WO2019015814A1 - Utilisation de protéine-1 de type chitinase-3 (chi3l1) en tant qu'auto-antigène dans des troubles auto-immuns du système digestif - Google Patents

Utilisation de protéine-1 de type chitinase-3 (chi3l1) en tant qu'auto-antigène dans des troubles auto-immuns du système digestif Download PDF

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WO2019015814A1
WO2019015814A1 PCT/EP2018/058219 EP2018058219W WO2019015814A1 WO 2019015814 A1 WO2019015814 A1 WO 2019015814A1 EP 2018058219 W EP2018058219 W EP 2018058219W WO 2019015814 A1 WO2019015814 A1 WO 2019015814A1
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chi3l1
disease
protein
autoantibodies
sample
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Dirk Roggenbuck
Peter SCHIERACK
Claudia DEUTSCHMANN
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GA Generic Assays GmbH
Brandenburgische Technische Universitaet Cottbus
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GA Generic Assays GmbH
Brandenburgische Technische Universitaet Cottbus
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/06—Gastro-intestinal diseases
    • G01N2800/065—Bowel diseases, e.g. Crohn, ulcerative colitis, IBS

Definitions

  • CHITINASE-3-LIKE PROTEIN 1 (CHI3L1) AS AN AUTOANTIGEN IN AUTOIMMUNE DISORDERS OF THE DIGESTIVE SYSTEM
  • the invention relates to an in vitro method for diagnosis, prognosis, risk assessment, monitoring, therapy guidance and/or therapy control of an autoimmune disorder of the digestive system, such as inflammatory bowel disease and autoimmune liver disease, in which autoantibodies that bind Chitinase-3-Like Protein 1 (CHI3L1 ) protein are detected as a marker of such dis- ease.
  • CHI3L1 Chitinase-3-Like Protein 1
  • Autoimmune diseases are commonly considered to be medical conditions arising from an ab- normal immune response to a normal body part, for example where so-called autoantibodies react to and damage parts of the patient's body. It is currently estimated that approximately 24 million people in the United States are affected by an autoimmune disease. Of the most common and debilitating autoimmune diseases are those of the digestive system, including for example inflammatory bowel disease and autoimmune liver disease.
  • CD Crohn's disease
  • UC ulcerative colitis
  • biopsies are collected especially from macroscopically conspicuous as well as inconspicuous areas.
  • IBD histopathological differential diagnostics it is, however, necessary to collect biopsies from at least five different anatomic segments of the entire colon, including the rectum, from the terminal ileum and upper gastrointestinal tract.
  • Such analyses are time intensive and invasive, providing significant discomfort to the patient.
  • Autoimmune liver diseases have been recognised as clinical entities for decades.
  • the three main categories of autoimmune liver disease are autoimmune hepatitis (AIH), primary biliary cirrhosis (PBC), and primary sclerosing cholangitis (PSC). All are well-defined entities with diagnosis based upon a constellation of clinical, serologic, and liver pathology findings. Although these diseases are considered autoimmune in nature, the etiology and possible environmental triggers of each remain obscure (42).
  • the characteristic morphologic patterns of autoimmune liver disease are a chronic hepatitis pattern of injury with prominent plasma cells in AIH, destruction of small intrahepatic bile ducts and canals of Hering in PBC, and periductal fibrosis and inflammation involving larger bile ducts with variable small duct damage in PSC.
  • Serological findings include the presence of antimitochon- drial antibodies in PBC, antinuclear, anti-smooth muscle, and anti-LKM antibodies in AIH, and pANCA in PSC (42).
  • Chitinase-3-like protein 1 (CHI3L1 ):
  • CHI3L1 The 40 kDa protein CHI3L1 was first described in 1990 as a protein secreted by synovial cells and was also referred to as HC gp-39 (1 ). Later, it was also termed YKL-40 because of the one- letter code of its three N-terminal amino acids, Tyrosine (Y)- Lysine (K)- Leucine (L), and its mo- lecular weight of 40 kDa (2).
  • the protein was first discovered to be secreted by synovial cells, it was also shown to be produced in chondrocytes (4), colonic epithelial cells (8), smooth muscle cells (9), osteosarcoma cells (10), as well as macrophages (5) and neutrophils (1 1 ).
  • CHI3L1 has no catalytic activity, it was shown to bind chitin, heparin and collagen.
  • CHI3L1 has a heparin-binding motif it seems to be more likely that heparan sulfate is a ligand for CHI3L1 , since heparan sulfate proteoglycans play a role in cell adhesion or growth factor interaction (3).
  • CHI3L1 was reported to be associated with tissue remodeling in vascular smooth muscle cells (12) and to act as a mitogen or growth factor in chondrocytes and synovial cells stimulating tissue remodeling (13).
