U.S. patent application number 12/058230 was filed with the patent office on 2010-10-07 for serological markers of inflammatory bowel disease phenotype and disease progression.
Invention is credited to Lee A. Denson, Bruce Colston Trapnell, Kanji Uchida.
Application Number | 20100255513 12/058230 |
Document ID | / |
Family ID | 42826492 |
Filed Date | 2010-10-07 |
United States Patent
Application |
20100255513 |
Kind Code |
A1 |
Denson; Lee A. ; et
al. |
October 7, 2010 |
SEROLOGICAL MARKERS OF INFLAMMATORY BOWEL DISEASE PHENOTYPE AND
DISEASE PROGRESSION
Abstract
Disclosed are novel biomarkers and methods related to diagnostic
tests for the detection and characterization of inflammatory bowel
diseases, such as Crohn's disease and ulcerative colitis. In
particular, the instant invention relates to novel biomarkers and
methods of using such biomarkers to predict disease behavior and
severity, to differentiate among disease types, and to optimize
selection of treatment options in individuals suspected of having
an inflammatory bowel disease.
Inventors: |
Denson; Lee A.; (Wyoming,
OH) ; Trapnell; Bruce Colston; (Hamilton, OH)
; Uchida; Kanji; (Tokyo, JP) |
Correspondence
Address: |
FROST BROWN TODD, LLC
2200 PNC CENTER, 201 E. FIFTH STREET
CINCINNATI
OH
45202
US
|
Family ID: |
42826492 |
Appl. No.: |
12/058230 |
Filed: |
March 28, 2008 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
60909153 |
Mar 30, 2007 |
|
|
|
Current U.S.
Class: |
435/7.92 ;
435/7.1 |
Current CPC
Class: |
C07K 2317/76 20130101;
G01N 33/686 20130101; C07K 2317/21 20130101; G01N 33/6863 20130101;
C07K 16/241 20130101; A61K 38/193 20130101; C07K 2317/24 20130101;
G01N 2800/065 20130101; G01N 33/68 20130101; G01N 2333/535
20130101; G01N 2800/50 20130101 |
Class at
Publication: |
435/7.92 ;
435/7.1 |
International
Class: |
G01N 33/53 20060101
G01N033/53 |
Claims
1. A diagnostic kit for the diagnosis and prognosis of inflammatory
bowel diseases in a mammalian subject comprising: a probe specific
for GM-CSF wherein the probe is capable of detecting a
concentration of anti-GM-CSF antibodies, such that a diagnosis or
prognosis of the subject may be made; and reactants for detecting
the concentration of anti-GM-CSF antibodies.
2. The diagnostic kit according to claim 1, wherein the reactants
for detecting the concentration of anti-GM-CSF antibodies function
in a method selected from the group consisting of in situ
hybridization, hybridization, and recognition by marked specific
antibodies, the method being conducted on filter, on solid support,
in solution, or on gel, by using at least one technique selected
from the group consisting of a sandwich method, Dot blot
hybridization, isotopic or non-isotopic labeling, cold probe
techniques, double immunodiffusion, counter-immunoelectrophoresis,
and hemagglutination.
3. The diagnostic kit according to claim 1, wherein the probe is an
antigen reactive to anti-GM-CSF antibodies.
4. The diagnostic kit according to claim 1, wherein the probe is
immobilized on a solid support.
5. The diagnostic kit according to claim 1, wherein the antigen
forms an antigen-antibody complex with the anti-GM-CSF
antibodies.
6. The diagnostic kit according to claim 1, wherein the detection
reactants comprise a reporter group conjugated to a binding
agent.
7. The diagnostic kit according to claim 1, wherein the binding
agent is selected from the group consisting of
anti-immunoglobulins, Protein G, Protein A and lectins.
8. The diagnostic kit according to claim 1, wherein the reporter
group is selected from the group consisting of radioisotopes,
fluorescent groups, luminescent groups, enzymes, biotin and dye
particles.
9. The diagnostic kit according to claim 1, wherein the probe is a
phage particle expressing an antigen specific for anti-GM-CSF
antibody.
10. A method for the diagnosis and prognosis of inflammatory bowel
diseases in a mammalian subject comprising the following steps: (a)
obtaining a biological sample from an individual suspected of
having an inflammatory bowel disease; (b) determining the
concentration of anti-GM-CSF antibodies in the sample; and (c)
correlating the concentration of anti-GM-CSF antibodies in the
sample to known standards.
11. The method according to claim 10, wherein the inflammatory
bowel disease is a pathology selected from the group consisting of
small bowel Crohn's disease (CD.sub.SB), Colonic Crohns disease
(CD.sub.C), and Ulcerative Colitis (UC).
12. The method according to claim 10, wherein the biological fluid
is serum.
13. The method according to claim 10, further comprising
correlating the concentration of anti-GM-CSF antibodies in the
sample to known standards to predict the severity of the
inflammatory bowel disease.
14. The method according to claim 10, further comprising
correlating the concentration of anti-GM-CSF antibodies in the
sample to known standards to predict whether the subject will
require surgery.
15. The method according to claim 10, further comprising
correlating the concentration of anti-GM-CSF antibodies in the
sample to known standards to select the appropriate therapeutic
treatment for the inflammatory bowel disease, wherein elevated
anti-GM-CSF antibodies indicate that the patient is a candidate for
therapies selected from anti-TNF.alpha. therapy, GM-CSF
administration, or combinations thereof.
16. The method according to claim 10, wherein correlating the
concentration of anti-GM-CSF antibodies in the sample to known
standards is used to distinguish between pathologies selected from
the group consisting of small bowel Crohn's disease (CD.sub.SB),
Colonic Crohns disease (CD.sub.C), and Ulcerative Colitis (UC).
17. The method according to claim 10, further comprising the step
of a determination of GM-CSF dependent up-regulation of cell
surface CD11b in cells of the sample.
18. The method according to claim 10, wherein determination of
GM-CSF dependent up-regulation of cell surface CD11b in cells of
the sample is used as a diagnostic assay to distinguish between UC
and CD patients.
19. The method according to claim 10, wherein the concentration of
anti-GM-CSF antibodies in the sample is determined using at least a
probe specific for GM-CSF wherein the probe is capable of detecting
a concentration of anti-GM-CSF antibodies, such that a diagnosis or
prognosis of the individual may be made.
20. The method according to claim 10, wherein the concentration of
anti-GM-CSF antibodies in the sample is determined by providing an
immobilized antigen reactive to anti-GM-CSF antibodies, contacting
the sample with the antigen to form an antigen-antibody complex,
washing the complex to remove non-specifically bound components,
followed by detecting the captured anti-GM-CSF antibodies.
21. A method for the diagnosis and prognosis of inflammatory bowel
diseases in a mammalian subject comprising the following steps: (a)
obtaining a biological sample from an individual suspected of
having an inflammatory bowel disease; (b) determining the
concentration of anti-GM-CSF antibodies in the sample relative to
neutrophil function, IBD phenotype, CARD15 variants or commercial
inflammatory bowel disease serology; (c) correlating the
concentration of anti-GM-CSF antibodies in the sample to known
standards and (d) diagnosing the individual based on these
results.
22. A method for the diagnosis of irritable bowel syndrome, the
method comprising: obtaining a blood or serum sample from a subject
presenting with symptoms common to inflammatory bowel disease and
irritable bowel syndrome; and determining whether the sample
contains an elevated level of anti-GM-CSF antibodies, wherein if
the sample contains an elevated level of anti-GM-CSF antibodies, a
diagnosis of inflammatory bowel disease is substantially
concluded.
23. The method according to claim 22, wherein the anti-GM-CSF
antibodies are qualitatively determined.
24. The method according to claim 22, wherein the step of
determining whether the sample contains an elevated level of
anti-GM-CSF antibodies includes contacting the sample with
immobilized polyclonal antibodies to human GM-CSF to create an
antibody bound sample.
25. A diagnostic assay for determining whether a blood or serum
sample contains an elevated level of anti-GM-CSF antibodies as
compared to a reference value for healthy control subjects, the
assay comprising: obtaining a human blood or serum sample from a
person presenting with symptoms common between inflammatory bowel
disease and irritable bowel syndrome; contacting the sample with
immobilized polyclonal antibodies to anti-GM-CSF antibodies to
create an antibody bound sample; contacting the treated sample with
enzyme-linked polyclonal antibodies such that the enzyme-linked
polyclonal antibodies are allowed to bind to capture human GM-CSF
to create an enzyme-linked antibody bound sample; and determining
whether the enzyme-linked antibody bound sample contains an
elevated level of anti-GM-CSF antibodies as compared to a reference
value for healthy control subjects, wherein if the enzyme-linked
antibody bound sample contains an elevated level of anti-GM-CSF
antibodies, a diagnosis of inflammatory bowel disease is
substantially concluded.
26. The diagnostic assay according to claim 22, wherein the assay
comprises an enzyme-linked immunoassay.
27. A method for the diagnosis and prognosis of inflammatory bowel
diseases in a mammalian subject comprising the following steps: (a)
obtaining a biological sample from an individual suspected of
having an inflammatory bowel disease; (b) determining the
concentration of a biological marker, the marker being a
cell-surface adhesion molecule present on myeloid cells or CD11b in
the sample; and (c) correlating the concentration of marker in the
sample to known standards.
28. The method according to claim 27, wherein the concentration of
a biological marker is determined by GM-CSF priming, which
increases cell-surface levels of CD11b on neutrophils contained in
the sample.
29. The method according to claim 27, wherein GM-CSF autoantibodies
block the GM-CSF-stimulated increase in cell surface CD11b
levels.
30. The method according to claim 27, wherein the addition of
exogenous GM-CSF to whole blood from the subject is used to measure
the ability to stimulate a change in neutrophil CD11b levels.
31. (b) placing the biological fluid in contact with at least
either: (i) a biological marker obtained from a mammalian cell, the
marker being a cell-surface adhesion molecule present on myeloid
cells or CD11b or (ii) an anti-marker antibody or antigen-binding
portion thereof specific for the CD11b.
