 0.2-fold or less), or by at least about 90% (about
0.1-fold or less), relative to a second value with which a
comparison is being made.
[0315] For example, a deviation may encompass an increase of a
first value by, without limitation, at least about 10% (about
1.1-fold or more), or by at least about 20% (about 1.2-fold or
more), or by at least about 30% (about 1.3-fold or more), or by at
least about 40% (about 1.4-fold or more), or by at least about 50%
(about 1.5-fold or more), or by at least about 60% (about 1.6-fold
or more), or by at least about 70% (about 1.7-fold or more), or by
at least about 80% (about 1.8-fold or more), or by at least about
90% (about 1.9-fold or more), or by at least about 100% (about
2-fold or more), or by at least about 150% (about 2.5-fold or
more), or by at least about 200% (about 3-fold or more), or by at
least about 500% (about 6-fold or more), or by at least about 700%
(about 8-fold or more), or like, relative to a second value with
which a comparison is being made.
[0316] Preferably, a deviation may refer to a statistically
significant observed alteration. For example, a deviation may refer
to an observed alteration which falls outside of error margins of
reference values in a given population (as expressed, for example,
by standard deviation or standard error, or by a predetermined
multiple thereof, e.g., .+-.1.times.SD or .+-.2.times.SD, or
.+-.1.times.SE or .+-.2.times.SE). Deviation may also refer to a
value falling outside of a reference range defined by values in a
given population (for example, outside of a range which comprises
.gtoreq.40%, .gtoreq.50%, .gtoreq.60%, .gtoreq.70%, .gtoreq.75% or
.gtoreq.80% or 85% or .gtoreq.90% or .gtoreq.95% or even
.gtoreq.100% of values in said population).
[0317] In a further embodiment, a deviation may be concluded if an
observed alteration is beyond a given threshold or cut-off. Such
threshold or cut-off may be selected as generally known in the art
to provide for a chosen sensitivity and/or specificity of the
prediction, diagnosis and/or prognosis methods, e.g., sensitivity
and/or specificity of at least 50%, or at least 60%, or at least
70%, or at least 80%, or at least 85%, or at least 90%, or at least
95%.
[0318] For example, in an embodiment, an elevated quantity of MCAM
in the sample from the subject--preferably at least about 1.1-fold
elevated, or at least about 1.2-fold elevated, more preferably at
least about 1.3-fold elevated, even more preferably at least about
1.4-fold elevated, yet more preferably at least about 1.5-fold
elevated, such as between about 1.1-fold and 3-fold elevated or
between about 1.5-fold and 2-fold elevated--compared to a reference
value representing the prediction or diagnosis of no AHF or
representing a good prognosis for AHF indicates that the subject
has or is at risk of having AHF or indicates a poor prognosis for
AHF in the subject.
[0319] When a deviation is found between the quantity of MCAM in a
sample from a subject and a reference value representing a certain
prediction, diagnosis and/or prognosis of AHF, said deviation is
indicative of or may be attributed to the conclusion that the
prediction, diagnosis and/or prognosis of AHF in said subject is
different from that represented by the reference value.
[0320] When no deviation is found between the quantity of MCAM in a
sample from a subject and a reference value representing a certain
prediction, diagnosis and/or prognosis of AHF, the absence of such
deviation is indicative of or may be attributed to the conclusion
that the prediction, diagnosis and/or prognosis of AHF in said
subject is substantially the same as that represented by the
reference value.
[0321] The above considerations apply analogously to biomarker
profiles.
[0322] When two or more different biomarkers are determined in a
subject, their respective presence, absence and/or quantity may be
together represented as a biomarker profile, the values for each
measured biomarker making a part of said profile. As used herein,
the term "profile" includes any set of data that represents the
distinctive features or characteristics associated with a condition
of interest, such as with a particular prediction, diagnosis and/or
prognosis of AHF. The term generally encompasses inter alia nucleic
acid profiles, such as for example genotypic profiles (sets of
genotypic data that represents the genotype of one or more genes
associated with a condition of interest), gene copy number profiles
(sets of gene copy number data that represents the amplification or
deletion of one or more genes associated with a condition of
interest), gene expression profiles (sets of gene expression data
that represents the mRNA levels of one or more genes associated
with a condition of interest), DNA methylation profiles (sets of
methylation data that represents the DNA methylation levels of one
or more genes associated with a condition of interest), as well as
protein, polypeptide or peptide profiles, such as for example
protein expression profiles (sets of protein expression data that
represents the levels of one or more proteins associated with a
condition of interest), protein activation profiles (sets of data
that represents the activation or inactivation of one or more
proteins associated with a condition of interest), protein
modification profiles (sets of data that represents the
modification of one or more proteins associated with a condition of
interest), protein cleavage profiles (sets of data that represent
the proteolytic cleavage of one or more proteins associated with a
condition of interest), as well as any combinations thereof.
[0323] Biomarker profiles may be created in a number of ways and
may be the combination of measurable biomarkers or aspects of
biomarkers using methods such as ratios, or other more complex
association methods or algorithms (e.g., rule-based methods). A
biomarker profile comprises at least two measurements, where the
measurements can correspond to the same or different biomarkers. A
biomarker profile may also comprise at least three, four, five, 10,
20, 30 or more measurements. In one embodiment, a biomarker profile
comprises hundreds, or even thousands, of measurements.
[0324] Hence, for example, distinct reference profiles may
represent the prediction of a risk (e.g., an abnormally elevated
risk) of having AHF vs. the prediction of no or normal risk of
having AHF. In another example, distinct reference profiles may
represent predictions of differing degrees of risk of having
AHF.
[0325] In a further example, distinct reference profiles can
represent the diagnosis of AHF vs. the diagnosis no AHF (such as,
e.g., the diagnosis of healthy, recovered from AHF, etc.). In
another example, distinct reference profiles may represent the
diagnosis of AHF of varying severity.
[0326] In a yet another example, distinct reference profiles may
represent a good prognosis for AHF vs. a poor prognosis for AHF. In
a further example, distinct reference profiles may represent
varyingly favourable or unfavourable prognoses for AHF.
[0327] Reference profiles used herein may be established according
to known procedures previously employed for other biomarkers.
[0328] For example, a reference profile of the quantity of MCAM and
the presence or absence and/or quantity of one or more other
AHF-related biomarkers for a particular prediction, diagnosis
and/or prognosis of AHF may be established by determining the
profile in sample(s) from one individual or from a population of
individuals characterised by said particular prediction, diagnosis
and/or prognosis of AHF (i.e., for whom said prediction, diagnosis
and/or prognosis of AHF holds true). Such population may comprise
without limitation.gtoreq.2, .gtoreq.10, .gtoreq.100, or even
several hundreds or more individuals.
[0329] Hence, by means of an illustrative example, reference
profiles for the diagnoses of AHF vs. no AHF may be established by
determining the biomarker profiles in sample(s) from one individual
or from a population of individuals diagnosed as, respectively,
having or not having AHF.
[0330] In an embodiment the present methods may include a step of
establishing such reference profile(s). In an embodiment, the
present kits and devices may include means for establishing a
reference profile for a particular prediction, diagnosis and/or
prognosis of AHF. Such means may for example comprise one or more
samples (e.g., separate or pooled samples) from one or more
individuals characterised by said particular prediction, diagnosis
and/or prognosis of AHF.
