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.

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 SB ), Colonic Crohns disease (CD 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-TNFα 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 SB ), Colonic Crohns disease (CD 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 SB ), Colonic Crohn's disease (CD 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-TNFα 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 SB ), Colonic Crohns disease (CD 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 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.