  • CHI3L1 was shown to interact with IGF-1 and can also promote growth of skin and fetal lung fibroblasts in a comparable way like IGF-1.
  • AKT-, ERK- and PKB-mediated mechanisms are involved in CHI3L1 mitogenic function (14). It acts as a chemoattractant in vascular endothelial cell (HUVEC) stimulating cell migration and adhesion (15)(16).
  • CHI3L1 has also an impact on bacterial adhesion and migration in colonic epithelial cells.
  • Mizoguchi showed that expression of CHI3L1 is upregulated in lamina limbal epithelial cells (CECs) of patients suffering from ulcerative colitis and Crohn's disease as well as in murine colitis models.
  • this upregulation enhances adhesion and invasion of Salmonella typhimurium and Adherent-invasive E. coli (AIEC) but not non-pathogenic E. coli (DH5a) in CEC and thus may act as a pathogenic mediator in acute colitis (8).
  • AIEC Adherent-invasive E. coli
  • DH5a non-pathogenic E. coli
  • CHI3L1 was found to be secreted by the human osteosarcoma cell line MG63 (17) leading to extensive studies on its function in different cancers. In addition, its divers biological function as adhesion, migration and growth factor, may indicate that CHI3L1 plays an important role in cancer development.
  • CHI3L1 levels were first discovered in synovial cells and cartilage of patients with rheumatoid arthritis ( A), a lot of efforts has been made in investigating its role in chronic inflammatory diseases.
  • serum CHI3L1 levels were significantly increased but when disease becomes inactive, serum levels decrease (18).
  • CHI3L1 levels correlate with two other proinflammatory markers, interleukin 6 (IL-6) and c-reactive protein (CRP) (19) and increased tissue expression was found in chondrocytes of arthritic cartilage as well as in lining and stromal cells (macrophages) in the synovium (20).
  • IL-6 interleukin 6
  • CPP c-reactive protein
  • CHI3L1 peptide-MHC com- plexes could be detected, but not in spondyloarthropathy (SpA) and psoriatic arthritis patients (22).
  • SpA spondyloarthropathy
  • psoriatic arthritis patients 212.
  • the use of antigen microarrays comprising CHI3L1 peptides for profiling of autoantibodies in the diagnosis of RA has been suggested (Hueber et al., Arthritis & Rheuma, vol. 52, no. 9, 1 September 2005, 2645-2655).
  • CHI3L1 An increased tissue and serum level of CHI3L1 was also reported in osteoarthritis (OA) but ap- pears not to be a good marker for monitoring disease progression. Depending on disease severity, CHI3L1 is expressed in different cartilage zones. It can be detected in the deep, mid and superficial zone in advanced OA but only in the superficial zone in mild OA, whereas it could not be detected in chondrocytes from normal articular cartilage (23)(24).
  • CHI3L1 has been reported to as a marker for autoimmune disorder of the diges- tive system, such as in Crohn's disease (Yusuf Erzin et al., Journal of Gastroenterology and Hepatology, vol. 23, 1 August 2008, e357-e362) and inflammatory bowel disease (Koutroubakis et al., International Journal of Colorectal Disease, vol. 18, no. 3, 1 May 2003, 254-259; A. Bu- isson et al., Alimentary Pharmacology & Therapeutics., vol. 43, no. 10, 8March 2016, 1069- 1079). It was shown in patients with Inflammatory Bowel Disease (IBD) that the serum CHI3L1 level correlates with the disease activity.
  • IBD Inflammatory Bowel Disease
  • CHI3L1 serum levels were also found to be increased in liver disorders like alcoholic liver cir- rhosis or hepatitis C virus (HCV)-mediated fibrosis, where serum CHI3L1 levels correlate with the degree of fibrosis but its potential to be a good marker depends on the cause of liver fibrosis (27)(28).
  • HCV hepatitis C virus
  • CHI3L1 is only one of different serum markers of liver fibrosis but it is a better marker in disease progression in liver fibrosis due to schistosomiasis japonica infection (29)(30).
  • CHI3L1 autoimmune hepatitis (AIH)- induced cirrhosis or primary biliary cirrhosis (PBC) (31 ). Elevated serum CHI3L1 levels were also found in atherosclerosis (32), pulmonary sarcoidosis (33) and systemic sclerosis (34).
  • CHI3L1 has been suggested as a marker for inflammatory and autoimmune disorders of the digestive system and of RA and the generation of auto reactivity against CHI3L1 has been suggested in RA (Coffman F D, Critical Reviews in Clinical Laboratory Science, vol. 45, no. 6, 1 January 2008, 531-562).