[0331] Further, art-known multi-parameter analyses may be employed
mutatis mutandis to determine deviations between groups of values
and profiles generated there from (e.g., between sample and
reference biomarker profiles).
[0332] When a deviation is found between the sample profile and a
reference profile representing a certain prediction, diagnosis
and/or prognosis of AHF, said deviation is indicative of or may be
attributed to the conclusion that the prediction, diagnosis and/or
prognosis of AHF in said subject is different from that represented
by the reference profile.
[0333] When no deviation is found between the sample profile and a
reference profile representing a certain prediction, diagnosis
and/or prognosis of AHF, the absence of such deviation is
indicative of or may be attributed to the conclusion that the
prediction, diagnosis and/or prognosis of AHF in said subject is
substantially the same as that represented by the reference
profile.
[0334] The present invention further provides kits or devices for
diagnosis of heart failure, more particularly of acute heart
failure, comprising means for detecting the level of the MCAM
marker in a sample of the patient. In a more preferred embodiment,
such a kit or kits of the invention can be used in clinical
settings or at home. The kit according to the invention can be used
for diagnosing Acute Heart Failure, for monitoring the
effectiveness of treatment of a subject suffering from AHF with an
agent, or for preventive screening of subjects for the occurrence
of AHF in said subject.
[0335] In a clinical setting, the kit or device can be in the form
of a bed-side device or in an emergency team setting, e.g. as part
of the equipment of an ambulance or other moving emergency vehicle
or team equipment or as part of a first-aid kit. The diagnostic kit
or device can assist a medical practitioner, a first aid helper, or
nurse to decide whether the patient under observation is developing
an acute heart failure, after which appropriate action or treatment
can be performed.
[0336] A home-test kit gives the patient a readout which he can
communicate to a medicinal practitioner, a first aid helper or to
the emergency department of a hospital, after which appropriate
action can be taken. Such a home-test device is of particular
interest for people having either a history of, or are at risk of
suffering from heart failure (e.g. chronic heart failure patients)
or have a history or are at risk of suffering from dyspnea
(shortness of breath), which may be caused by e.g. acute heart
failure, infections, lung-problems, sepsis, etc. Such subjects with
a high risk for heart failure or having a history of dyspnea could
certainly benefit from having a home test device or kit according
to the invention at home, because they can then easily distinguish
between an acute heart failure event and another event causing the
dyspnea, resulting in an easier way of determining the actions to
be taken to resolve the problem.
[0337] Typical kits or devices according to the invention comprise
the following elements:
[0338] a) a means for obtaining a sample from the subject
[0339] b) a means or device for measuring the amount of the MCAM
marker in said sample and visualizing whether the amount of the
MCAM marker in said sample is below or above a certain threshold
level or value, indicating whether the subject is suffering from
acute heart failure or not.
[0340] In any of the embodiments of the invention, the kits or
devices can additionally comprise c) means for communicating
directly with a medical practitioner, an emergency department of
the hospital or a first aid post, indicating that a person is
suffering from acute heart failure or not.
[0341] The term "threshold level or value" or "reference value" is
used interchangeably as a synonym and is as defined herein. It can
also be a range of base-line (e.g. "dry weight") values determined
in an individual patient or in a group of patients with highly
similar disease conditions.
[0342] In any of the embodiments of the invention, the device or
kit or kits of the invention can additionally comprise means for
detecting the level of an additional marker for heart failure or
acute heart failure in the sample of said patient. Additional
markers could for example be BNP or NT-pro-BNP or fragments of BNP
or NT-pro-BNP.
[0343] Any of kits as defined herein can be used as a bed-side
device for use by the subject himself or by a clinical
practitioner.
[0344] In said kit of the invention, the means for obtaining a
sample from the subject (a) can be any means for obtaining a sample
from the subject known in the art. Examples for obtaining e.g. a
blood sample are known in the art and could be any kind of finger
or skin prick or lancet based device, which basically pierces the
skin and results in a drop of blood being released from the skin.
When a urine sample is used, the means for obtaining a sample from
the subject can be in the form of an absorbent strip such as the
ones used in home pregnancy tests known in the art. In analogy, a
saliva sample could be obtained using a mount swab known in the
art. Example of blood sampling devices or other sampling devices
are for example given in U.S. Pat. Nos. 4,802,493, 4,966,159,
5,099,857, 6,095,988, 5,944,671, 4,553,541, 3,760,809, 5,395,388,
5,212,879, 5,630,828, 5,133,730, 4,653,513, 5,368,047, 5,569,287,
4,360,016, 5,413,006 and U.S. Pat. Applic. 2002/111565,
2004/0096959, 2005/143713, 2005/137525, 2003/0153900, 2003/0088191,
WO9955232, WO2005/049107, WO2004/060163, WO02/056751, WO02/100254,
WO2003/022330, WO2004/066822, WO97/46157, WO2004/039429, or
EP0364621, EP0078724, EP1212138, EP0081975, or EP0292928.
[0345] In said kit of the invention, the means or device for
measuring the amount of the MCAM marker in said sample (b) can be
any means or device that can specifically detect the amount of the
MCAM protein in the sample. Examples are systems comprising MCAM
specific binding molecules attached to a solid phase, e.g. lateral
flow strips or dipstick devices and the like well known in the art.
One non-limiting example to perform a biochemical assay is to use a
test-strip and labelled antibodies which combination does not
require any washing of the membrane. The test strip is well known,
for example, in the field of pregnancy testing kits where an
anti-hCG antibody is present on the support, and is carried
complexed with hCG by the flow of urine onto an immobilised second
antibody that permits visualisation. Other non-limiting examples of
such home test devices, systems or kits can be found for example in
the following U.S. Pat. No. 6,107,045, U.S. Pat. Nos. 6,974,706,
5,108,889, 6,027,944, 6,482,156, 6,511,814, 5,824,268, 5,726,010,
6,001,658 or U.S. patent applications: 2008/0090305 or
2003/0109067.
[0346] In a preferred embodiment, the invention provides a lateral
flow device or dipstick. Such dipstick comprises a test strip
allowing migration of a sample by capillary flow from one end of
the strip where the sample is applied to the other end of such
strip where presence of an analyte in said sample is measured.
[0347] In another embodiment, the invention provides a device
comprising a reagent strip. Such reagent strip comprises one or
more test pads which when wetted with the sample, provide a color
change in the presence of an analyte and/or indicate the
concentration of the protein in said sample.
[0348] In one preferred embodiment of the kit of the invention, the
means or device (1) for measuring the amount of protein in a sample
(b) is a solid support (7) having a proximal (2) and distal (3)
end, comprising: [0349] a sample application zone (4) in the
vicinity of the proximal end, [0350] a reaction zone (5) distal to
the sample application zone (4), and [0351] a detection zone (6)
distal to the reaction zone (5), whereby said support has a
capillary property that directs a flow of fluid sample applied in
the application zone in a direction from the proximal end to the
distal end, [0352] optionally, the means or device also comprises a
source of fluid, e.g. in a container, dropper pipette or vial,
enabling viscous samples to flow easier through the strip.