32. A method for the diagnosis and prognosis of inflammatory bowel
diseases in a mammalian subject comprising the following steps: (a)
obtaining a biological sample from an individual suspected of
having an inflammatory bowel disease; (b) determining the
concentration of a biological marker, the marker being a
cell-surface adhesion molecule present on myeloid cells or CD11b in
the sample; and (c) correlating the concentration of marker in the
sample to known standards.
33. A method for the diagnosis and prognosis of inflammatory bowel
diseases in a mammalian subject comprising the following steps: (a)
obtaining a biological fluid from a subject suspected of having an
inflammatory bowel disease; (b) placing the biological fluid in
contact with at least either: (i) a marker obtained from an animal
cell, the marker being a cell-surface adhesion molecule present on
myeloid cells or CD11b or (ii) an anti-marker antibody or
antigen-binding portion thereof specific for in order to obtain
either a biological binding in vitro between the antibody present
in the biological fluid and the marker, or a competitive
immunological binding in vitro between the antibody present in the
biological fluid and the anti-marker antibody or the
antigen-binding portion thereof specific for the CD11b; (c)
detecting binding obtained; and (d) correlating the binding
antibodies in the sample to known standards.
34. The method according to claim 33, wherein the inflammatory
bowel disease is a pathology selected from the group consisting of
small bowel Crohn's disease (CD.sub.SB), Colonic Crohn's disease
(CD.sub.C), and Ulcerative Colitis (UC).
35. The method according to claim 33, wherein the biological fluid
is serum.
36. The method according to claim 33, further comprising
correlating the binding of the biomarker in the sample to known
standards to predict the severity of the inflammatory bowel
disease.
37. The method according to claim 33, further comprising
correlating the binding of the biomarker in the sample to known
standards to predict whether the subject will require surgery.
38. The method according to claim 33, further comprising the
binding of the biomarker in the sample to known standards to select
the appropriate therapeutic treatment for the inflammatory bowel
disease, wherein elevated binding of the biomarker indicates that
the patient is a candidate for therapies selected from
anti-TNF.alpha. therapy, GM-CSF administration, or combinations
thereof.
39. The method according to claim 33, wherein correlating the
binding of the biomarker in the sample to known standards is used
to distinguish between pathologies selected from the group
consisting of small bowel Crohn's disease (CD.sub.SB), Colonic
Crohns disease (CD.sub.C), and Ulcerative Colitis (UC).
40. The method according to claim 10, wherein if the subject
exhibits low levels of anti-GM-CSF antibody levels determined,
relative to patients with colonic CD and healthy controls, the
subject is diagnosed with Ulcerative Colitis (UC).
41. The method according to claim 10, wherein if the subject
exhibits high levels of anti-GM-CSF antibody levels determined,
relative to patients with colonic CD and healthy controls, the
subject is diagnosed with small bowel CD (CD.sub.SB).
42. The method according to claim 21, wherein the commercial IBD
serology includes antibodies selected from the group consisting of
ASCA, pANCA, CBir1, I2, OmpC or mixtures thereof.
43. The method according to claim 27, wherein the CD11b activity in
response to exogenous GM-CSF may be used as a biomarker to classify
subtypes of inflammatory bowel disease.
44. The method according to claim 43, wherein individuals having
reduced CD11b activity are more likely to have CD, and less likely
to have UC.
45. The method according to claim 27, wherein the CD11b activity on
monocytes is measured to distinguish between CD and UC
patients.
46. The method according to claim 27, wherein the CD11b activity on
neutrophils is determined to distinguish between CD and UC
patients.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Patent
Application Ser. No. 60/909,153, which was filed on Mar. 30, 2007,
the entirety of which is incorporated herein by reference for all
purposes.
BACKGROUND OF THE INVENTION
[0002] The incidence of the Inflammatory Bowel Diseases (IBD),
Crohn's Disease (CD) and Ulcerative Colitis (UC) has increased
dramatically over the past four decades with approximately five
million individuals affected in North America and Europe. While
therapeutic options have increased over the past decade, the
ability to classify subtypes of IBDs, predict disease progression
and behavior, and target newer biologic therapies to specific
subgroups of patients has lagged behind. This has led to an empiric
step-up approach to therapy, in which increasingly more potent
agents are offered until an effective regimen is identified.
[0003] The inflammatory bowel diseases are believed to be caused by
a complex interaction between genetic susceptibility and
environmental triggers leading to chronic relapsing intestinal
inflammation. (Bouma, G. 2003). CD is characterized by
discontinuous transmural inflammation which can involve any part of
the gastrointestinal (GI) tract, although the terminal ileum and
proximal colon are most commonly involved. (Hanauer S. B. 2006).
Conversely, UC is characterized by continuous superficial
inflammation limited to the colon, beginning in the rectum and
extending proximally (Hanauer S. B. 2006).
[0004] IBDs can be difficult to diagnose, a diagnosis being
obtained only after extensive, costly, and often invasive
procedures have been performed. An initial diagnosis, made on the
basis of medical history and physical examination, is generally
confirmed via imaging of the intestines and laboratory culture
tests to rule out bacterial, viral and parasitic infections.
Colonoscopy may also be used to image the intestines and colon,
such that the entire intestines can be evaluated to distinguish
between IBDs on the basis of the location of ulcerations. (Crohn's
disease affects some areas of the intestines and skips over others,
while ulcerative colitis is more indiscriminate.) Endoscopy may
also be used to biopsy the intestinal tissue, which can be used to
identify the deep inflammation of the bowel that is characteristic
of Crohn's disease. Further, X-rays (after oral or rectal ingestion
of Barium), computed tomography (CT) scan, and magnetic resonance
imaging (MRI) may be helpful in locating fistulas and assisting in
the diagnosis. A stool analysis (including a test for blood in the
stool) is often performed, depending on symptoms, to look for blood
and signs of bacterial infection. Blood and urine tests may be done
to check for anemia, high white cell counts, or malnutrition--all
signs of IBDs. Finally, the conditions may go undiagnosed for years
as symptoms usually develop gradually and less than all of the
intestines may be involved. As such, while there is currently no
reliable biochemical test available for IBD or to distinguish
subtypes of the disease, such a test would be highly valuable in
curtailing cost and the physical discomfort endured by individuals
in obtaining an IBD diagnosis.
[0005] With current diagnostic approaches, approximately 60% of IBD
patients are classified as CD, 30% as UC, and 10% as indeterminate
colitis (IC) (Kugathasan S. 2003). Patients with IC have intestinal
inflammation limited to the colon, without specific endoscopic or
histological features diagnostic of either CD or UC. A diagnosis of
IC limits the ability to predict clinical disease behavior and
outcome following surgery, and prevents patients with refractory
disease from entering into clinical trials of new agents.
[0006] IBD causes substantial morbidity including frequent
hospitalizations and surgeries, and longstanding disease is
complicated by cancer (Hanauer S. B. 2006). It is likely that there
are several immunogenetic sub-types of IBD, with CD and UC
representing the broadest clinical classifications (Dubinsky M. C.
2006). Current evidence suggests that CD results when mucosal
tolerance to the enteric flora is lost (Bouma G. 2003, Lodes M. J.
2004). CARD15/NOD2, an intra-cellular sensor for the bacterial
product muramyl dipeptide, is expressed in intestinal epithelial
cells (IEC) and macrophages and participates in anti-microbial
defenses (Bouma G. 2003, Kobayashi K. S. 2005). Loss of function
mutations in NOD2 increases susceptibility to CD primarily
involving the ileum (Tomer G. 2003).
[0007] A variety of defects in neutrophil function have also been
described in CD (Korzenik J. R. 2000). These primary defects in
mucosal innate immunity may then lead to activation of intestinal
antigen presenting cells (APG) and expansion of T.sub.eff reactive
to the host flora (Bouma G. 2003, Lodes M. J. 2004). The
immunogenetic basis for differences in ileal versus colonic
involvement in CD is not known, although mounting evidence suggests
that defects in innate immunity and loss of tolerance to bacterial
antigens are predominately associated with small bowel disease.
Mutations in CARD15 are associated with ileal disease and growth
failure in children, while seroreactivity to microorganism
components (ASCA, OmpC, CBir1, and I2 antibodies) is associated
with small bowel, stricturing disease (Tomer G. 2003), (Mow W. S.
2004). This does not appear to be the case for UC, in which defects
in epithelial repair and xenobiotic detoxification are implicated
(Dignass A. U. 2004). The introduction of biologic therapies
targeting specific components of the mucosal immune response has
improved outcomes for patients with IBD. However, sustained
remissions have not been observed in more than fifty percent of
individuals with any new biologic agent.
Current Therapies for Inflammatory Bowel Diseases
[0008] Current options for induction of remission in IBD include
mesalamine, corticosteroids, methotrexate, and infliximab. Options
for maintenance of remission include mesalamine, the
immunomodulators 6-mercaptopurine/azathioprine (6-MP/AZA),
methotrexate, and infliximab. The most common first line regimen
includes induction of remission with prednisone, and maintenance of
remission with 6-MP/AZA. In the absence of a reliable test to
predict response to therapy, patients are empirically offered
agents for induction and maintenance of remission largely based
upon disease severity and location. As the effectiveness of any one
agent is typically on the order of 50% to 80%, this leads to a
substantial number of patients receiving a series of ineffective
agents, with attendant side effects, before an effective regimen is
identified.
[0009] Despite the recent advent of the biologic therapies,
corticosteroids remain the predominant first line choice for
induction of remission in moderate to severe IBD. Typically, 50% of
patients will go into remission and then be able to wean
corticosteroids within three months. However, 20% will not respond,
and 30% will not be able to discontinue steroids; these groups are
termed steroid refractory (SR) and steroid dependent (SD),
respectively.
Inflammatory Bowel Disease Markers for Diagnosis and Progression Of
Disease
[0010] In recent years, serological markers which largely comprise
antibodies reactive to the host flora have been extensively studied
in an attempt to improve IBD classification and prediction of
disease progression. However, these markers have not proven
effective in definitively characterizing IBD patients as CD or UC,
and have not been able to predict with sufficient accuracy which
patients are most likely to require surgery for the condition.