  • serological tests may be performed and various (auto) antibodies examined, as described in several review articles (Conrad et al., Autoimmunity Reviews, vol. 13, no. 4-5, 1 April 2014, 463-466; Laass et al., Autoimmunity Reviews, vol. 13, no. 4-5, 1 April 2014, 467-471 ; Hennes et al., Hepatology, vol. 48, no. 1 , 1 July 2008, 169-176) and the patent application EP2913675A2.
  • ASCA anti-Saccharomyces cerevisiae antibodies
  • p-ANCA perinuclear antineutrophil cytoplasmic antibodies
  • AIH autoimmune hepatitis
  • IAIHG Interna- tional Autoimmune Hepatitis Group
  • CHI3L1 protein levels are used in the diagnosis of various diseases, and conclusions are drawn about the intensity and course of the disease. Due to the incidence of elevated CHI3L1 protein levels in multiple diseases, the simple detection of the protein is not suitable for determining specific diseases.
  • autoimmune disorders of the digestive system such as inflammatory bowel disease and autoimmune liver disease
  • more straightforward and effective means for diagnosis are required, in particular means for disease diagnosis that are closely based on the underlying pathology of the respective diseases.
  • the technical problem underlying the present invention is the provision of means for diagnosing autoimmune diseases of the digestive system, such as inflammatory bowel disease and/or autoimmune liver disease, that do not exhibit the disadvantages of the prior art.
  • a further problem to be solved may be considered the provision of means for differentiating between CD and UC, for example in patients with similar symptoms of disease.
  • the invention is intended to improve the serological (differential) diagnosis of inflammatory bowel diseases and autoimmune liver diseases.
  • the invention relates to Chitinase-3-Like Protein 1 (CHI3L1 ) protein as an autoantigen in immune disorders of the digestive system, and corresponding methods and kits for diagnosis and associated embodiments.
  • CHI3L1 Chitinase-3-Like Protein 1
  • the invention therefore relates to an in vitro method for diagnosis, prognosis, risk assessment, monitoring, therapy guidance and/or therapy control of an autoimmune disorder of the digestive system, comprising:
  • detecting autoantibodies from said sample that bind to said CHI3L1 protein wherein the presence of said autoantibodies and/or increased levels of said autoantibodies compared to an appropriate control, such as a sample from a healthy subject, indicate the presence an autoimmune disorder of the digestive system.
  • the invention therefore relates to the surprising and unexpected finding that CHI3L1 protein is a target (autoantigen) for autoantibodies that are associated with different autoimmune diseases of the digestive system.
  • This surprising finding makes it possible to diagnose autoimmune disorder of the digestive system.
  • Furthermore, on the basis of the method of diagnosis it is also possible to provide a prognosis and a risk assessment for the subject exhibiting symptoms of an autoimmune disorder of the digestive system depending on the result of a method.
  • said subject In case of presence of the autoantibodies against CHI3L1 protein it may be concluded that said subject the symptoms are related to an autoimmune reaction, which allows it to categorize the subject as a patient suffering from an autoimmune disorder of the digestive system.
  • Such categorization of the patients allows conclusions about the prognosis and risk of the patient with respect to disease progression and duration of the symptoms.
  • the diagnosis of an autoimmune disease of the digestive system enables conclusions with respect to suitable therapeutic measures that should be initiated or that are already ongoing and may be modified. Such suitable therapeutic measures may differ on the basis of the result of the method of the invention, since autoimmune disorders of the digestive system may require different therapeutic approaches than other disorders of the Gl tract that cause similar symptoms. Accordingly, the method of the invention can be used for therapy guidance and therapy control. Additionally, the method of the present invention enables quantification of the amount of autoantibodies against CHI3L1 , which may be used as a measure for the current severity of the autoimmune disease in a patient and therefore is an indicator of the disease status of the patient. Accordingly, the method of the present invention can be used for monitoring the status of a subject or patient.
  • the in vitro method described herein is characterised in that the autoimmune disorder of the digestive system to be diagnosed is an inflammatory intestinal disease in which autoantibodies bind components of the gastrointestinal tract of said subject.
  • the in vitro method described herein is characterised in that the inflammatory intestinal disease to be diagnosed is Crohn's disease (CD) and/or ulcerative colitis (UC).
  • CD Crohn's disease
  • UC ulcerative colitis
  • the in vitro method described herein is characterised in that the autoimmune disorder of the digestive system to be diagnosed is an inflammatory liver disease in which autoantibodies bind components of the liver of said subject.
  • the in vitro method described herein is characterised in that the inflammatory liver disease to be diagnosed is autoimmune hepatitis and/or primary scle- rosing cholangitis.
  • the components of the gastrointestinal tract, to which autoantibodies may bind include, but are not limited to, the mucosa of the small intestine or other small-bowel tissue, the villous extracellular matrix, intestinal epithelial cells, in particular villous epithelial cells, the endomysium or other tissues or cells of the stomach, small intestine, and co- Ion, in particular the cells lining of the stomach, small intestine, and colon.