[0353] The reaction zone (5) comprises one or more bands (10) of
MCAM binding molecule conjugated to a detection agent (e.g.
colloidal gold) which MCAM binding molecule conjugate is disposed
on the solid support such that it can migrate with the capillary
flow of fluid i.e. it is not immobilised. The detection zone (6)
comprises one or more capture bands (11) comprising a population of
MCAM binding molecules immobilised on the solid support.
[0354] When a sample is applied to the sample application zone (4),
it migrates towards the reaction zone (5) by capillary flow. Any
MCAM present in the sample reacts with the MCAM labelled binding
molecule conjugate, and the complex so formed is carried by
capillary flow to the detection zone (6). The detection zone (6),
having MCAM binding molecules permanently immobilised thereon,
captures and immobilises any complex, resulting in a localised
concentration of conjugate that can be visualised.
[0355] The two zones (5 and 6) as described herein (one zone with
the non-fixed conjugates and one zone with the fixed capture
antibodies) generally do not overlap. They may be adjacently
arranged with an absence or presence of an intervening gap of solid
support devoid of band. A band may be disposed on a solid support
by any means, for example, absorbed, adsorbed, coated, covalently
attached or dried, depending on whether the reagent is required to
be mobilised or not.
[0356] In order to obtain a semi-quantitative test strip in which
only a signal is formed once the MCAM protein level in the sample
is higher than a certain predetermined threshold level or value,
the reaction zone (5) comprising the non-fixed conjugated MCAM
binding molecules, could also comprise a predetermined amount of
fixed MCAM capture antibodies. This enables to capture away a
certain amount of MCAM protein present in the sample, corresponding
to the threshold level or value as predetermined. The remaining
amount of MCAM protein (if any) bound by the conjugated or labelled
binding molecules can then be allowed to migrate to the detection
zone (6).
[0357] In this case, the reaction zone (6) will only receive
labelled binding molecule-MCAM complexes and subsequently only
produce a signal if the level of the MCAM protein in the sample is
higher than the predetermined threshold level or value.
[0358] Another possibility to determine whether the amount of the
MCAM protein in the sample is below or above a certain threshold
level or value, is to use a primary capturing antibody capturing
all MCAM protein present in the sample, in combination with a
labeled secondary antibody, developing a certain signal or color
when bound to the solid phase. The intensity of the color or signal
can then either be compared to a reference color or signal chart
indicating that when the intensity of the signal is above a certain
threshold signal, the test is positive (i.e. AHF is imminent).
Alternatively, the amount or intensity of the color or signal can
be measured with an electronic device comprising e.g. a light
absorbance sensor or light emission meter, resulting in a numerical
value of signal intensity or color absorbance formed, which can
then be displayed to the subject in the form of a negative result
if said numerical value is below the threshold value or a positive
result if said numerical value is above the threshold value.
[0359] This embodiment is of particular relevance in monitoring the
MCAM level in a patient over a period of time.
[0360] The reference value or range can e.g. be determined using
the home device in a period wherein the subject is free of AHF,
giving the patient an indication of his base-line MCAM level.
Regularly using the home test device will thus enable the subject
to notice a sudden change in MCAM levels as compared to the
base-line level, which can enable him to contact a medical
practitioner.
[0361] Alternatively, the reference value can be determined in the
subject suffering from AHF, which then indicates his personal MCAM
"risk level", i.e. the level of MCAM which indicates he is or will
soon be exposed to an AHF event. This risk level is interesting for
monitoring the disease progression or for evaluating the effect of
the treatment. Reduction of the MCAM level as compared to the risk
level indicates that the condition of the patient is improving.
[0362] Furthermore, the reference value or level can be established
through combined measurement results in subjects with highly
similar disease states or phenotypes (e.g. all in non-AHF condition
or all in AHF condition).
[0363] Non-limiting examples of such semi-quantitative tests known
in the art, the principle of which could be used for the home test
device according to the present invention are the HIV/AIDS test or
Prostate Cancer tests sold by Sanitoets. The home prostate test is
a rapid test intended as an initial semi-quantitative test to
detect PSA blood levels higher than 4 ng/ml in whole blood. The
typical home self-test kit comprises the following components: a
test device to which the blood sample is to be administered and
which results in a signal when the protein level is above a certain
threshold level, an amount of diluent e.g. in dropper pipette to
help the transfer of the analytes (i.e. the protein of interest)
from the sample application zone to the signal detection zone,
optionally an empty pipette for blood specimen collection, a finger
pricking device, optionally a sterile swab to clean the area of
pricking and instructions of use of the kit.
[0364] Similar tests are also known for e.g. breast cancer
detection and CRP-protein level detection in view of cardiac risk
home tests. The latter test encompasses the sending of the test
result to a laboratory, where the result is interpreted by a
technical or medical expert. Such telephone or internet based
diagnosis of the patient's condition is of course possible and
advisable with most of the kits, since interpretation of the test
result is often more important than conducting the test. When using
an electronic device as mentioned above which gives a numerical
value of the level of protein present in the sample, this value can
of course easily be communicated through telephone, mobile
telephone, satellite phone, E-mail, internet or other communication
means, warning a hospital, a medicinal practitioner or a first aid
team that a person is suffering from an acute heart failure. A
non-limiting example of such a system is disclosed in U.S. Pat. No.
6,482,156.
[0365] Reference is made in the description below to the drawings
which exemplify particular embodiments of the invention; they are
not at all intended to be limiting. The skilled person may adapt
the device and substituent components and features according to the
common practices of the person skilled in the art.
[0366] FIGS. 6A and B shows a preferred embodiment of a test strip
of the invention. The strip (1) includes a proximal end (2) and a
distal end (3). A sample application zone (4) is provided in the
proximal end (2), a reaction zone (5) is adjacent thereto and a
detection zone (6) is in the vicinity of the distal end (3). A
sample may be deposited onto the solid support (7) at the
application zone (4) to transfer by capillary action to the
detection zone (6). A protective layer (8) that covers either or
both the surfaces of the solid support (7), except for a region of
the sample application zone (4) may be provided. Such protective
layer protects the sample and chemical constituency of the strip
from contamination and evaporation. One or more absorbent pads (9)
in capillary contact with the sample application zone (4) of the
solid support (7) may absorb and release sample as necessary; such
pad (9) is typically placed on the surface of the solid support (7)
that is the same or opposing the sample application zone (4). In
FIG. 5B, the absorbent pad (9) is part of the sample application
zone (4). One or more other absorbent pads (9') in capillary may be
placed in contact with the detection zone (6) of the solid support
(7), distal to any capture bands (11), (14). These pads (9') may
absorb fluid that has passed through the solid support; such pad
(9') is typically placed on the surface of the solid support (7)
that is the same or opposing the sample application zone (4). The
solid support (7) may made from any suitable material that has a
capillary action property, and may have the same properties as
described above. It should also be capable of supporting a
substance (e.g. non-immobilised MCAM binding molecule), which, when
hydrated, can migrate across the solid support by a capillary
action fluid flow.
[0367] The solid support (7) may also comprise a band of MCAM
binding molecule conjugate (10), located in the reaction zone (5),
at a position distal to the sample application zone (4). Any MCAM
in the sample is carried by capillary action towards this band
(10), where it reacts with the permanently immobilised MCAM binding
molecule conjugate.