[0011] The first serological markers to be described in IBD were
the anti-saccharomyces cervisiae antibody (ASCA) and
anti-neutrophil cytoplasmic antibody with perinuclear staining
(pANCA) (Austin G. L. 2006). Subsequently, additional markers
including antibodies to the outer membrane porin protein C (OmpC)
of E. Coli, to Pseudomonas fluorescens (I2) and to flagellin
(CBir1) have been described (Dubinsky M. C. 2006). Whether any of
these antibodies are pathogenic is not known, although recent
evidence points to bacterial flagellin as a critical antigen in CD
(Lodes M. J. 2004). pANCA has been associated with UC, while the
other markers have been associated with CD. A recent series of
well-characterized adult CD patients demonstrated a frequency of
37% for OmpC, 52% for I2, 39% for ASCA, and 14% for pANCA (Arnott
I. D. 2004). The frequency of pANCA in UC is typically 60% to 70%
(Austin G. L. 2006). Multiple studies have been performed to
determine whether levels of these antibodies can be used to screen
for IBD in patients with suggestive symptoms, and to discriminate
CD from UC, thereby reducing the number of cases of IC. However,
the sensitivity of pANCA for UC in recent adult and pediatric
series has ranged from 63% to 70%, while the sensitivity of ASCA
for CD ranged from 44% to 72% (Linskens R. K. 2002), (Zholudev A.
2004), (Gupta S. K. 2004). Thus, these results demonstrate that
these markers are not sufficiently sensitive to be used as
effective screening tools. In terms of specificity, these assays
perform substantially better, and range from 86% to 95% for pANCA
for UC and from 82% to 95% for ASCA for CD (Linskens R. K. 2002,
Zholudev A. 2004, Gupta S. K. 2004). From 10% to 18% of CD patients
will be positive for pANCA, these typically have Crohn's colitis
(Zholudev A. 2004). In cohorts of patients with a high prevalence
of IBD (42% to 68%), this has led to a reported positive predictive
value (PPV) of 90% to 96% for both CD and UC, and a negative
predictive value (NPV) of 50% to 80% (Austin G. L. 2006, Gupta S.
K. 2004). Therefore, as recently reviewed by Austin et al., these
tests perform best when used in a patient population with a high
pre-test probability of having IBD (Austin G. L. 2006). As such,
there is a need for improved markers having improved sensitivity
and selectivity that can be used as effective screening tools to
diagnose and differentiate inflammatory bowel diseases.
[0012] IBD patients further have variable risk of needing surgery
to treat the disease. Current standard clinical approaches are not
able to further reduce the number of diagnoses of IC, or to predict
which patients will be most likely to progress to surgery. From
about 30% to 50% of IBD patients will fail medical therapy and
progress to surgery within 3 to 10 years of diagnosis (Austin G. L.
2006). Most CD patients initially present with inflammatory disease
(80%), and over the next 10 years may progress to stricturing
and/or penetrating behavior, categorized as B2/B3-type disease.
(Hanauer S. B. 2006). Patients with stricturing/penetrating disease
then frequently require surgery.
[0013] Several groups have performed studies to determine whether
IBD serology can predict disease progression and the need for
surgery (Mow W. S. 2004). Consistently, high titer ASCA+ CD
patients have been more likely to have fibrostenosing small bowel
disease and require ileocecal resection (36% vs. 13% in one recent
pediatric series) (Arnott I. D. 2004). Conversely, pANCA+ CD
patients are more likely to have colonic disease and not require
resection. Recent reports demonstrated that the combined magnitude
of reactivity to microbial components (ASCA, OmpC, and I2)
predicted a phenotype of more severe small bowl CD with progression
to surgery in adult patients (Arnott I. D. 2004). However, the
magnitude of the serological response did not predict the need for
6-MP or infliximab. Importantly, the association was also
independent of the CARD15 genotype, which has also been associated
with small bowel CD and progression to surgery (Arnott I. D. 2004,
Walker L. K. 2004).
[0014] Recent reports in pediatrics have demonstrated very similar
predictive characteristics. High titer ASCA+ CD patients were more
likely to require surgery sooner than ASCA- patients (Amre D. K.
2006). In a study measuring ASCA, OmpC, I2, and CBir1 reactivity,
the presence and magnitude of response of these was associated with
a higher rate of progression to a stricturing or penetrating
complication over the first 18 months following diagnosis (Dubinsky
M. C. 2006). Potentially this sub-group of CD patients would
benefit from earlier institution of targeted biologic therapies
more likely to prevent disease progression. In this regard, Mow et
al have recently reported a trend towards improved responses to
antibiotics in CD patients sero-reactive to OmpC and/or I2, and
decreased responses to a steroid preparation, budesonide (Mow W. S.
2004).
[0015] A limitation of this approach is the substantial number of
patients who experience disease progression and require surgery who
are sero-negative for these markers. For example, in the Arnott
report, 96 CD patients were negative for ASCA or OmpC/I12, and
fifty percent of those experienced disease progression and required
surgery (Arnott I. D. 2004). This compared to 40 CD patients who
were sero-positive, with rates of disease progression and surgery
of 90% and 75%, respectively (Arnott I. D. 2004). Therefore,
additional approaches are needed to complement current serological
assays in defining clinically important sub-types of IBD with
respect to disease behavior and progression.
[0016] Current biomarkers have failed to be effectively used to
provide diagnostic tests for classifying, characterizing or
predicting the outcome of inflammatory bowel diseases. In
particular, current biomarkers have not been efficacious in
distinguishing among the various subtypes of inflammatory bowel
diseases. Accordingly, there is a need for biomarkers that can form
the basis for diagnostic tools that can effectively classify,
characterize and predict the severity of inflammatory bowel
disease
[0017] There is further the need for biomarkers that can be used to
predict disease progression, including the likelihood that an
individual will require surgery despite treatment with first line
therapies would be invaluable. Such markers would allow the
clinician to identify the subset of patients at an early stage of
disease progression who would benefit from earlier introduction of
second or third line therapies, or therapies particularly effective
in treatment of a particular subset of IBD, such as anti-TNF.alpha.
(for example, Remicade or Humira) and/or GM-CSF (Leukine) or the
like. The instant invention satisfies these needs and provides
related advantages as well.
BRIEF SUMMARY OF THE INVENTION
[0018] The present invention relates to biological markers and
methods of using biological markers for the diagnosis and prognosis
of inflammatory bowel diseases.
[0019] The present invention further relates to biological markers
that may be used to predict and characterize disease behavior in
individuals having an inflammatory bowel disease.
[0020] The present invention further relates to biological markers
that may be used to predict disease progression, such as the
likelihood that an individual having an inflammatory bowel disease,
particularly Crohn's disease, will require surgery.
[0021] The present invention further relates to a method of
classifying an individual with a particular subtype of inflammatory
bowel disease, comprising the steps of obtaining a biological
sample from an individual suspected of having an inflammatory bowel
disease; determining the concentration of anti-GM-CSF antibodies in
the sample; and correlating the concentration of anti-GM-CSF
antibodies in the sample to known standards.
[0022] In yet another aspect, the instant invention provides a
method of diagnosing an individual with a particular subtype of
inflammatory bowel disease, comprising the steps of obtaining a
biological sample from an individual suspected of having an
inflammatory bowel disease, determining the concentration of
anti-GM-C SF relative to neutrophil function, IBD phenotype, CARD15
variants or commercial inflammatory bowel disease serology;
correlating the results to known standards; and diagnosing the
individual based on these results.
[0023] In yet a further aspect, the present invention relates to a
method for predicting the severity of an inflammatory bowel disease
in an individual, particularly whether the individual will require
surgery, comprising the steps of obtaining a biological sample from
the individual suspected of having or having an inflammatory bowel
disease; determining the concentration of anti-GM-CSF antibodies in
the sample; and correlating the concentration of anti-GM-C SF
antibodies in the sample to known standards to predict the severity
of the inflammatory bowel disease, particularly whether an
individual will require surgery. In this aspect of the invention,
the method may be used to identify the appropriate therapy for an
individual based on the determination of elevated anti-GM-CSF
antibodies.
[0024] In yet another aspect, the present invention relates to a
method for predicting the appropriate therapeutic treatment for an
inflammatory bowel disease, comprising the steps of obtaining a
biological sample from an individual suspected of having an
inflammatory bowel disease; determining the concentration of
anti-GM-CSF antibodies in the sample; and correlating the
concentration of anti-GM-CSF antibodies in the sample to known
standards to select the appropriate therapeutic treatment for the
inflammatory bowel disease, wherein elevated anti-GM-CSF antibodies
indicate that the patient is a candidate for therapies selected
from anti-TNF.alpha. therapy, GM-CSF administration, or
combinations thereof.
[0025] In another aspect of the present invention, determination of
GM-CSF dependent up-regulation of cell surface CD11b may be used as
a diagnostic assay to distinguish between UC and CD patients.
[0026] The present invention further relates to kits embodying the
above described methods, such as a kit for determining a diagnosis
or a prognosis of an individual suspected of having or having an
inflammatory bowel disease, which comprises at least a probe
specific for GM-CSF wherein the probe is capable of detecting a
concentration of anti-GM-CSF antibodies, such that a diagnosis or
prognosis of the individual may be made.
[0027] The present invention further relates to immunoassays for
the qualitative or quantitative determination, in a sample from an
individual, of anti-GM-CSF antibodies wherein the immunoassay
comprises the steps of obtaining a sample from an individual
suspected of having or having an inflammatory bowel disease,
providing an immobilized antigen reactive to anti-GM-CSF
antibodies, contacting the sample with the antigen to form an
antigen-antibody complex, washing the complex to remove
non-specifically bound components, followed by detecting the
captured anti-GM-CSF antibodies.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] FIG. 1. Anti-GM-CSF Antibody Concentration and IBD
Phenotype
[0029] FIG. 2. Function of anti GM-CSF antibodies and Neutrophil
Phagocytosis in Healthy Individuals and Individuals with
Inflammatory Bowel Disease and PAP.
[0030] FIG. 3. CM-CSF activation of CD11b in Inflammatory Bowel
Disease.