  • the components of the liver, to which autoantibodies may bind include, but are not limited to hepatocytes and/or biliary epithelial cells.
  • CHI3L1 protein could be used as an epitope detect the presence or absence of different autoimmune diseases, preferably those characterised by autoantibodies that bind components of the gastrointestinal tract or liver of a subject.
  • the method thereby allows diagnosis and in some cases differentiation between such diseases on the basis of their distinct autoantibody profiles.
  • the use of CHI3L1 protein as an autoantigen in diagnosis of autoimmune diseases of the digestive tract thereby represents a novel and inventive concept in light of the prior art with respect to the diagnosis of autoimmune diseases.
  • the CHI3L1 protein comprises or consists of the amino acid sequence according to SEQ ID NO. 1 (see below). Potential isoforms of CHI3L1 are also intended for employment in embodiments of the present invention.
  • Isoforms of CHI3L1 relate to proteins comprising or consisting of substantially the same or similar amino acid se- quences as SEQ ID NO 1.
  • Potential isoforms may also refer to one or more amino acid sequences that is similar, but not identical to, the amino acid sequences provided explicitly herein.
  • Sequences according to SEQ ID NO 2, 3 and/or 4 may also be employed in the invention.
  • CHI3L1-similar sequences employed are functionally equivalent to CHI3L1 itself, in other words, such functional equivalence is define by the ability to de- tect/bind autoantibodies indicative of the herein mentioned diseases.
  • Variation in length of the amino acid sequences as described herein is also encompassed by the present invention.
  • a skilled person is capable of providing artificial amino acid sequence variants that are longer or shorter than the specific sequence of SEQ ID NO 1 , 2, 3 and/or 4, which will still exhibit sufficient similarity to the natural form in order to provide the diagnostic outcomes described herein.
  • shorter variants of SEQ ID NO 1 , 2 and/or 3 comprising 10, 20, 30, 40, 50 or up to 100 amino acids less than the full length form may also enable effective diagnostic outcomes, as described herein. Fragments of CHI3L1 are therefore also considered.
  • longer variants of SEQ ID NO 1 , 2, 3 and/or 4 comprising 10, 20, 30, 40, 50 or up to 100 amino acids any given additional sequence more than the natural CHI3L1 sequence may also enable effective diagnostic outcomes, as described herein.
  • the CHI3L1 protein employed may comprise or consist of an amino acid sequence with at least 50%, 60%, 70%, 80%, 90% or 95% sequence identity to one or more of SEQ ID NO 1 , 2, 3 and/or 4.
  • sequence variant comprises at least 80%, 90% or 95% sequence identity to one of SEQ ID NO 1 , 2, 3 and/or 4 and preferably exhibits functional identity to SEQ ID NO 1 , 2, 3 and/or 4 with respect to the binding and subsequent identification of autoantibodies directed against a natural form of CHI3L1.
  • the provision of the sample to be analysed may relate to either obtaining a sample from a patient, or providing a pre-prepared sample already having been obtained, preferably from a patient exhibiting symptoms and/or suspecting of having an autoimmune disorder, preferably an autoimmune disorder associated with autoantibodies that bind components of the digestive or intestinal tract or liver of said subject.
  • the sample of the present invention relates preferably to a sample obtained from a patient, such as a bodily fluid, preferably a blood, plasma or serum sample, but may also relate to stool sample. Tissue samples may also be used in the method of the invention. Any particular processing of the sample is not intended to be limiting to the scope of the invention, essentially any given sample obtained from the patient may be used, with or without additional processing steps before administration in the method described herein.
  • the in vitro method described herein is characterised in that the sample of a subject is as a bodily fluid selected from the group consisting of blood, a sample derived from blood, such as a plasma or serum sample, a stool sample or sample derived from stool, mucous secretions, such as tears, saliva, sweat or colostrum, and secretions from the genitourinary tract, gastrointestinal tract, prostate and respiratory epithelium.
  • a sample derived from blood such as a plasma or serum sample
  • a stool sample or sample derived from stool a mucous secretions, such as tears, saliva, sweat or colostrum, and secretions from the genitourinary tract, gastrointestinal tract, prostate and respiratory epithelium.
  • the contacting of a sample to CHI3L1 protein may take place in any given setting.
  • a solid phase, to which the isoforms are attached is used.
  • the sample is preferably provided as a liquid sample and is brought into contact with the CHI3L1 protein, thereby allowing the autoantibodies of the sample to interact with the CHI3L1 protein under conditions that allow binding of said antibodies to the CHI3L1 protein.
  • Such conditions are known to a skilled person and may represent biological conditions, in which the relevant proteins are capable of forming their native or near-native structures, in order to allow the binding properties of the antibodies to enable interaction with said isoforms.