[0368] The MCAM binding molecule conjugate may be associated with
or attached to a detection agent to facilitate detection. Examples
of lab detection agents include, but are not limited to,
luminescent labels; colorimetric labels, such as dyes; fluorescent
labels; or chemical labels, such as electroactive agents (e.g.,
ferrocyanide); enzymes; radioactive labels; or radiofrequency
labels. More commonly, the detection agent is a particle. Examples
of particles useful in the practice of the invention include, but
are not limited to, colloidal gold particles; colloidal sulphur
particles; colloidal selenium particles; colloidal barium sulfate
particles; colloidal iron sulfate particles; metal iodate
particles; silver halide particles; silica particles; colloidal
metal (hydrous) oxide particles; colloidal metal sulfide particles;
colloidal lead selenide particles; colloidal cadmium selenide
particles; colloidal metal phosphate particles; colloidal metal
ferrite particles; any of the above-mentioned colloidal particles
coated with organic or inorganic layers; protein or peptide
molecules; liposomes; or organic polymer latex particles, such as
polystyrene latex beads. Preferable particles are colloidal gold
particles. Colloidal gold may be made by any conventional means,
such as the methods outlined in G. Frens, 1973 Nature Physical
Science, 241:20 (1973). Alternative methods may be described in
U.S. Pat. Nos. 5,578,577, 5,141,850; 4,775,636; 4,853,335;
4,859,612; 5,079,172; 5,202,267; 5,514,602; 5,616,467;
5,681,775.
[0369] The solid support (7) further comprises one or more capture
bands (11) in the detection zone (6). A capture band comprises a
population of MCAM binding molecule permanently immobilised
thereon. The MCAM: MCAM-binding molecule conjugate complex formed
in the reaction zone (5) migrates towards the detection zone (6)
where said band (11) captures migrating complex, and concentrates
it, allowing it to be visualised either by eye, or using a machine
reader. The MCAM binding molecule present in the reaction zone (5)
and in the detection zone (6) may reaction to the same part of MCAM
or may react to different parts of MCAM.
[0370] One or more controls bands (12) may be present on the solid
support (7). For example, a non-immobilised peptide (12) might be
present in the sample application zone (4), which peptide does not
cross-react with any of bands of MCAM binding molecule (13) or
(14). As the sample is applied, it migrates towards the reaction
zone (5), where an anti-peptide antibody conjugate is disposed
(13), and where a complex peptide-antibody complex is formed. Said
complex migrates towards the detection zone (6), where a capture
band (14) of anti-peptide antibody is immobilised on the solid
support, and which concentrates said complex enabling
visualisation. The control capture band (14) is located separately
from the MCAM capture band (11), therefore, a positive reaction can
be seen distinct from the detection reaction if the assay is
working correctly.
[0371] A particular advantage of a control according to the
invention is that they are internal controls--that is, the control
against which the MCAM measurement results may be compared is
present on the individual solid support. Therefore, the controls
according to the invention may be used to correct for variability
in the solid support, for example. Such correction would be
impractical with external controls that are based, for example, on
a statistical sampling of supports. Additionally, lot-to-lot, and
run-to-run, variations between different supports may be minimized
by use of control binding agents and control agents according to
the invention. Furthermore, the effects of non-specific binding may
be reduced. All of these corrections would be difficult to
accomplish using external, off-support, controls.
[0372] During the assay, MCAM from the sample and the MCAM binding
molecule conjugate combine and concentrate on the solid support
(7). This combination results in a concentration of compounds that
may can be visualised above the background colour of the solid
support (7). The compounds may be formed from a combination of
above-mentioned compounds, including antibodies, detection agents,
and other particles associated with the reaction and detection
zones. Based on the particular assay being performed, the reaction
and detection zones may be selectively implemented to achieve an
appropriate dynamic range which may be linear or non-linear.
[0373] A solid support (7) for performing the assay may be housed
within the cartridge (20) as shown, for example, in FIG. 6. The
cartridge is preferably watertight against urine, except for one or
more openings. The solid support (7) may be exposed through an
opening (21) in the cartridge to provide an application zone (4) in
proximal end (2), and another opening (22) to enable reading of
detection zone (6) close to the distal end (3). Cartridge (20) may
include a sensor code (23) for communicating with a reading
device.
[0374] The presence and/or concentration of MCAM in a sample can be
measured by surface plasmon resonance (SPR) using a chip having
MCAM binding molecule immobilized thereon, fluorescence resonance
energy transfer (FRET), bioluminescence resonance energy transfer
(BRET), fluorescence quenching, fluorescence polarization
measurement or other means known in the art. Any of the binding
assays described can be used to determine the presence and/or
concentration of MCAM in a sample. To do so, MCAM binding molecule
is reacted with a sample, and the concentration of MCAM is measured
as appropriate for the binding assay being used. To validate and
calibrate an assay, control reactions using different
concentrations of standard MCAM and/or MCAM binding molecule can be
performed. Where solid phase assays are employed, after incubation,
a washing step is performed to remove unbound MCAM. Bound, MCAM is
measured as appropriate for the given label (e.g., scintillation
counting, fluorescence, antibody-dye etc.). If a qualitative result
is desired, controls and different concentrations may not be
necessary. Of course, the roles of MCAM and MCAM binding molecule
may be switched; the skilled person may adapt the method so MCAM
binding molecule is applied to sample, at various concentrations of
sample.
[0375] A MCAM binding molecule according to the invention is any
substance that binds specifically to MCAM. Examples of a MCAM
binding molecule useful according to the present invention,
includes, but is not limited to an antibody, a polypeptide, a
peptide, a lipid, a carbohydrate, a nucleic acid, peptide-nucleic
acid, small molecule, small organic molecule, or other drug
candidate. A MCAM binding molecule can be natural or synthetic
compound, including, for example, synthetic small molecule,
compound contained in extracts of animal, plant, bacterial or
fungal cells, as well as conditioned medium from such cells.
Alternatively, MCAM binding molecule can be an engineered protein
having binding sites for MCAM. According to an aspect of the
invention, a MCAM binding molecule binds specifically to MCAM with
an affinity better than 10.sup.-6 M. A suitable MCAM binding
molecule e can be determined from its binding with a standard
sample of MCAM. Methods for determining the binding between MCAM
binding molecule and MCAM are known in the art. As used herein, the
term antibody includes, but is not limited to, polyclonal
antibodies, monoclonal antibodies, humanised or chimeric
antibodies, engineered antibodies, and biologically functional
antibody fragments (e.g. scFv, nanobodies, Fv, etc) sufficient for
binding of the antibody fragment to the protein. Such antibody may
be commercially available antibody against MCAM, such as, for
example, a mouse, rat, human or humanised monoclonal antibody.
[0376] In a preferred embodiment, the binding molecule or agent is
capable of binding both the mature membrane- or cell-bound MCAM
protein or fragment. In a more preferred embodiment, the binding
agent or molecule is specifically binding or detecting the soluble
form, preferably the plasma circulating form of MCAM, as defined
herein.