DETAILED DESCRIPTION OF THE INVENTION
[0031] Current care and clinical trials for patients with IBD is
hampered by a lack of biomarkers to predict and monitor response to
therapy or progression of disease. Without biomarkers that allow
prediction of a patient's responsiveness to current therapies,
patients are offered therapies via an empiric approach with results
in, at best, a 50% rate of sustained remission, while subjecting
many patients to unnecessary side effects and expense, further
delaying recovery and improved quality of life. As such,
identification of biomarkers offers the opportunity to define
distinct immunogenetic sub-types of disease which may benefit from
specific treatment approaches.
[0032] For example, the availability of sensitive biomarker would
permit the treating physician to evaluate a patient's likely
responsiveness to first line or second line therapeutics. The
prediction of the patient's likely response permits the physician
to select therapies likely to be most efficacious for a given
individual, avoiding treatment with less useful therapies. This, in
turn, avoids subjecting the patient to unnecessary side effects and
expense, while improving the patient's quality of life and creating
opportunities to delay or prevent disease progression.
DEFINITIONS
[0033] Unless defined otherwise, technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs.
Singleton et al., Dictionary of Microbiology and Molecular Biology
2nd ed., J. Wiley & Sons (New York, N.Y. 1994), provide one
skilled in the art with a general guide to many of the terms used
in the present application.
[0034] For purposes of the present invention, the following terms
are defined below.
[0035] The articles "a" and "an" are used herein to refer to one or
to more than one (i.e., to at least one) of the grammatical object
of the article. By way of example, "an element" means one element
or more than one element.
[0036] A "biological sample" encompasses any sample obtained from a
living system or subject. The definition encompasses blood, serum,
tissue, and other samples of biological origin that can be
collected from a living system, subject or individual. In one
embodiment, biological samples are obtained through sampling by
minimally invasive or non-invasive approaches (e.g., urine
collection, stool collection, blood drawing, needle aspiration, and
other procedures involving minimal risk, discomfort or effort).
Biological samples can be gaseous (e.g., exhaled breath).
Biological samples are often liquid (sometimes referred to as a
"biological fluid"). Liquid biological samples include, but are not
limited to, urine, blood, interstitial fluid, edema fluid, saliva,
lacrimal fluid, inflammatory exudates, synovial fluid, abscess,
empyema or other infected fluid, cerebrospinal fluid, sweat,
pulmonary secretions (sputum), seminal fluid, feces, bile,
intestinal secretions, and others. Biological samples include
samples that have been manipulated in any way after their
procurement, such as by treatment with reagents, solubilization, or
enrichment for certain components, such as proteins or
polynucleotides. The term "biological sample" also encompasses a
clinical sample such as serum, plasma, other biological fluid, or
tissue samples, and also includes cells in culture, cell
supernatants and cell lysates.
[0037] As used herein, the term "biomarker" refers to a physical,
biochemical, or physiologic measurement from or on the organism
that represents a true or intended mechanistic target of a compound
or a mechanistic event believed to be responsible for, or
contributing in, a causal manner to the initiation, progression,
severity, pathology, aggressiveness, grade, activity, disability,
mortality, morbidity, disease sub-classification or other
underlying pathogenic or pathologic feature of one or more
diseases. A biomarker may be the target for monitoring the outcome
of a therapeutic intervention (i.e., the functional or structural
target of a drug agent). "Biomarker" refers to biochemical
processes that are involved in, or are believed to be involved in,
the etiology or progression of a disease or disorder. The
biochemical process (i.e., the flow of molecules through a targeted
metabolic pathway or network) is the focus of analysis (as
disclosed herein) since it is the underlying changes of the
biochemical process (i.e., molecular flux rates) that may be the
significant or authentic target for treatment or diagnostic
monitoring of the disease or disorder.
[0038] The terms "drug," "pharmaceutically active agent,"
"bioactive agent," "therapeutic agent," and "active agent" may be
used interchangeably and refer to a substance, such as a chemical
compound or complex, that has a measurable beneficial physiological
effect on the body, such as a therapeutic effect in treatment of a
disease or disorder, when administered in an effective amount.
Further, when these terms are used, or when a particular active
agent is specifically identified by name or category, it is
understood that such recitation is intended to include the active
agent per se, as well as pharmaceutically acceptable,
pharmacologically active derivatives thereof, or compounds
significantly related thereto, including without limitation, salts,
pharmaceutically acceptable salts, N-oxides, prodrugs, active
metabolites, isomers, fragments, analogs, solvates hydrates,
radioisotopes, etc.
[0039] The phrase "effective amount" refers to that amount of a
substance that produces some desired local or systemic effect at a
reasonable benefit/risk ratio applicable to any treatment. The
effective amount of such substance will vary depending upon the
individual and disease condition being treated, the weight and age
of the individual, the severity of the disease condition, the
manner of administration and the like, which can readily be
determined by one of ordinary skill in the art.
[0040] An "individual" is a vertebrate, preferably a mammal, more
preferably a human.
[0041] The term "prophylactic" or "therapeutic" treatment is
art-recognized and refers to administration to the host of one or
more of the subject compositions. If it is administered prior to
clinical manifestation of the unwanted condition (e.g., disease or
other unwanted state of the host animal) then the treatment is
prophylactic, i.e., it protects the host against developing the
unwanted condition, whereas if administered after manifestation of
the unwanted condition, the treatment is therapeutic (i.e., it is
intended to diminish, ameliorate or maintain the existing unwanted
condition or side effects therefrom).
[0042] The phrase "therapeutic effect" is art-recognized and refers
to a local or systemic effect in animals, particularly mammals, and
more particularly humans caused by a pharmacologically active
substance. The term thus means any substance intended for use in
the diagnosis, cure, mitigation, treatment or prevention of disease
or in the enhancement of desirable physical or mental development
and/or conditions in an animal or human. The phrase
"therapeutically-effective amount" means that amount of such a
substance that produces some desired local or systemic effect at a
reasonable benefit/risk ratio applicable to any treatment. The
therapeutically effective amount of such substance will vary
depending upon the individual and disease condition being treated,
the weight and age of the individual, the severity of the disease
condition, the manner of administration and the like, which can
readily be determined by one of ordinary skill in the art.
[0043] The term "treatment" refers to both therapeutic treatment
and prophylactic or preventative measures, wherein the object is to
prevent or slow down (lessen) the targeted pathologic condition or
disorder. Those in need of treatment include those already with the
disorder as well as those prone to have the disorder or those in
whom the disorder is to be prevented.
[0044] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of molecular biology
(including recombinant techniques), microbiology, cell biology and
biochemistry, which are within the skill of the art.
[0045] All references cited herein are incorporated in their
entirety by reference.
[0046] The present invention relates to biomarkers that may be used
to characterize individuals having an inflammatory bowel disease.
In one aspect, the present invention provides novel biomarkers for
inflammatory bowel disease that may be used to diagnose subtypes of
the disease. In particular, the biomarkers as disclosed herein may
be used to characterize inflammatory bowel disease behavior and
progression. In yet another aspect, the invention provides novel
biomarkers that may be used to predict the likelihood that an
individual diagnosed with an inflammatory bowel disease will
require surgical intervention.
[0047] Further, the biomarkers and methods disclosed herein may be
used in the targeted care of individuals having inflammatory bowel
disease. In this aspect of the invention, Crohn's disease patients
having a high anti-GM-CSF level are identified as high risk for
disease progression and surgery, and may be offered second or third
line therapies earlier in their treatment. For example, Crohn's
disease patients having elevated anti-GM-CSF antibodies would be
candidates for earlier treatment with anti-TNF.alpha. therapy
(Remicade or Humira), GM-CSF therapy (Leukine) or similar
therapies.
[0048] A variety of defects in neutrophil function have been
described in CD, without a clear etiology (Korzenik J. R. 2000).
These have included reduced phagocytosis, adhesion, chemotaxis, and
oxidative burst. Functionally, this has been shown to paradoxically
reduce neutrophil accumulation at sites of acute ileal or rectal
injury (Harbord M. W. 2006, Mow W. S. 2004). This may in turn
promote accumulation of bacterial products and stimulation of the
mucosal adaptive immune system. Granulocyte-macrophage
colony-stimulating factor (GM-CSF) is a protein secreted by
macrophages that functions as a white blood cell growth factor.
GM-CSF and its Role in the Regulation of Myeloid Cell
Functions.
[0049] GM-CSF is a 23 kD, homodimeric cytokine expressed present in
serum and tissues with pleiotropic regulatory effects on the
functions of myeloid cells (neutrophils, monocytes, eosinophils,
tissue macrophages and dendritic cells). The biological effects of
GM-CSF are mediated by cell-surface receptors, comprised of .alpha.
(CDw116) and .beta. chains (CD131) and, in macrophages and
neutrophils include increased phagocytosis, cell-surface levels of
CD11b (an adhesion molecule), CD11b, cell adhesion, bacterial
killing, surfactant catabolism (in alveolar macrophages) (Trapnell,
B. et al., NEJM, 2003, incorporated herein by reference). GM-CSF
also primes phagocytosis, oxidative burst, and bactericidal
activity in neutrophils. In vitro stimulation with GM-CSF restores
the function of CD neutrophils, and GM-CSF administration reduces
mucosal injury in murine colitis due to trinitrobenzenesulfonic
acid (TNBS) or dextran sodium sulfate (DSS) administration (Hanna
E. 2006), (Fiorucci S. 2006) (Biagioni C. 2006).
[0050] While it has been known for some time that GM-CSF could
modulate the functions of myeloid cells, a critical role for GM-CSF
in myeloid cell function was discovered by the serendipitous
observation that mice deficient in GM-CSF due to gene ablation
(GM-CSF knockout mice) develop a lung phenotype that is
histologically, biochemically, ultrastructurally and
physiologically similar to the human disease known as pulmonary
alveolar proteinosis (PAP) (Trapnell, NEJM, 2003). PAP in mice is
caused by abrogation of the effects of GM-CSF signaling on myeloid
cell functions, due to ablation of the genes encoding either GM-CSF
or its receptor. Either genetic manipulation results in a reduction
in the ability of alveolar macrophages to degrade or catabolize
surfactant. (Shibata, et al., Immunity, 2001, incorporated herein
by reference.) Abrogation of GM-CSF signaling also increases the
risk of infection due to loss of the stimulatory effects of GM-CSF
on neutrophils (Uchida, et al, NEJM, 2007). The stimulatory effects
of GM-CSF on macrophages and neutrophils are known as `priming`,
and are known to augment the antimicrobial functions of these
cells, and thus, also augments innate immune host defenses.