  • the contacting and detection steps may in further embodiments be carried out as follows: allowing the antibody to bind CHI3L1 , thereby forming a complex (CHI3L1-autoantibody complex), contacting the complex with a label, such as a labeled indicator antibody, preferably an antibody that binds human immunoglobulin, to form a labeled complex; and detecting the presence or absence of the labeled complex, and preferably associating the presence of the detected antibodies in the sample with the autoimmune disease.
  • a label such as a labeled indicator antibody, preferably an antibody that binds human immunoglobulin
  • the detection of bound antibodies may be carried out in any given suitable manner, including but not limited to the use of a spectrophotometer to detect colour from a chromogenic substrate, a radiation counter to detect radiation such as a gamma counter for detection of 1251, or a fluo- rometer to detect fluorescence in the presence of light of a certain wavelength.
  • a spectrophotometer to detect colour from a chromogenic substrate
  • a radiation counter to detect radiation such as a gamma counter for detection of 1251
  • a fluo- rometer to detect fluorescence in the presence of light of a certain wavelength.
  • Washing of the bound antibodies may be carried out as is commonly known in the art, for example as is carried out in a standard immunoassay, such as an ELISA assay. Additional detection means are described herein.
  • a further aspect of the invention relates to a solid phase-immobilised CHI3L1 protein, and its use in the assay described herein.
  • the in vitro method is characterized in that the autoantibody detected is an IgG, IgA, IgM and/or secretory IgA autoantibody.
  • the detection of different subclasses of autoantibody is possible according to the knowledge of a skilled person, for example IgG-, IgA-, IgM- and/or secretory IgA autoantibody-specific secondary antibodies may be em- ployed in an ELISA assay or similar.
  • the in vitro method is characterized in that the autoimmune disorder of the digestive system is Crohn's disease (CD), ulcerative colitis (UC) or primary sclerosing cholangitis, and wherein the autoantibody detected is an IgA and/or secretory IgA autoantibody that binds CHI3L1 protein.
  • CD Crohn's disease
  • UC ulcerative colitis
  • primary sclerosing cholangitis characterized in that the autoimmune disorder of the digestive system is Crohn's disease (CD), ulcerative colitis (UC) or primary sclerosing cholangitis
  • the autoantibody detected is an IgA and/or secretory IgA autoantibody that binds CHI3L1 protein.
  • IgA and/or secretory IgA autoantibodies that bind CHI3L1 protein are detected, preferably in elevated levels, in subjects with Crohn's disease (CD), ulcerative colitis (UC) or primary sclerosing cholangitis.
  • CD Crohn's disease
  • UC ulcerative colitis
  • sclerosing cholangitis This finding is novel and enables a preferred embodiment of the invention based on entirely unexpected experimental findings.
  • the in vitro method is characterized in that the autoimmune disorder of the digestive system is autoimmune hepatitis, and wherein the autoantibody detected is an IgG autoantibody that binds CHI3L1 protein.
  • the in vitro method is used in the differential diagnosis between Crohn's disease (CD) and ulcerative colitis (UC), wherein the amount of IgA and/or secretory IgA autoantibodies from said sample that bind to said CHI3L1 protein indicates the pres- ence of Crohn's disease (CD), when said amount is above a reference level, wherein said reference level corresponds to amounts of autoantibodies that bind to said CHI3L1 protein in patients with ulcerative colitis (UC).
  • CD Crohn's disease
  • UC ulcerative colitis
  • the presence of IgA and/or secretory IgA autoantibodies that bind CHI3L1 protein are detected in elevated levels in subjects with CD, compared to the levels in samples from patients with UC.
  • This finding is novel and enables a preferred embodiment of the invention based on entirely unexpected experimental findings.
  • This embodiment enables the use of reference values or reference samples representing amounts of autoantibodies that bind to said CHI3L1 protein in patients with ulcerative colitis (UC), and comparison to these levels, such that when levels are above a particular reference (potential a cut-off value, or population average), the presence of CD may be diagnosed.
  • a further aspect of the invention relates to a kit for diagnosing an autoimmune disorder of the digestive system by detecting autoantibodies from a sample that bind to CHI3L1 protein, comprising: CHI3L1 protein immobilized on a solid phase, and optionally
  • human anti-immunoglobulin antibodies wherein said human anti-immuno- globulin antibodies bind autoantibodies of Ig-subtypes IgG, IgA and/or IgM,
  • a label for detection of said one or more human anti-immunoglobulin antibodies either capable of binding said human anti-immunoglobulin antibody or linked to said anti-immunoglobulin antibody, and means for detecting said label.
  • the kit further comprises reference data, such as a reference level, cut-off values, populations averages, or similar, corresponding to autoantibody amounts in healthy subjects, in subjects with Crohn's disease (CD), with ulcerative colitis (UC), autoimmune hepatitis and/or primary sclerosing cholangitis.