[0377] According to one aspect of the invention, the MCAM binding
molecule is labelled with a tag that permits detection with another
agent (e.g. with a probe binding partner). Such tags can be, for
example, biotin, streptavidin, his-tag, myc tag, maltose, maltose
binding protein or any other kind of tag known in the art that has
a binding partner. Example of associations which can be utilised in
the probe:binding partner arrangement may be any, and includes, for
example biotin:streptavidin, his-tag:metal ion (e.g. Ni.sup.2+),
maltose:maltose binding protein.
[0378] In another embodiment, the invention provides a simple and
accurate colorimetric reagent strip and method for measuring
presence of MCAM in a sample. More in particular, the present
invention also relates to a device comprising a reagent strip. The
present reagent strip comprises a solid support which is provided
with at least one test pad for measuring the presence of MCAM in a
sample. Said test pad preferably comprises a carrier matrix
incorporating a reagent composition capable of interacting with
MCAM to produce a measurable response, preferably a visually or
instrumentally measurable response. The reagent strip may be
manufactured in any size and shape, but in general the reagent
strip is longer than wide. The solid support may be composed of any
suitable material and is preferably made of firm or stiff material
such as cellulose acetate, polyethylene terephthalate,
polypropylene, polycarbonate or polystyrene. In general, the
carrier matrix is an absorbent material that allows the urine
sample to move, in response to capillary forces, through the
carrier matrix to contact the reagent composition and produce a
detectable or measurable color transition. The carrier matrix can
be any substance capable of incorporating the chemical reagents
required to perform the assay of interest, as long as the carrier
matrix is substantially inert with respect to the chemical
reagents, and is porous or absorbent relative to the soluble
components of the liquid test sample. The expression "carrier
matrix" refers to either bibulous or nonbibulous matrices that are
insoluble in water and other physiological fluids and maintain
their structural integrity when exposed to water and other
physiological fluids. Suitable bibulous matrices include filter
paper, sponge materials, cellulose, wood, woven and nonwoven
fabrics and the like. Nonbibulous matrices include glass fiber,
polymeric films, and preformed or microporous membranes. Other
suitable carrier matrices include hydrophilic inorganic powders,
such as silica gel, alumina, diatomaceous earth and the like;
argillaceous substances; cloth; hydrophilic natural polymeric
materials, particularly cellulose material, like cellulosic beads,
and especially fibercontaining papers such as filter paper or
chromatographic paper; synthetic or modified naturally-occurring
polymers, such as crosslinked gelatin, cellulose acetate, polyvinyl
chloride, polyacrylamide, cellulose, polyvinyl alcohol,
polysulfones, polyesters, polyacrylates, polyurethanes, crosslinked
dextran, agarose, and other such crosslinked and noncrosslinked
water-insoluble hydrophilic polymers. Hydrophobic and nonabsorptive
substances are not suitable for use as the carrier matrix of the
present invention. The carrier matrix can be of different chemical
compositions or a mixture of chemical compositions. The matrix also
can vary in regards to smoothness and roughness combined with
hardness and softness. However, in every instance, the carrier
matrix comprises a hydrophilic or absorptive material. The carrier
matrix is most advantageously constructed from bibulous filter
paper or nonbibulous polymeric films. A preferred carrier matrix is
a hydrophilic, bibulous matrix, including cellulosic materials,
such as paper, and preferably filter paper or a nonbibulous matrix,
including polymeric films, such as a polyurethane or a crosslinked
gelatin. A reagent composition which produces a colorimetric change
when reacted with MCAM in a sample can be homogeneously
incorporated into the carrier matrix, and the carrier matrix then
holds the reagent composition homogeneously throughout the carrier
matrix while maintaining carrier matrix penetrability by the
predetermined component of the test sample. Examples of suitable
reagent compositions may include for instance a MCAM binding
molecule in case of an antibody-based technique, or pH buffer in
case of enzymatic detection. The reagent composition is preferably
dried and stabilized onto a test pad adhered to at least one end of
a solid support. The test pad onto which the reagent composition is
absorbed and dried, is preferably made of a membrane material that
shows minimal background color. Preferably, the test pad may be
constructed of acid or base washed materials in order to minimize
background color. In another embodiment the reagent composition
which is dried onto the reagent strip further comprises wetting
agents to reduce brittleness of the test pad. Non-limiting examples
of preferred wetting agents include TritonX-100, Bioterg, glycerol,
0 Tween, and the like. The reagent composition can be applied to
the reagent strip by any method known in the art. For example, the
carrier matrix from which the test pads are made may be dipped into
a solution of the reagent composition and dried according to
techniques known in the art. A reagent strip according to the
invention may be provided with multiple test pads to assay for more
than one analyte in a urine sample. A reagent strip may be provided
comprising a solid support provided with one or more test pads
including test pads for measuring the presence of one or more
analytes selected from the group comprising proteins such as AHF
markers BNP, NT-pro-BNP or fragments thereof, blood, leukocytes,
nitrite, glucose, ketones, creatinine, albumin, bilirubin,
urobilinogen and/or a pH test pad, and/or a test pad for measuring
specific gravity.
[0379] A possible embodiment of a reagent strip 101 according to
the invention is depicted diagrammatically in FIG. 8 A-B. The strip
101 includes a proximal end 102 and a distal end 103. Various test
pads 109, 109', 109'' on which the reagent compositions are
provided at the proximal end 102 on a solid support 107 of the
reagent strip. The strip must be designed in such a way that it can
be wetted with a sufficiently large amount of sample, optionally
diluted by a physiological fluid improving the capillary flow of a
viscous sample such as blood or saliva and the like.
[0380] A reagent strip as defined herein is used as follows.
Briefly, one or more test pad areas of the reagent strip of the
invention is dipped into a sample or a small amount of sample is
applied to the reagent strip onto the test pad area(s). A color
development which can be analyzed visually or by reflectometry
occurs on the reagent strip within a short time, usually within 0.5
to 10 minutes. The change in color of the reagent area on the test
pad upon reacting with MCAM is preferably directly proportional to
the concentration of MCAM in the patient sample. The color
intensity that develops on the test pad may be determined visually
or by a reflectance-based reader, for example. Color development at
the test pad area(s) is compared to a reference color or colors to
determine an estimate of the amount of MCAM present in the sample.
The color intensity that develops on the test pad is compared to at
least one, and preferably at least two standard color shades that
correspond to a range of MCAM concentration determined by
application of a correction factor.
[0381] The reagent strip may further comprises a fluorescent or
infrared dye, applied either to the support strip or incorporated
into a test pad, which ensures proper alignment of the reagent
strip in an apparatus having a detection system for the detectable
or measurable response.
[0382] In another embodiment, the invention also relates to a test
pad for measuring the presence of MCAM in a sample. Preferably said
test pad comprises a carrier matrix incorporating a reagent
composition capable of interacting with MCAM to produce a
measurable response, preferably a visually or instrumentally
measurable response. In another preferred embodiment the invention
provides a test pad according as define herein for use in on a
reagent strip, preferably on a reagent strip as defined herein.
[0383] The specific-binding agents, peptides, polypeptides,
proteins, biomarkers etc. in the present kits may be in various
forms, e.g., lyophilised, free in solution or immobilised on a
solid phase. They may be, e.g., provided in a multi-well plate or
as an array or microarray, or they may be packaged separately
and/or individually. The may be suitably labelled as taught herein.
Said kits may be particularly suitable for performing the assay
methods of the invention, such as, e.g., immunoassays, ELISA
assays, mass spectrometry assays, and the like.