GM-CSF Autoantibodies and their Role in Myeloid Cell Functions.
[0051] Human primary PAP is a rare disorder in which surfactant
within pulmonary alveoli accumulates, thereby causing respiratory
insufficiency (Trapnell et al, NEJM, 2007). Primary PAP is
specifically associated with high levels of autoantibodies against
granulocyte/macrophage-colony stimulating factor (GM-CSF) that
neutralize the biological activity of GM-CSF and are present in
vast excess of GM-CSF in vivo. Based on studies in mice, it is
believed that pulmonary GM-CSF stimulates the terminal
differentiation of alveolar macrophages including many of the
effects of GM-CSF on alveolar macrophages. A recent study has now
established that GMCSF autoantibodies abrogate the effects of
GM-CSF on neutrophils in patients with primary PAP (Uchida et al,
NEJM, 2007, incorporated herein by reference). Loss of the effects
of GM-CSF on neutrophil function caused by GM-CSF autoantibodies
provides a mechanistic explanation for the morbidity and mortality
from infection in patients with primary PAP.
[0052] The molecular basis for the defects in neutrophil function
which have been described in CD, but not UC, is not known. However,
GM-CSF and GM-CSF receptor levels are typically elevated in
patients with active CD, suggesting the role of a serum inhibitory
factor. Recent reports demonstrating that GM-CSF can correct CD
neutrophil function in vitro, and that GM-CSF administration can
relieve symptoms and reduce intestinal inflammation in a sub-set of
CD patients, have also pointed to a potential role for alterations
in GM-CSF bioactivity (Biagioni C. 2006, Korzenik J. R. 2005). It
has also been determined that very high levels of anti-GM-CSF
auto-antibodies impair neutrophil function and cause the rare lung
disease, PAP (Uchida K., et al., Blood 2004, incorporated herein by
reference). Neutralizing anti-GM-CSF auto-antibodies have been
characterized in patients with the rare lung disease Pulmonary
Alveolar Proteinosis (PAP). (Uchida K. 2004). Higher levels of
anti-GM-CSF inhibit myeloid cell function in patients with the rare
lung disease PAP. It has been reported that anti-GM-CSF
auto-antibodies suppress systemic innate immunity, by preventing
GM-CSF priming of monocyte and neutrophil anti-microbial functions.
(Vindi I. 2006). These auto-antibodies have been linked to defects
in macrophage and neutrophil function and development of disease
(Uchida K. 2004).
[0053] GM-CSF is currently undergoing phase II clinical trials in
CD, with clinical responses reported in approximately fifty percent
of patients (Korzenik J. R. 2005). However, a pro-inflammatory role
for GM-CSF has also been invoked for autoimmune diseases including
rheumatoid arthritis, and neutralizing anti-GM-CSF antibodies are
also in development as potential therapeutic agents (Krinner E. M.
2006). Moreover, most patients with active CD or UC have increased
production of GM-CSF in the inflamed intestine, and increased local
expression of the GM-CSF receptors. Endogenous anti-GM-CSF
antibodies may regulate local GM-CSF bioactivity. It thus is
critically important to define the sub-groups of IBD patients who
will benefit from therapeutic modulation of GM-CSF
bio-activity.
CD11b as a Biomarker of GM-CSF Bioactivity In Vivo.
[0054] CD11b is a cell-surface adhesion molecule present on myeloid
cells, the concentration of which increases in response to a wide
variety of inflammatory stimuli. GM-CSF priming increases
cell-surface levels of CD11b on neutrophils, which promotes their
adhesion to vascular endothelium, a critical event in recruitment
of neutrophils into infected tissues. GM-CSF autoantibodies block
the GM-CSF-stimulated increase in cell surface CD11b levels in PAP
patients (Uchida et al, NEJM, 2007). Addition of exogenous GM-CSF
to whole blood from a patient can be used to measure the ability to
stimulate this change in neutrophil CD11b levels and is the basis
of an assay known as the CD11b stimulation index (Uchida et al,
NEJM, 2007, incorporated herein by reference).
[0055] The present invention provides novel kits and methods for
the classification, characterization, and prediction of outcome in
patients having inflammatory bowel diseases. The present invention
further relates to kits and methods that may be used to determine
the prognosis of an individual having an inflammatory bowel
disease, or to determine the optimal choice of treatment for such
individuals.
Anti-GM-CSF Antibody Concentration and IBD Phenotype
[0056] In one aspect of the invention, levels of anti-GM-CSF
antibody can be measured and used to classify individuals having an
inflammatory bowel disease. It has been found that IBD patients
exhibit a range of anti-GM-CSF antibody levels, with patients with
small bowel CD (CD.sub.SB) exhibiting high levels, and patients
with UC exhibiting low levels, relative to patients with colonic CD
and healthy controls. The elevated anti-GM-CSF levels in patients
with CD.sub.SB are in the range which inhibits neutrophil function.
Serum levels of anti-GM-CSF independently regulate neutrophil
function and IBD phenotype, relative to CARD15 genotype and current
IBD serological markers. Comparing patients with ileal or
ileo-colonic disease grouped together as small bowel CD
(CD.sub.SB), and patients with colonic CD grouped as CD.sub.C, we
have found for the first time that CD.sub.SB patients have
increased circulating levels of anti-GM-CSF antibodies, while UC
patients have decreased levels of anti-GM-CSF antibodies, relative
to CD.sub.C or healthy controls.
[0057] FIG. 1 depicts the relative anti-GM-CSF antibody
concentrations in the various IBD subgroups compared to healthy
control patients. Serum anti-GM-CSF antibody concentrations were
determined by ELISA in IBD sub-groups and healthy controls (NL).
FIG. 1 depicts anti-GM-CSF antibody concentration in patients with
small bowel Crohn's disease (CD.sub.SB), patients with Colonic
Crohns disease (CD.sub.C), and Ulcerative Colitis (UC). The
differences between groups were compared by Kruskal-Wallis with
post hoc Bonferroni multiple comparisons test. p<0.0001 vs. NL
& UC; *p=0.006 vs. CD.sub.C. The mean value was as indicated
for each group.
[0058] It was therefore surprisingly discovered that inflammatory
bowel disease patients having small bowel Crohn's disease
(CD.sub.SB) had statistically significant increases in anti-GM-CSF
antibody concentration as compared to individuals with colonic
Crohn's disease, individuals with ulcerative colitis, and normal
individuals. As such, determination of anti-GM-CSF antibody
concentration can be used to distinguish subclasses of patients
diagnosed with Crohn's disease, providing improved selection of
therapeutic treatments and prediction of disease behavior and
progression.
[0059] In fact, mean (.+-.SE) serum anti-GM-CSF concentration in
ileal CD.sub.SB and ileo-colonic CD.sub.SB individuals was equal to
2.2 (n=20, and 86, respectively), versus 0.7 mcg/ml (n=51) in
CD.sub.C, 0.7 mcg/ml (n=43) in UC, and 0.4 mcg/ml (n=20) in healthy
controls. See Table 1.
[0060] Thus, in one aspect of the present invention, a method of
classifying individuals having an inflammatory bowel disease is
provided comprising the steps of obtaining a sample from the
individual, determining the concentration of serum anti-GM-CSF
concentration in the sample, a mean (.+-.SE) serum anti-GM-CSF
concentration of greater than or equal to about 1 mcg/ml, or
greater than or equal to about 1.5 mcg/ml, or greater than or equal
to about 2.0 mcg/ml, or within the range of about 1.0 mcg/ml to 2.5
mcg/ml, indicates that the individual is likely to have ileal or
ileocolonic Crohn's disease.
[0061] Importantly, anti-GM-CSF levels did not change in CD
patients who had achieved clinical remission with anti-TNF therapy
(2.4.+-.0.8 mcg/ml vs 3.3.+-.1.3 mcg/ml, n=6), indicating that
these are a stable feature of their disease, and do not simply
reflect a response to mucosal inflammation.
[0062] The instant invention further relates to a method of
modulating IBD phenotype or treating individuals having an IBD
using exogenously or endogenously administered GM-CSF.
[0063] As shown in FIG. 2, anti-GM-CSF antibodies in the range
observed in CD.sub.SB can regulate (i.e., neutralize) GM-CSF
bioactivity and neutrophil function, thereby creating a functional
GM-CSF deficiency in the individual.
[0064] We assessed the potential significance of this finding by
quantifying neutrophil phagocytosis with a sensitive assay. FIG. 2
depicts the results of this study. FIG. 2a depicts neutrophil
phagocytosis measured in whole blood. Each dot represents four
determinations per individual. .alpha.GM-CSF-antibody levels were
done in triplicate for each individual. Results demonstrated that
neutrophil phagocytic capacity correlated inversely with
.alpha.GM-CSF antibody levels, over the range observed in IBD
patients (n=17; R.sup.2=0.51; FIG. 2a). We have found that
neutrophil phagocytosis in whole blood is significantly reduced in
Crohn's Disease patients, relative to healthy controls (see FIG.
2b). This reduction was intermediate between healthy controls and
that observed in PAP patients, and suggests that the elevated
anti-GM-CSF antibodies observed in the Crohn's Disease patients do
have a significant effect upon neutrophil phagocytosis.
[0065] We then asked whether GM-CSF bioactivity, as measured by
CD11b activation on neutrophils in whole blood, would also be
reduced in Crohn's Disease patients. As shown in FIG. 3, we have
found that GM-CSF dependent CD11b activation on neutrophils is
significantly reduced in Crohn's Disease patients, relative to
healthy controls.