  • reference data such as a reference level, cut-off values, populations averages, or similar, corresponding to autoantibody amounts in healthy subjects, in subjects with Crohn's disease (CD), with ulcerative colitis (UC), autoimmune hepatitis and/or primary sclerosing cholangitis.
  • said reference data is stored on a computer readable me- dium and/or is present in the form of computer executable code configured for comparing the determined amounts of autoantibodies that bind to said CHI3L1 protein in said sample to said reference data.
  • the kit also comprises a computer program.
  • the computer program is adapted to perform the method of the present invention.
  • a skilled person is capable of developing reference levels for either healthy or diseased subjects using methods established in the art.
  • the invention therefore also relates to the use of a kit as described herein for the diagnosis of an autoimmune disorder as described herein.
  • the invention further relates to a system for diagnosing an autoimmune disorder of the digestive system by detecting autoantibodies from a sample that bind to CHI3L1 protein.
  • a microscopic device configured to determine the presence and/or intensity of signal produced by a label bound to an autoantibody, and/or
  • An optionally networked computer processing device configured to perform executable instructions; and a computer program, the computer program comprising a software module executed by the computer processing device to apply a model or algorithm for analyzing said autoantibodies, And optionally:
  • human anti-immunoglobulin antibodies wherein said human antiimmunoglobulin antibodies bind autoantibodies of Ig-subtypes IgG, IgA and/or IgM, and a label for detection of said one or more human anti-immunoglobulin antibodies, either capable of binding said human anti-immunoglobulin antibody or linked to said anti-immunoglobulin antibody, and means for detecting said label.
  • system and/or kit described herein comprises an optionally networked computer processing device configured to perform executable instructions; and a computer pro- gram, the computer program comprising a software module executed by the computer processing device to apply a model or algorithm for analyzing said autoantibodies.
  • system is characterised in that the computer program further comprises a software module executed by the computer processing device to designate a treatment regimen for the individual.
  • the computer program is adapted to perform the method of the invention.
  • the computer program of the system of the invention is adapted to perform the method of the invention and comprises a software module executed by the computer processing device to apply a model or algorithm for analyzing said autoantibodies,
  • the system is characterised in that a patient from which said sample has been taken is identified as providing said sample and is optionally treated for the autoimmune disease.
  • the invention therefore relates to a method for treating an autoimmune disease of the digestive system, comprising carrying out the method as described herein and subsequently treating said subject.
  • Treatment for an autoimmune disease of the gastrointestinal tract may relate to any appropriate treatment known to a skilled medical practitioner.
  • Medical treatment of IBD may be individualized to each patient. The choice of which drugs to use and by which route to administer them (oral, rectal, injection, infusion) depends on factors including the type, distribution, and severity of the patient's disease, as well as other historical and biochemical prognostic factors, and pa- tient preferences. For example, mesalazine may be administered. Generally, depending on the level of severity, autoimmune IBD may require immunosuppression to control the symptoms, such as prednisone, TNF inhibition, azathioprine (Imuran), methotrexate, or 6-mercaptopurine administration.
  • anti-inflammatory steroids are used to control disease flares. Crohn's disease and ulcerative colitis patients may receive TNF inhibitors. Severe cases may require sur- gery, such as bowel resection or a temporary or permanent colostomy or ileostomy. Surgery can cure ulcerative colitis if the large intestine is removed. A pouch can be created from the small intestine when required, this serves as the rectum and prevents the need for a permanent ileostomy.
  • Treatment for an autoimmune disease of the liver may relate to any appropriate treatment known to a skilled medical practitioner.
  • Medical treatment of autoimmune liver disease may be individualized to each patient.
  • the choice of which drugs to use and by which route to administer them depends on factors including the type, distribution, and severity of the patient's disease, as well as other historical and biochemical prognostic factors, and patient preferences.
  • drugs and medication may be administered.
  • Immunosuppressant drugs can be used to stop the immune system's attack.
  • Such drugs include, for example, 6-mercaptopurine and azathioprine.
  • Corticosteroids may be administered, usually in the form of prednisone, and can directly treat liver inflammation. Such medications typically serve as immunosuppressants.
  • a liver transplant (replacing your liver with a donor organ) can treat autoimmune liver disease.
  • NDDIC National Digestive Diseases Information Clearinghouse
  • the invention also relates to a method for the treatment of the autoimmune disease mentioned herein, comprising the following steps:
  • CHI3L1 protein in accordance with SEQ ID NO 1 recognizes autoantibodies directed against intestinal and/or liver tissue.
  • This treatment embodiment has the advantageous effect that diagnosis of autoimmune dis- eases by identifying autoantibodies, underlying the pathology of the disease, can be simply and effectively removed from body fluids without drug treatment interventions frequently involving stress to the patient.