[0384] The above aspects and embodiments are further supported by
the following non-limiting examples.
EXAMPLES
Example 1
MASSterclass Targeted Protein Quantitation for Early Validation of
Candidate Markers Derived from Discovery
MASSterclass Experimental Setup
[0385] MASSterclass assays use targeted tandem mass spectrometry
with stable isotope dilution as an end-stage peptide quantitation
system (also called Multiple Reaction Monitoring (MRM) and Single
Reaction Monitoring (SRM)). The targeted peptide is specific (i.e.,
proteotypic) for the specific protein of interest. i.e., the amount
of peptide measured is directly related to the amount of protein in
the original sample. To reach the specificity and sensitivity
needed for biomarker quantitation in complex samples, peptide
fractionations precede the end-stage quantitation step.
[0386] A suitable MASSterclass assay may include the following
steps: [0387] Plasma/serum sample [0388] Depletion of human albumin
and IgG (complexity reduction on protein level) using affinity
capture with anti-albumin and anti-IgG antibodies using ProteoPrep
spin columns (Sigma Aldrich) [0389] Spiking of known amounts of
isotopically labelled peptides. This peptide has the same amino
acid sequence as the proteotypic peptide of interest, typically
with one isotopically labelled amino acid built in to generate a
mass difference. During the entire process, the labelled peptide
has identical chemical and chromatographic behaviour as the
endogenous peptide, except during the end-stage quantitation step
which is based on molecular mass. [0390] Tryptic digest. The
proteins in the depleted serum/plasma sample are digested into
peptides using trypsin. This enzyme cleaves proteins C-terminally
from lysine and argninine, except when a proline is present
C-terminally of the lysine or arginine. Before digestion, proteins
are denatured by boiling, which renders the protein molecule more
accessible for the trypsin activity during the 16 h incubation at
37.degree. C. [0391] First peptide-based fractionation: Free Flow
Electrophoresis (FFE; BD Diagnostic) is a gel-free, fluid
separation technique in which charged molecules moving in a
continuous laminar flow are separated through an electrical field
perpendicular to the flow. The electrical field causes the charged
molecules to separate in the pH gradient according to their
isoelectric point (pl). Only those fractions containing the
monitored peptides are selected for further fractionation and
LC-MS/MS analysis. Each peptide of interest elutes from the FFE
chamber at a specific fraction number, which is determined during
protein assay development using the synthetic peptide homologue.
Specific fractions or fraction pools (multiplexing) proceed to the
next level of fractionation. [0392] Second peptide-based
fractionation: Phenyl HPLC (XBridge Phenyl; Waters) separates
peptides according to hydrophobicity and aromatic nature of amino
acids present in the peptide sequence. Orthogonality with the
back-end C18 separation is achieved by operating the column at an
increased pH value (pH 10). As demonstrated by Gilar et al. 2005, J
Sep Sci 28(14): 1694-1703), pH is by far the most drastic parameter
to alter peptide selectivity in RP-HPLC. Each peptide of interest
elutes from the Phenyl column at a specific retention time, which
is determined during protein assay development using the synthetic
peptide homologue. The use of an external control system, in which
a mixture of 9 standard peptides is separated upfront a batch of
sample separations, allows adjusting the fraction collection in
order to correct for retention time shifts. The extent of
fractionation is dependent on the concentration of the protein in
the sample and the complexity of that sample. [0393] LC-MS/MS based
quantitation, including further separation on reversed phase (C18)
nanoLC (PepMap C18; Dionex) and MS/MS: tandem mass spectrometry
using MRM (4000 QTRAP; ABI)/SRM (Vantage TSQ; Thermo Scientific)
mode. The LC column is connected to an electrospray needle
connected to the source head of the mass spectrometer. As material
elutes from the column, molecules are ionized and enter the mass
spectrometer in the gas phase. The peptide that is monitored is
specifically selected to pass the first quadrupole (Q1), based on
its mass to charge ratio (m/z). The selected peptide is then
fragmented in a second quadrupole (Q2) which is used as a collision
cell. The resulting fragments then enter the third quadrupole (Q3).
Depending on the instrument settings (determined during the assay
development phase) only a specific peptide fragment or specific
peptide fragments (or so called transitions) are selected for
detection. [0394] The combination of the m/z of the monitored
peptide and the m/z of the monitored fragment of this peptide is
called a transition. This process can be performed for multiple
transitions during one experiment. Both the endogenous peptide
(analyte) and its corresponding isotopically labelled synthetic
peptide (internal standard) elute at the same retention time, and
are measured in the same LC-MS/MS experiment. [0395] The
MASSterclass readout is defined by the ratio between the area under
the peak specific for the analyte and the area under the peak
specific for the synthetic isotopically labelled analogue (internal
standard). MASSterclass readouts are directly related to the
original concentration of the protein in the sample. MASSterclass
readouts can therefore be compared between different samples and
groups of samples.
[0396] A typical MASSterclass protocol followed in the present
study is given here below: [0397] 25 .mu.L of plasma is subjected
to a depletion of human albumin and IgG (ProteoPrep spin columns;
Sigma Aldrich) according to the manufacturer's protocol, except
that 20 mM NH.sub.4HCO.sub.3 was used as the binding/equilibration
buffer. [0398] The depleted sample (225 .mu.L) is denatured for 15
min at 95.degree. C. and immediately cooled on ice [0399] 500 fmol
of the isotopically labelled peptide (custom made `Heavy AQUA`
peptide; Thermo Scientific) is spiked in the sample [0400] 20 .mu.g
trypsin is added to the sample and digestion is allowed for 16 h at
37.degree. C. [0401] The digested sample was first diluted 1/8 in
solvent A (0.1% formic acid) and then 1/20 in the same solvent
containing 250 amol/.mu.L of all isotopically labelled peptides
(custom made `Heavy AQUA` peptide; Thermo Scientific) of interest.
[0402] 20 .mu.L of the final dilution was separated using
reverse-phase NanoLC with on-line MS/MS in MRM/SRM mode: [0403]
Column: PepMap C18, 75 .mu.m I.D..times.25 cm L, 100 .ANG. pore
diameter, 5 .mu.m particle size [0404] Solvent A: 0.1% formic acid
[0405] Solvent B: 80% acetonitrile, 0.1% formic acid [0406]
Gradient: 30 min; 2%-55% Solvent B [0407] MS/MS in MRM mode: method
contains the transitions for the analyte as well as for the
synthetic, labelled peptide. [0408] The used transitions were
experimentally determined and selected during protein assay
development [0409] Each of the transitions of interest was measured
for a period starting 3 minutes before and ending 3 minutes after
the determined retention time of the peptide of interest, making
sure that each peak had at least 15 datapoints. [0410] The raw data
was analysed and quantified using the LCQuan software (Thermo
Scientific): the area under the analyte (=the MCAM peptide) peak
and under the internal standard (the labelled, synthetic MCAM
peptide) peak at the same C18 retention time was determined by
automatic peak detection. These were checked manually. [0411] The
MASSterclass readout was defined by the ratio of the analyte peak
area and the internal standard peak area
MASSterclass Statistical Analysis
[0412] The measured ratios are differential quantitations of
peptides. In other words a ratio is the normalised concentration of
a peptide. The concentration of a peptide is proportional to the
ratio measured with mass spectrometry.