[0066] Taken together, these studies have identified anti-GM-CSF as
a novel regulator of neutrophil function and IBD phenotype. Further
these studies establish the predictive value of the serum
anti-GM-CSF antibody with regard to IBD phenotype. The risk
associated with the serum anti-GM-CSF antibody level with regard to
IBD phenotype and behavior is at least comparable to that
associated with CARD15 SNP carriage, suggesting a functional
interaction. More importantly, as both GM-CSF administration and
GM-CSF neutralization are in development as therapies for
inflammatory bowel diseases and other autoimmune disorders, this
raises the possibility that determination of the anti-GM-CSF level
in an individual patient may guide biologic therapy, in terms of
whether augmentation or inhibition of GM-CSF bioactivity would be
beneficial.
[0067] CD11b activity in response to exogenous GM-CSF may be used
as a biomarker to classify subtypes of inflammatory bowel disease,
wherein individuals having reduced CD11b activity are more likely
to have CD, and less likely to have UC. Thus, in this aspect of the
present invention, determination of CD11b activity, particularly in
response to GM-CSF, may be used as a diagnostic assay to classify
subtypes of inflammatory bowel disease, particularly ulcerative
colitis and Crohn's disease. In one aspect, CD11b activity on
monocytes is measured to distinguish between CD and UC patients. In
another aspect, CD11b activity on neutrophils is determined to
distinguish between CD and UC patients. Such classification of IBD
patients permits targeted therapy and prediction of disease
characteristics, behavior and likely outcome.
[0068] In yet another aspect, the instant invention provides a
method of performing an overall risk assessment, comprising the
steps of obtaining a serum sample from a individual suspected of
having an IBD, determining the concentration of anti-GM-CSF
relative to neutrophil function, IBD phenotype, CARD15 variants or
commercial IBD serology to obtain a ratio, then using the
determined ratio to determine whether the individual has Crohn's
disease or whether an individual having an IBD has a high
likelihood of disease progression and surgery. The commercial IBD
serology includes, for example, ASCA, pANCA, CBir1, I2, or OmpC
antibodies. The disclosure of U.S. Pat. No. 6,218,129 Walsh et al.,
U.S. Pat. No. 6,183,951, Plevy et al., and U.S. Pat. No. 5,932,429
Targan et al., describing methods of diagnosing IBD are all
incorporated herein by reference.
[0069] In another aspect of the instant invention, the novel
biomarkers and methods disclosed herein may be used to predict or
determine inflammatory bowel disease location and behavior via
measurement or detection of anti-GM-CSF antibody concentrations
with or without the presence of the CARD15 single nucleotide
polymorphism.
[0070] Table 1 depicts data representing the interactions between
the CARD15 Crohn's Disease (CD) susceptibility gene and anti-GM-CSF
antibody with respect to disease location and behavior. In Table 1,
the demographic characteristics and CD location for a cohort of
pediatric onset IBD patients are shown, together with the frequency
of CARD15 SNP carriage, stricturing or penetrating behavior
(B2/B3), and surgery. Serum anti-GM-CSF (mcg/ml) is given as the
median (25th, 75th percentile), a, b, c, d, e=p<0.05 by
Kruskal-Wallis test with Dunn's multiple comparison test versus the
indicated group. The patients have been followed for a median
(25th, 75th percentile) of 4 (2, 6) years from diagnosis. The
diagnosis of CD or UC is made based upon established endoscopic,
histological, and radiographic criteria (Hanauer S. B. 2006). In
this system, ileal involvement is classified as L1, colonic as L2,
and ileo-colonic as L3. CD phenotype is assigned as per the
Montreal classification, with patients with ileal or ileo-colonic
disease grouped together as small bowel CD (CD.sub.SB), and
patients with colonic CD grouped as CD.sub.C (Satsangi J.
2006).
[0071] The individuals have been genotyped for the three
predominant CARD15 CD susceptibility SNPs (R702w, G908R and
1007fs). Carriage of at least one SNP has been recorded as positive
with respect to the current analysis. Serum anti-GM-CSF
concentrations may be determined using an ELISA as described in the
Examples herein. Genotyping for the NOD2/CARD15 variants associated
with CD, R702W (SNP8), G908R (SNP12), and 1007fs (SNP13) is
performed using the protocol published in Tomer G. 2003,
incorporated herein by reference. Titers of the ASCA, OmpC, 12, and
CBir1 antibodies which are associated with small bowel CD, and
pANCA, which is associated with UC and colonic CD, may be
determined by a commercial laboratory (Prometheus Laboratories, San
Diego, Calif.).
[0072] In our studies, 40% to 45% of the CD individuals with ileal
or ileocolonic disease exhibited CARD15 SNP carriage and
stricturing or penetrating disease behavior over time (B2/B3), with
30% requiring at least one surgery. Consistent with our prior
results, we have confirmed that serum anti-GM-CSF is up regulated
in CD patients with ileal or ileo-colonic involvement, relative to
patients with Crohn's colitis, UC, or healthy controls.
TABLE-US-00001 TABLE 1 Anti-GM-CSF and Disease Location and
Behavior CARD15 Anti-GM-CSF Location n Age Male SNP (mcg/ml) B2/B3
Surgery L1: Ileal, 20 13 75% 45% 2.2 (0.9, 6.7) 45% 30% a d, e L3:
Ileo-colonic, 86 12 52% 47% 2.2 (0.7, 9.4) 37% 33% b c, d, e L2:
Colonic, 51 11 61% 16% 0.7 (0.4, 1.6) 14% 16% c b UC, d 43 12 60%
16% 0.7 (0.2, 1.3) N/A 16% a, b Control, e 20 13 70% Not done 0.4
(0.2, 0.6) N/A N/A a, b
[0073] Referring to Table 1, the mean (95% CI) anti-GM-CSF level
was equal to 10 (4, 16) mcg/ml in CD patients carrying at least one
CARD15 SNP compared to 9 (3, 15) mcg/ml in those without a CARD15
SNP. Similarly, 47% of patients with elevated anti-GM-CSF
(.gtoreq.2 mcg/ml) carried a CARD15 SNP, while 53% did not. This
suggested that increased anti-GM-CSF was not simply a function of
CARD15 SNP carriage.
[0074] The relative risk (RR) for ileal or ileo-colonic involvement
(CD.sub.SB) in a CD patient carrying a CARD15 susceptibility SNP
was equal to 5.2, while the RR for a CD patient with an anti-GM-CSF
level .gtoreq.2 mcg/ml was equal to 4, and the RR for a CD patient
with both an anti-GM-CSF level .gtoreq.2 mcg/ml and CARD15 SNP was
equal to 10.2 (p<0.001 for each). Therefore, elevated
anti-GM-CSF levels appear to interact with CARD15 SNPs to increase
susceptibility for CD.sub.SB. Importantly, anti-GM-CSF levels did
not change over one year in patients who had achieved clinical
remission with anti-TNF therapy (2.4.+-.0.8 mcg/ml vs 3.3.+-.1.3
mcg/ml, n=6), indicating that these are a stable feature of their
disease, and do not simply reflect a response to mucosal
inflammation.
[0075] The chi-square test of independence has found a
significantly different prevalence of L1/L3 (vs. L2), CARD15
mutation, and high anti-GM-CSF (.gtoreq.2 mcg/ml) in patients with
CD compared to patients with UC (p-values <0.0001, 0.0155,
0.0032 respectively). Hence, we have investigated the association
of anti-GM-CSF with the disease location, behavior and surgery
while controlling for CARD15 mutation, separately in patients with
CD and in patients with UC, the results of which are shown in Table
1. The median anti-GM-CSF level was increased two-fold in CD
patients with penetrating or stricturing behavior (B2/B3), compared
to those with inflammatory behavior (B1). After stratifying for
individuals with ileal or ileo-colonic CD, the median anti-GM-CSF
level was equal to 1.8 (0.7, 7.7) in those who did not require
surgery, versus 6.6 (0.7, 10) in those who did require surgery
(p=0.04). Consistent with this, the odds ratio (OR) for surgery in
CD patients with elevated anti-GM-CSF was equal to 3.2
(p<0.01).
[0076] Using the Cochran-Mantel-Haenszel (CMH) test controlling for
CARD15 SNP carriage, it was found that anti-GM-CSF level (high vs.
low) is significantly associated with the disease location
distribution (L2 vs. L1/L3), the disease behavior distribution (B1
vs. B2/B3) and surgery status (p-values=0.0005, 0.0076, 0.0038
respectively). More specifically, for a CD patient with a high
anti-GM-CSF level (.ltoreq.2 mcg/ml), the disease is less likely to
be only colonic (L2) (Relative Risk=0 with CARD15 mutation, 0.63
with no CARD15 mutation), less likely to be non-stricturing,
non-penetrating B1 (Relative Risk=0.7366), and more likely treated
with a surgery (Relative Risk=2.155), independent of the
presence/absence of CARD 15 mutation. The mean elapsed time since
diagnosis is not significantly different in the patients with high
anti-GM-CSF level than in the patients with a low anti-GM-CSF
level. Therefore, these results indicate a significant effect of
anti-GM-CSF level, independent of CARD15 mutation effect.
[0077] In contrast, using the CMH test anti-GM-CSF level (high vs.
low) controlling for CARD 15 SNP status, is not significantly
associated with the surgery status in patients with UC.
[0078] As such, one aspect of the present invention provides kits
and methods of classifying IBD patients by determining the
concentration of anti-GM-CSF antibody in blood or serum samples
from individuals suspected of having or diagnosed with IBD. In
particular, anti-GM-CSF levels may be used to distinguish CD
patients with ileal or ileo-colonic disease (in which surgery is a
more likely outcome) from colonic CD patients, UC patients, and
normal (healthy) patients. By classifying the IBD patient into a
particular subgroup, the appropriate course of therapy may be
determined, reducing the likelihood of administering treatments
that will not be successful, thereby stemming disease progression
and improving the individual's overall quality of life.
[0079] For example, determination of anti-GM-CSF antibody levels in
an individual also be used to guide biologic therapy, in terms of
whether augmentation or inhibition of GM-CSF bioactivity would be
beneficial, comprising the steps of obtaining a sample from the
individual, determining the concentration of serum anti-GM-CSF
antibody concentration in the sample, wherein a mean serum
anti-GM-CSF antibody concentration of greater than or equal to
about 1 mcg/ml, or great than or equal to about 1.5 mcg/ml, or
greater than or equal to about 2.0 mcg/ml, or within the range of
about 1.0 mcg/ml to 2.5 mcg/ml indicates an increased risk for
disease progression and surgery. In patients having elevated risk
of surgery, treatments such as anti-TNF alpha (such as Humira or
Remicade), or GM-CSF therapy would be indicated as the preferred
therapy. In this regard, the instant methods, and kits embodying
these methods, allow for improved treatment of individuals likely
to require surgery.