  • Chitinase-3-Like Protein 1 (CHI3L1 ) is a 40 kDa protein and was first described in 1990 as a protein secreted by synovial cells and was also referred to as HC gp-39 (1 ). Later, it was also termed YKL-40 because of the one-letter code of its three N-terminal amino acids, Tyrosine (Y)- Lysine (K)- Leucine (L), and its molecular weight of 40 kDa (2).
  • CHI3L1 lacks chitinase activity (1 )(3)(4)(5)(6).
  • the gene for CHI3L1 consists of 10 exons, spanning 8kb genomic DNA and is located on chromosome 1 q31-q32 (7).
  • CHI3L1 may also be known as GP39; ASRT7; GP-39; YKL40; CGP-39; YKL-40; YYL-40; HC- gp39; HCGP-3P; or hCGP-39.
  • the CHI3L1 protein comprises or consists of the amino acid sequence of SEQ ID NO 1 (Accession No. AAH08568, EAW91467.1 , EAW91468.1 BAG35757,
  • Protein Name Chitinase 3-like 1 (cartilage glycoprotein-39) [Homo sapiens] GenBank Number GenBank: AAH08568.1
  • Natural variants of CHI3L1 are also known, which may be employed in the invention, such as: Natural variant VAR_019838, aa 145, R ⁇ G2, Corresponds dbSNP:rs880633 Ensembl.
  • the CHI3L1 protein preferably according to sequences disclosed herein, may comprise a 0 to 10 amino acid addition or deletion at the N and/or C terminus of a sequence.
  • a 0 to 10 amino acid addition or deletion at the N and/or C terminus of a sequence means that the polypeptide may have a) 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional amino acids at its N terminus and 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids deleted at its C terminus or b) 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional amino acids at its C terminus and 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides deleted at its N terminus, c) 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional amino acids at its N terminus and 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional amino ac- ids at its N terminus or d) 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids deleted at its N terminus and 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids deleted at its C terminus.
  • peptidomimetics are also contemplated.
  • Peptide analogs are commonly used in the pharmaceutical and diagnostic industry as non-peptide analogues with properties analogous to those of the template peptide. These types of non-peptide compound are termed “peptide mimetics” or “peptidomimetics” (Fauchere
  • the amino acid sequence of the CHI3L1 protein is as described according to one or more of SEQ ID NO 1 to 4.
  • sequence variants of these peptides are also contemplated, in which protein sequences with at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or preferably 95% sequence identity to any one or more of SEQ ID NO 1-4 are employed, preferably those with functionally analogy, in particular when the CHI3L1 variant protein is capable of binding autoantibodies indicative of disease.
  • Protein modifications to the polypeptides of the present invention which may occur through substitutions in amino acid sequence, and nucleic acid sequences encoding such molecules, are also included within the scope of the invention.
  • Substitutions as defined herein are modifications made to the amino acid sequence of the protein, whereby one or more amino acids are replaced with the same number of (different) amino acids, producing a protein which contains a different amino acid sequence than the primary protein. In some embodiments this amendment will not significantly alter the function of the protein.
  • substitutions may be natural or artificial. It is well known in the art that amino acid substitutions may be made without significantly altering the protein's function.
  • This list is not exhaustive. For example, it is well known that Ala, Gly, Ser and sometimes Cys can substitute for each other even though they belong to different groups.
  • An autoantibody is an antibody (a type of protein) manufactured by the immune system that is directed against one or more of the individual's own proteins. Many autoimmune diseases are associated with and/or caused by such autoantibodies.
  • autoimmune disease refers to any given disease associated with and/or caused by the presence of autoantibodies. Autoimmune diseases arise from an abnormal immune response of the body against substances and tissues normally present in the body (autoimmun- ity). This may be restricted to certain organs or involve a particular tissue.
  • autoimmune disorder of the digestive system relates to an immune disease in which parts of the digestive system are attacked by autoreactive antibodies.
  • the digestive system consists of the gastrointestinal tract plus accessory organs of digestion, such as the liver.
  • Autoimmune disorders of the digestive system comprise systemic autoimmune disorders with gas- trointestinal manifestation or autoimmune disorders that manifest in particular in the Gl tract (Andrew Campbell. Autoimmunity and the Gut. Autoimmune Diseases Volume 2014, Article ID 152428, 12 pages; Cojocaru et al. Maedica (Buchar). 201 1 Jan; 6(1 ): 45-51 ).
  • inflammatory bowel disease As used herein the terms “inflammatory bowel disease”, “inflammatory autoimmune gastrointestinal disorder”, and “inflammatory intestinal disease in which autoantibodies bind components of the gastrointestinal tract”, and the like, may be used interchangeably.