[0413] A statistical analysis is conducted in order to determine
the diagnostic accuracy of a specific protein. To do so, sample
classes are compared pairwise. The analysis defines the ability of
a protein to discriminate two sample populations.
[0414] The diagnostic performance of a specific protein was
determined by measuring the area under the
Receiver-Operating-Characteristics (ROC) curves (AUC) (cf. Sullivan
Pepe M, The statistical evaluation of medical tests for
classification and prediction. 1993 Oxford University Press New
York). The estimated and confidence intervals (CI) for AUCs were
also computed using a nonparametric approach, namely bootstrapping
(cf. Efron B, Tibshirani RJ. Nonparametric confidence intervals. An
introduction to the bootstrap. Monographs on statistics and applied
probability. 1993; 57:75-90 Chapman & Hall New York).
Example 2
Verification of Diagnostic Value of Candidate Marker MCAM Using
MASSterclass
[0415] Clinical samples were collected prospectively across 3
different medical centres from patients presenting to emergency
department (ED) with acute dyspnea (n=100) either related to acute
heart failure or related to other causes (=dyspnea non AHF).
[0416] For all included patients a comprehensive case report file
(CRF) was completed with details on medical background, admission
diagnosis and medications.
[0417] Receiver-operating characteristics (ROC) analysis
demonstrated MCAM to be highly sensitive and specific for
diagnosing AHF in dyspneic patients presenting to the ED, as
indicated by an overall median AUC of 0.91 with 95% CI 0.85-0.96
(cf. FIG. 4). This diagnostic performance is equivalent to BNP and
NT-proBNP, the current gold standard biomarkers for diagnosing AHF
in an acute dyspnea population. Table 1 lists the results.
TABLE-US-00001 TABLE 1 BNP NT-proBNP MCAM Median AUC 0.88 0.85 0.91
95% CI 0.82-0.95 0.77-0.92 0.85-0.96
[0418] Combining MCAM with BNP has a significant impact on the
overall diagnostic accuracy, reaching a maximum of 86% in the
current dataset (cf. FIG. 4). The diagnostic accuracy of MCAM and
BNP at a single cut-off and the combination of the two markers is
summarized in Table 2 below. Taking into account that 100 pg/mL is
the clinically used "rule-out" cut-off for BNP, using the MCAM
level at a single cut-off can greatly improve on the diagnostic
accuracy of BNP. MCAM values can compensate for the lack of
specificity of BNP when values are above 100 pg/mL.
TABLE-US-00002 TABLE 2 Accuracy BNP at 100 pg/mL = 71% Accuracy
MCAM = 84% Accuracy BNP (rule-out) + 86% MCAM =
Example 3
Verification of MCAM as a Marker of Disease Progression: Comparison
of Levels at Admission Versus Discharge
[0419] Patients diagnosed with acute heart failure were sampled
both at admission to the ED as well as at discharge from the
hospital, i.e. when patients were deemed to have recovered and to
be stable. On average the discharge sample was taken 9-11 days
after the admission sample. Levels of MCAM were measured using
MASSterclass in both samples and levels were compared within the
same patient. For the majority of patients there was a significant
decrease of MCAM when admission and discharge levels were compared
(FIG. 5). A very similar picture is obtained when BNP levels at
admission versus discharge are compared. This data supports the
idea that MCAM levels are a reflection of disease status and thus
could be used to monitor and/or predict an acute event.
[0420] Furthermore the main treatment given to these AHF patients
are diuretics and as a consequence the patients lose fluids. Hence
a drop in MCAM levels is reflective of a change in filling status
of the patients.
Example 4
MCAM Levels Associate with Weight Gain and Weight Loss in Acute
Dyspnea Patients
[0421] Clinical samples from acute dyspnea patients (BASEL V cohort
as described in Potocki et al., Journal of Internal Medicine 2010
January; 267(1):119-29), either diagnosed with acute decompensated
heart failure or dyspnea due to other causes were screened for MCAM
using MASSterclass. All clinical data pertaining the samples was
obtained via the clinical collaborator and added to the
MASSterclass data analysis pipeline.
[0422] Associations of MCAM levels with all available clinical
parameters were computed using univariate statistical tests.
Spearman's ranked test was used to compute correlation coefficients
and Wilcoxon rank sum test for assessing whether two independent
samples of observation originate from the same population.
[0423] This analysis showed a clear association of MCAM with weight
gain prior to admission to the hospital and weight loss after
therapeutic diuretics use as indicated by the low Wilcoxon p-values
(summarized in Table 3).
TABLE-US-00003 TABLE 3 population MCAM p-values weight loss
admission to discharge AHF 0.00383 weight gain prior to admission
AHF 0.00058
[0424] FIG. 9 illustrates the effect weight gain on MCAM levels.
AHF patients that put on weight prior to admission to the hospital
(fluid build-up) have clearly increased levels of MCAM.
Example 5
MCAM Levels are Increased in AHF Patients with Systolic
Dysfunction
[0425] The effect of systolic versus diastolic dysfunction in heart
failure patients on MCAM levels was investigated based on the
MASSterclass screening results of the BASEL V cohort. This cohort
contains a sufficient number of AHF patients with either reduced
left ventricular ejection fraction (LVEF<55) or preserved LVEF
(LVEF>55). MCAM levels are significantly higher in AHF patients
with reduced ejection fractions (p<0.001). FIG. 10 shows box and
whisker plots for MCAM in these two AHF subpopulations.
[0426] Patients with a systolic dysfunction (reduced EF) are more
resistant to fluid build-up and will accumulate more volume
compared to patients with diastolic dysfunction before symptoms of
dyspnea occur.