[0080] Thus, the present invention is directed to a highly
sensitive method of diagnosing, classifying, or characterizing
disease in individuals having an inflammatory bowel disease, such
that disease progression and/or the appropriate course of treatment
may be readily determined, and kits utilizing this method.
[0081] A variety of assay formats can be used to determine
anti-GM-CSF antibody levels in a sample.
[0082] Flow cytometry can be used to determine anti-GM-CSF antibody
levels according to a method of the invention. Such flow cytometric
assays, including bead based immunoassays, can be used to determine
anti-GM-CSF antibody levels in the same manner as used to detect
serum antibodies to Candida albicans and serum antibodies to HIV
proteins (see, for example, Bishop and Davis, J. Immunol. Methods
210:79-87 (1997); McHugh et al., J. Immunol. Methods 116:213
(1989); Scillian et al., Blood 73:2041 (1989), each of which is
incorporated by reference herein).
[0083] Phage display technology for expressing a recombinant
antigen specific for anti-GM-CSF antibodies also can be used to
determine the level of anti-GM-CSF antibody. Phage particles
expressing the antigen specific for anti-GM-CSF antibody, or an
antigen specific for anti-GM-CSF antibody, can be anchored, if
desired, to a multiwell plate using an antibody such as an
antiphage monoclonal antibody (Felici et al., "Phage-Displayed
Peptides as Tools for Characterization of Human Sera" in Abelson
(Ed.), Methods in Enzymol. 267, San Diego: Academic Press, Inc.
(1996), which is incorporated by reference herein).
[0084] A variety of immunoassay formats including competitive and
non-competitive immunoassay formats also are useful the methods of
the invention (Self and Cook, Curr. Opin. Biotechnol. 7:60-65
(1996), which is incorporated by reference). Immunoassays encompass
capillary electrophoresis based immunoassays (CEIA) and can be
automated, if desired. Immunoassays also can be used in conjunction
with laser induced fluorescence (see, for example, Schmalzing and
Nashabeh, Electrophoresis 18:2184-93 (1997)); Bao, J. Chromatogr.
B. Biomed. Sci. 699:463-80 (1997), each of which is incorporated
herein by reference). Liposome immunoassays, such as flow-injection
liposome immunoassays and liposome immunosensors, also can be used
to determine anti-GM-CSF antibody concentration.
[0085] Immunoassays, such as enzyme-linked immunosorbent assays
(ELISAs), can be particularly useful in a method of the invention.
An ELISA, for example, can be useful for determining whether a
sample is positive for anti-GM-CSF antibodies or for determining
the anti-GM-CSF antibody level in a sample. An enzyme such as
horseradish peroxidase (HRP), alkaline phosphatase (AP),
.beta.-galactosidase or urease can be linked to a secondary
antibody selective for anti-GM-CSF antibody, or to a secondary
antibody selective for anti-GM-CSF antibody for use in a method of
the invention. A horseradish-peroxidase detection system can be
used, for example, with the chromogenic substrate
tetramethylbenzidine (TMB), which yields a soluble product in the
presence of hydrogen peroxide that is detectable at 450 nm. An
alkaline phosphatase detection system can be used with the
chromogenic substrate p-nitrophenyl phosphate, for example, which
yields a soluble product readily detectable at 405 nm. Similarly, a
.beta.-galactosidase detection system can be used with the
chromogenic substrate o-nitrophenyl-.beta.-D-galactopyranoside
(ONPG), which yields a soluble product detectable at 410 nm, or a
urease detection system can be used with a substrate such as
urea-bromocresol purple (Sigma Immunochemicals, St. Louis, Mo.). A
useful secondary antibody linked to an enzyme can be obtained from
a number of commercial sources; goat F(ab').sub.2 anti-human
IgG-alkaline phosphatase, for example, can be purchased from
Jackson Immuno-Research (West Grove, Pa.).
[0086] A radioimmunoassay also can be useful for determining the
level of anti-GM-CSF antibodies in a sample. A radioimmunoassay
using, for example, an iodine.sup.125 labeled secondary antibody
(Harlow and Lane, Antibodies A Laboratory Manual Cold Spring Harbor
Laboratory: New York, 1988, which is incorporated herein by
reference) is encompassed within the invention.
[0087] A secondary antibody labeled with a chemiluminescent marker
also can be useful in the methods of the invention. Such a
chemiluminescent secondary antibody is convenient for sensitive,
non-radioactive detection of anti-GM-CSF antibodies and can be
obtained commercially from various sources such as Amersham
Lifesciences, Inc. (Arlington Heights, Ill.).
[0088] In addition, a detectable reagent labeled with a
fluorochrome can be useful in the methods of the invention for
determining the levels of anti-GM-C SF antibody in a sample.
Appropriate fluorochromes include, for example, DAPI, fluorescein,
Hoechst. 33258, R-phycocyanin, B-phycoerythrin, R-phycoerythrin,
rhodamine, Texas red or lissamine. A particularly useful
fluorochrome is fluorescein or rhodamine. Secondary antibodies
linked to fluorochromes can be obtained commercially. For example,
goat F(ab').sub.2 anti-human IgG-FITC is available from Tago
Immunologicals (Burlingame, Calif.).
[0089] A signal from the detectable reagent can be analyzed, for
example, using a spectrophotometer to detect color from a
chromogenic substrate; a radiation counter to detect radiation,
such as a gamma counter for detection of iodine-125; or a
fluorometer to detect fluorescence in the presence of light of a
certain wavelength. For detection of enzyme-linked reagents, a
quantitative analysis of the amount of anti-GM-CSF antibody can be
made using a spectrophotometer such as an EMAX Microplate Reader
(Molecular Devices, Menlo Park, Calif.) in accordance with the
manufacturer's instructions. If desired, the assays of the
invention can be automated or performed robotically, and the signal
from multiple samples can be detected simultaneously.
[0090] Immunoassays using a secondary antibody selective for
anti-GM-CSF antibodies are particularly useful in the methods of
the invention. As used herein, the term "antibody" means a
population of immunoglobulin molecules, which can be polyclonal or
monoclonal and of any isotype. As used herein, the term "antibody"
encompasses an immunologically active fragment of an immunoglobulin
molecule. Such an immunologically active fragment contains the
heavy and light chain variable regions, which make up the portion
of the antibody molecule that specifically binds an antigen. For
example, an immunologically active fragment of an immunoglobulin
molecule known in the art as Fab, Fab' or F(ab').sub.2 is included
within the meaning of the term antibody.
[0091] Using the above-described methods, an individual may be
identified as being an optimal candidate for GM-CSF treatment.
"GM-CSF" refers to a protein that stimulates the production of
granulocytes and macrophages by stem cells. GM-CSF used in the
practice of the invention includes any pharmaceutically safe and
effective human GM-CSF (e.g., the human GM-CSF with amino acid
having the following sequence: Ala Pro Ala Arg Ser Pro Ser Pro Ser
Thr Gln Pro Trp Glu His Val Asn Ala Ile Gln Glu Ala Arg Arg Leu Leu
Asn Leu Ser Arg Asp Thr Ala Ala Glu Met Asn Glu Thr Val Glu Val Ile
Ser Glu Met Phe Asp Leu Gln Glu Pro Thr Cys Leu Gln Thr Arg Leu Glu
Leu Tyr Lys Gln Gly Leu Arg Gly Ser Leu Thr Lys Leu Lys Gly Pro Leu
Thr Met Met Ala Ser His Tyr Lys Gln His Cys Pro Pro Thr Pro Glu Thr
Ser Cys Ala Thr Gln Ile Ile Thr Phe Glu Ser Phe Lys Glu Asn Leu Lys
Asp Phe Leu Leu Val Ile Pro Phe Asp Cys Trp Glu Pro Val Gln Glu
(SEQ ID NO:1)), or any derivative thereof having the biological
activity of human GM-CSF. Derivatives of GM-CSF may be (i) one in
which one or more of the amino acid residues of the protein are
substituted with a conserved or non-conserved amino acid residue,
and such substituted amino acid residue may or may not be one
encoded by the genetic code, (ii) one in which one or more of the
amino acid residues of the protein include a substituent group,
(iii) one in which the mature protein is fused with another
compound, such as a compound to increase the half-life or the
polypeptide (for example, polyethyleneglycol), (iv) one in which
additional amino acids are fused to the mature protein, such as a
leader or secretory sequence or a sequence which is employed for
purification of the mature protein, or (v) one in which the protein
is fused with a larger protein, i.e., an antibody or Fc. Examples
of GM-CSFs include, but are not limited to, native GM-CSF,
molgramostim (bacteria-derived GM-CSF), ragramostim (CHO-derived
GM-CSF), sargramostim (Leukine--yeast-derived GM-CSF), and
pegylated GMCSF (i.e., pegylated native GM-CSF or pegylated GMCSF
derived from any source).
[0092] Recombinant GM-CSF may be used with the methods of the
instant invention. "Recombinant GM-CSF" refers to either to GM-CSF
synthesized in a cell into which a nucleic acid encoding exogenous
GM-CSF has been introduced, or a cell in which the endogenous
GM-CSF gene has been stimulated to overproduce GM-CSF by the
introduction of regulatory elements that induce a high rate of
transcription of the endogenous GM-CSF gene.
[0093] The GM-CSF used may be recombinant human GM-CSF (rhu
GM-CSF), such as Leukine.RTM.. (Berlex Inc., Bothell, Wash.).
Leukine.RTM.. (generically termed "sargramostim") is a
biosynthetic, yeast-derived, recombinant human GM-CSF, consisting
of a single 127 amino acid glycoprotein that differs from the
endogenous human GM-CSF shown having the sequence as set forth
above in which arginine is substituted with leucine at position 23.