Sequence CWU 1
1
61646PRTHomo sapiens 1Met Gly Leu Pro Arg Leu Val Cys Ala Phe Leu
Leu Ala Ala Cys Cys 1 5 10 15 Cys Cys Pro Arg Val Ala Gly Val Pro
Gly Glu Ala Glu Gln Pro Ala 20 25 30 Pro Glu Leu Val Glu Val Glu
Val Gly Ser Thr Ala Leu Leu Lys Cys 35 40 45 Gly Leu Ser Gln Ser
Gln Gly Asn Leu Ser His Val Asp Trp Phe Ser 50 55 60 Val His Lys
Glu Lys Arg Thr Leu Ile Phe Arg Val Arg Gln Gly Gln 65 70 75 80 Gly
Gln Ser Glu Pro Gly Glu Tyr Glu Gln Arg Leu Ser Leu Gln Asp 85 90
95 Arg Gly Ala Thr Leu Ala Leu Thr Gln Val Thr Pro Gln Asp Glu Arg
100 105 110 Ile Phe Leu Cys Gln Gly Lys Arg Pro Arg Ser Gln Glu Tyr
Arg Ile 115 120 125 Gln Leu Arg Val Tyr Lys Ala Pro Glu Glu Pro Asn
Ile Gln Val Asn 130 135 140 Pro Leu Gly Ile Pro Val Asn Ser Lys Glu
Pro Glu Glu Val Ala Thr 145 150 155 160 Cys Val Gly Arg Asn Gly Tyr
Pro Ile Pro Gln Val Ile Trp Tyr Lys 165 170 175 Asn Gly Arg Pro Leu
Lys Glu Glu Lys Asn Arg Val His Ile Gln Ser 180 185 190 Ser Gln Thr
Val Glu Ser Ser Gly Leu Tyr Thr Leu Gln Ser Ile Leu 195 200 205 Lys
Ala Gln Leu Val Lys Glu Asp Lys Asp Ala Gln Phe Tyr Cys Glu 210 215
220 Leu Asn Tyr Arg Leu Pro Ser Gly Asn His Met Lys Glu Ser Arg Glu
225 230 235 240 Val Thr Val Pro Val Phe Tyr Pro Thr Glu Lys Val Trp
Leu Glu Val 245 250 255 Glu Pro Val Gly Met Leu Lys Glu Gly Asp Arg
Val Glu Ile Arg Cys 260 265 270 Leu Ala Asp Gly Asn Pro Pro Pro His
Phe Ser Ile Ser Lys Gln Asn 275 280 285 Pro Ser Thr Arg Glu Ala Glu
Glu Glu Thr Thr Asn Asp Asn Gly Val 290 295 300 Leu Val Leu Glu Pro
Ala Arg Lys Glu His Ser Gly Arg Tyr Glu Cys 305 310 315 320 Gln Gly
Leu Asp Leu Asp Thr Met Ile Ser Leu Leu Ser Glu Pro Gln 325 330 335
Glu Leu Leu Val Asn Tyr Val Ser Asp Val Arg Val Ser Pro Ala Ala 340
345 350 Pro Glu Arg Gln Glu Gly Ser Ser Leu Thr Leu Thr Cys Glu Ala
Glu 355 360 365 Ser Ser Gln Asp Leu Glu Phe Gln Trp Leu Arg Glu Glu
Thr Gly Gln 370 375 380 Val Leu Glu Arg Gly Pro Val Leu Gln Leu His
Asp Leu Lys Arg Glu 385 390 395 400 Ala Gly Gly Gly Tyr Arg Cys Val
Ala Ser Val Pro Ser Ile Pro Gly 405 410 415 Leu Asn Arg Thr Gln Leu
Val Asn Val Ala Ile Phe Gly Pro Pro Trp 420 425 430 Met Ala Phe Lys
Glu Arg Lys Val Trp Val Lys Glu Asn Met Val Leu 435 440 445 Asn Leu
Ser Cys Glu Ala Ser Gly His Pro Arg Pro Thr Ile Ser Trp 450 455 460
Asn Val Asn Gly Thr Ala Ser Glu Gln Asp Gln Asp Pro Gln Arg Val 465
470 475 480 Leu Ser Thr Leu Asn Val Leu Val Thr Pro Glu Leu Leu Glu
Thr Gly 485 490 495 Val Glu Cys Thr Ala Ser Asn Asp Leu Gly Lys Asn
Thr Ser Ile Leu 500 505 510 Phe Leu Glu Leu Val Asn Leu Thr Thr Leu
Thr Pro Asp Ser Asn Thr 515 520 525 Thr Thr Gly Leu Ser Thr Ser Thr
Ala Ser Pro His Thr Arg Ala Asn 530 535 540 Ser Thr Ser Thr Glu Arg
Lys Leu Pro Glu Pro Glu Ser Arg Gly Val 545 550 555 560 Val Ile Val
Ala Val Ile Val Cys Ile Leu Val Leu Ala Val Leu Gly 565 570 575 Ala
Val Leu Tyr Phe Leu Tyr Lys Lys Gly Lys Leu Pro Cys Arg Arg 580 585
590 Ser Gly Lys Gln Glu Ile Thr Leu Pro Pro Ser Arg Lys Ser Glu Leu
595 600 605 Val Val Glu Val Lys Ser Asp Lys Leu Pro Glu Glu Met Gly
Leu Leu 610 615 620 Gln Gly Ser Ser Gly Asp Lys Arg Ala Pro Gly Asp
Gln Gly Glu Lys 625 630 635 640 Tyr Ile Asp Leu Arg His 645
215PRTArtificialsynthetic peptide 2Gly Ala Thr Leu Ala Leu Thr Gln
Val Thr Pro Gln Asp Glu Arg 1 5 10 15 3134PRTHomo sapiens 3Met Asp
Pro Gln Thr Ala Pro Ser Arg Ala Leu Leu Leu Leu Leu Phe 1 5 10 15
Leu His Leu Ala Phe Leu Gly Gly Arg Ser His Pro Leu Gly Ser Pro 20
25 30 Gly Ser Ala Ser Asp Leu Glu Thr Ser Gly Leu Gln Glu Gln Arg
Asn 35 40 45 His Leu Gln Gly Lys Leu Ser Glu Leu Gln Val Glu Gln
Thr Ser Leu 50 55 60 Glu Pro Leu Gln Glu Ser Pro Arg Pro Thr Gly
Val Trp Lys Ser Arg 65 70 75 80 Glu Val Ala Thr Glu Gly Ile Arg Gly
His Arg Lys Met Val Leu Tyr 85 90 95 Thr Leu Arg Ala Pro Arg Ser
Pro Lys Met Val Gln Gly Ser Gly Cys 100 105 110 Phe Gly Arg Lys Met
Asp Arg Ile Ser Ser Ser Ser Gly Leu Gly Cys 115 120 125 Lys Val Leu
Arg Arg His 130 4108PRTHomo sapiens 4His Pro Leu Gly Ser Pro Gly
Ser Ala Ser Asp Leu Glu Thr Ser Gly 1 5 10 15 Leu Gln Glu Gln Arg
Asn His Leu Gln Gly Lys Leu Ser Glu Leu Gln 20 25 30 Val Glu Gln
Thr Ser Leu Glu Pro Leu Gln Glu Ser Pro Arg Pro Thr 35 40 45 Gly
Val Trp Lys Ser Arg Glu Val Ala Thr Glu Gly Ile Arg Gly His 50 55
60 Arg Lys Met Val Leu Tyr Thr Leu Arg Ala Pro Arg Ser Pro Lys Met
65 70 75 80 Val Gln Gly Ser Gly Cys Phe Gly Arg Lys Met Asp Arg Ile
Ser Ser 85 90 95 Ser Ser Gly Leu Gly Cys Lys Val Leu Arg Arg His
100 105 576PRTHomo sapiens 5His Pro Leu Gly Ser Pro Gly Ser Ala Ser
Asp Leu Glu Thr Ser Gly 1 5 10 15 Leu Gln Glu Gln Arg Asn His Leu
Gln Gly Lys Leu Ser Glu Leu Gln 20 25 30 Val Glu Gln Thr Ser Leu
Glu Pro Leu Gln Glu Ser Pro Arg Pro Thr 35 40 45 Gly Val Trp Lys
Ser Arg Glu Val Ala Thr Glu Gly Ile Arg Gly His 50 55 60 Arg Lys
Met Val Leu Tyr Thr Leu Arg Ala Pro Arg 65 70 75 632PRTHomo sapiens
6Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Lys Met Asp 1
5 10 15 Arg Ile Ser Ser Ser Ser Gly Leu Gly Cys Lys Val Leu Arg Arg
His 20 25 30
* * * * *
References