Leukine.RTM. is produced in the yeast Saccharomyces cerevisiae.
[0094] Leukine.RTM. has been shown to exhibit the same
hematopoietic effects as those induced by endogenous GM-CSF,
namely, the stimulation of progenitor cells committed along the
granulocyte-macrophage pathway to form neutrophils, monocytes,
macrophages, and eosinophils (Technical Product Report:
Leukine.RTM. Liquid, Immunex Corporation, Seattle, Wash., 1997,
which is herein incorporated by reference). Leukine.RTM., like
endogenous GM-CSF, also promotes the differentiation of progenitor
cells giving rise to erythrocytes and megakaryocytes. In addition
to stimulating hematopoiesis, Leukine.RTM. enhances many of the
functional activities of mature neutrophils, monocytes and
macrophages, such as chemotaxis, growth factor secretion,
anti-tumor activity, antibacterial and antifungal activities, and
so on.
[0095] Leukine.RTM. Liquid is a sterile injectable aqueous solution
generally sold in 1 ml vials containing 500 .mu.g/ml
(2.8.times.10.sup.6 IU) sargramostim; 40 mg/ml mannitol; 10 mg/ml
sucrose; 1.2 mg/ml tromethamine; sterile water; and 1.15% benzyl
alcohol. LEUKINE.RTM. Lyohphilized is also sold, and typically is
packaged in vials containing a sterile lyophilized powder for
reconstitution with 1 ml sterile water. LEUKINE.RTM. Lyophilized
may contain 250 .mu.g or 500 .mu.g sargramostim (1.4 or
2.8.times.10.sup.6 IU); 40 mg mannitol; 10 mg sucrose; and 1.2 mg
tromethamine. LEUKINE.RTM. Liquid and reconstituted solutions of
LEUKINE.RTM. Lyophilized are stored refrigerated at 2-8.degree.
C.
[0096] Typically, the aqueous solution of GM-CSF is administered by
subcutaneous injection or intravenous infusion. However, other
methods such as oral, intraperitoneal, subdermal, and intramuscular
administrations may be used. Doses delivered may be the same as
those delivered to stimulate an immune response in humans for other
disease purposes. In certain embodiments, doses may be about 100 to
about 1500 .mu.g (including any values therebetween, such as about
250, 500, and 1000 .mu.g) once per week when administered via
subcutaneous injection.
[0097] The methods of administration of GM-CSF as described in US
2007/0041938, Pettit, et al. are incorporated herein by
reference.
[0098] In addition, using the above-described methods, patients may
be identified that would benefit from administration of anti-TNF.
This therapy is sold under the trade name Humira.RTM., and is
described in U.S. Pat. No. 6,090,382, Salfeld et al., incorporated
herein by reference. This therapy is administered using standard
methods as known to one of ordinary skill in the art.
[0099] The following examples are intended to illustrate but not
limit the present invention.
Example I
Determining Anti-GM-CSF Antibody Activity for Classification of
Subtypes
[0100] GM-CSF concentration in whole blood or serum samples
obtained from a patient suspected of having an inflammatory bowel
disease is determined using the methods as described in Uchida, et
al. Blood, 2004, incorporated herein by reference, and described
herein.
[0101] Anti-GM-CSF antibodies are assayed using serum samples that
are either immediately obtained or previously collected and stored
at--70.degree. C. until analysis. Anti-GM-CSF antibody
concentration of a serum sample from an individual of interest is
then measured. The serum sample is diluted 1:100, 1:1000 and 1:3000
with phosphate buffered saline (PBS) containing 1% bovine serum
albumin and 0.1% Tween 20. Separate 50 .mu.l aliquots of diluted
serum and the affinity-purified anti-GM-CSF antibody isolated from
the serum of patients with pulmonary alveolar proteinosis as a
standard (0-50 ng/ml) were incubated at room temperature for 40
minutes in ELISA plates previously coated overnight at 4.degree. C.
with 1 .mu.g/ml rhGM-CSF (Leukine.RTM. Berlex) and blocked for 1
hour with StabilCoat.RTM. (SurModics). After washing five times
with PBS 0.1% Tween 20 (PBST), anti-GM-CSF antibody captured by
rhGM-CSF is detected by peroxidase labeled anti-human IgG
F(ab).sub.2 antibody (Sigma). Tetramethylbenzidine is used as a
substrate and absorbance is measured at 450 nm after stopping the
reaction with 1N sulfuric acid. Assays are performed in duplicate
and the mean of the two results used.
Example II
Anti-GM-CSF Concentration as a Predictor of Disease Behavior
[0102] The methods of Example 1 are carried out essentially as
described above. Following performance of the above-described
methods, the values obtained from the assay are then used to
classify the individual as either having colonic, or ileal or
ileo-colonic. Values greater than or equal to about 2 mcg/ml
indicate that the disease behavior is less likely to be only
colonic (L2), less likely to be non-stricuring, non-penetrating
(B1), and more likely to be treated with a surgery. Assessment of
the presence or absence of the CARD 15 mutation is not necessary
for this determination.
Example III
Determining CD11b Stimulation Index for Classification of
Subtypes
[0103] The CD11b Stimulation Index on neutrophils from whole blood
samples obtained from an individual suspected of having an
inflammatory bowel disease is determined using the methods as
described in Uchida, et al. NEJM, 2007, incorporated herein by
reference, and described herein. Briefly, this assay is performed
using whole blood samples from IBD patients or healthy controls in
the absence or presence of stimulation with exogenously added
GM-CSF and then cell surface CD11b is quantified on neutrophils
(and/or also on monocytes and eosinophils) is then measured by flow
cytometry. Neutrophils can be identified with the high expression
level of CD16 determined by fluorescein isothiocyanate
(FITC)-conjugated anti-CD16 antibody. Monocytes can be determined
APC-conjugated anti-CD14 antibody. Eosinophils can be determined
moderate expression of CD16 and specific cell size and complexity.
This assay provides a method to quantify the level of neutrophil
(as well as monocyte and eosinophil) innate immune function in IBD
patients.
[0104] The phycoerythrin (PE)-conjugated monoclonal antibodies
against CD11b is purchased from BD Biosciences (San Jose, Calif,
USA). CD11b modulates the adherence of polymorphonuclear
neutrophils and monocytes to fibrinogen and positive ICAM-1
endothelia.
[0105] Preparation for Flow-Cytometry Analyses
[0106] Heparinized whole blood is incubated for 30 minutes in the
presence or absence of 10 ng of human GM-CSF per milliliter
(Leukine, Berlex) or 10 ng of mouse GM-CSF per milliliter (R&D
Systems) and incubated for 30 minutes. The samples (50 .mu.L) are
then mixed with 50 .mu.L PBS in polystyrene round-bottom tubes (5
mL Falcon, BD-Pharmingen, Heidelberg, Germany) and incubated with
incubated with 5 .mu.l, undiluted FITC-, APC- and PE-conjugated
antibodies for 30 minutes on ice in the dark. The reaction is
stopped and cells are fixed by washing the suspension with 3 ml of
BD FACS Lysing solution (BD Biosciences).
[0107] Flow cytometry is performed with a FACS Calibur flow
cytometer (BD Biosciences) equipped with a 15-mW argon laser
emitting at 488 nm. The fluorescence signals of FITC and PE are
detected with 530/30 nm (channel 1) and 585/42 nm band pass filters
(channel 2) with correction of the spectral overlap by color
compensation. Calibration of the flow cytometer is performed using
standard fluorescent microbeads (CaliBRITE, BD Biosciences).
Analysis of the fluorescence properties of 50,000 events is
performed using CellQuest software (BD Biosciences). The increase
in CD11b levels after GM-CSF priming (CD11b stimulation index) is
calculated as the mean fluorescence intensity of CD11b primed by
GM-CSF minus that of CD11b on nonprimed samples, divided by the
mean fluorescence intensity of nonprimed samples and multiplied by
100.
[0108] GM-CSF stimulation of CD11b on neutrophils (and/or monocytes
or eosinophils) is (are) compared to the corresponding cell types
in a control group (such as normal, healthy controls) or known
standards such as patients diagnosed with small bowel Crohn's
disease or ulcerative colitis. A determination that reduced or
absent GM-CSF-mediated increase in cell surface CD11b levels in a
sample is one mechanism to confirm that the increased level of
GM-CSF autoantibodies have functional significance with respect to
GM-CSF bioactivity in a given IBD patient.
[0109] The foregoing description of examples and embodiments of the
subject application has been presented for purposes of illustration
and description. It is not intended to be exhaustive or to limit
the subject application to the precise form disclosed. Obvious
modifications or variations are possible in light of the above
teachings. The embodiments were chosen and described to provide the
best illustration of the principles of the subject application and
its practical application to thereby enable one of ordinary skill
in the art to use the subject application in various embodiments
and with various modifications as suited to the particular use
contemplated. All such modifications and variations are within the
scope of the subject application as determined by the appended
claims when interpreted in accordance with the breadth to which
they are fairly, legally and equitably entitled.
Sequence CWU 1
1
11127PRTUknownHuman GM-CSF Amino Acid Sequence 1Ala Pro Ala Arg Ser
Pro Ser Pro Ser Thr Gln Pro Trp Glu His Val1 5 10 15Asn Ala Ile Gln
Glu Ala Arg Arg Leu Leu Asn Leu Ser Arg Asp Thr 20 25 30Ala Ala Glu
Met Asn Glu Thr Val Glu Val Ile Ser Glu Met Phe Asp 35 40 45Leu Gln
Glu Pro Thr Cys Leu Gln Thr Arg Leu Glu Leu Tyr Lys Gln 50 55 60Gly
Leu Arg Gly Ser Leu Thr Lys Leu Lys Gly Pro Leu Thr Met Met65 70 75
80Ala Ser His Tyr Lys Gln His Cys Pro Pro Thr Pro Glu Thr Ser Cys
85 90 95Ala Thr Gln Ile Ile Thr Phe Glu Ser Phe Lys Glu Asn Leu Lys
Asp 100 105 110Phe Leu Leu Val Ile Pro Phe Asp Cys Trp Glu Pro Val
Gln Glu 115 120 125
* * * * *