  • a preferred autoimmune disease of the invention is inflammatory bowel disease.
  • IBD inflammatory bowel disease
  • CD Crohn's disease
  • UC ulcerative colitis
  • IC indeterminate colitis
  • a preferred autoimmune disease of the invention is therefore an autoimmune disease of the di- gestive or intestinal tract of said subject.
  • Such diseases are characterised in that the autoimmune disorder exhibits autoantibodies that bind components of the digestive or intestinal tract of said subject.
  • Such components of the digestive or intestinal tract may be any organ, tissue, cell or protein found in said area of the subject.
  • the digestive or intestinal tract may be understood as the gastrointestinal tract (Gl tract), which refers to the stomach and intestine, and is divided into the upper and lower gastrointestinal tracts, and may include all the structures from the mouth to the anus.
  • the tract may also be divided into foregut, midgut, and hindgut, reflecting the embryological origin of each segment of the tract.
  • Gastrointestinal (Gl)-related autoantibodies can be evaluated in autoimmune diseases such as inflammatory bowel disease, autoimmune hepatitis and celiac disease.
  • Such antibodies may relate to ANCA (anti-neutrophil cytoplasmic antibodies) and/or autoantibodies to glycoprotein 2.
  • ANCA anti-neutrophil cytoplasmic antibodies
  • Anti-GP2 IgA and IgG can be detected in sera from patients with Crohn's disease and may be used in order to differentiate Crohn's disease from UC.
  • CD Crohn's disease
  • Ulcerative colitis is a disease of the large intestine characterized by chronic diarrhea with cramping, abdominal pain, rectal bleeding, loose discharges of blood, pus, and mucus.
  • the manifestations of UC vary widely.
  • a pattern of exacerbations and remissions typifies the clinical course for about 70% of UC patients, although continuous symptoms without remission are present in some patients with UC.
  • Local and systemic complications of UC include arthritis, eye in- flammation such as uveitis, skin ulcers, and liver disease.
  • UC and especially the long-standing, extensive form of the disease is associated with an increased risk of colon carcinoma.
  • autoimmune liver disease and “inflammatory liver disease in which autoantibodies bind components of the liver” may be used interchangeably.
  • a preferred autoimmune disease of the invention is therefore an autoimmune disease of the liver of a subject.
  • Such diseases are characterised in that the autoimmune disorder exhibits autoantibodies that bind components of the liver of said subject.
  • AIH autoimmune hepatitis
  • PBC primary biliary cirrhosis
  • PSC primary sclerosing cholangitis
  • AIH is typically characterized as an unresolving hepatitis usually associated with hypergammaglobulinemia and tissue-directed autoantibodies and responding in most cases to immunosuppressive therapy.
  • PBC is typically characterized as a chronic cholestatic liver disease in which the intrahepatic bile ducts are progressively destroyed by a nonsuppurative inflammatory process.
  • PSC is typically characterized as a chronic cholestatic disorder characterized by fibrosis and inflammation of the ex- tra- and intrahepatic biliary tree.
  • sample includes any biological specimen obtained from an individual. Suitable samples for use in the present invention include, without limitation, whole blood, plasma, serum, saliva, urine, stool, tears, any other bodily fluid, pure pancreatic juices or duodenal juices, tissue samples (e.g., biopsy) and cellular extracts thereof (e.g., red blood cellular extract). In a pre- ferred embodiment, the sample is a serum sample.
  • tissue samples e.g., biopsy
  • cellular extracts thereof e.g., red blood cellular extract
  • the sample is a serum sample.
  • samples such as serum, saliva, and urine is well known in the art (see, e.g., Hashida et al., J. Clin. Lab. Anal., 1 1 :267-86 (1997)).
  • samples such as serum samples can be diluted prior to analysis.
  • subject typically refers to humans, but also to other animals including, e.g., other primates, rodents, canines, felines, equines, ovines, porcines, and the like.
  • substantially the same or similar amino acid sequence includes an amino acid sequence that is similar, but not identical to, the naturally-occurring amino acid sequence.
  • an amino acid sequence i.e., polypeptide
  • a particularly useful modification of a polypeptide of the present invention, or a fragment thereof, is a modification that confers, for example, increased stability or reactivity.
  • Incorpora- tion of one or more D-amino acids is a modification useful in increasing stability of a polypeptide or polypeptide fragment.
  • deletion or substitution of lysine residues can increase stability by protecting the polypeptide or polypeptide fragment against degradation.
  • the amino acid sequences may also comprise 0 to 100, 2 to 50, 5 to 20, or for example 8 to 15, or any value from 0 to 20, amino acid additions or deletions at either the N- and/or C-terminus of the proteins.
  • the termini may also be modified with additional linker sequences, or removal of sequences, as long as the autoantibody binding properties and immunoreactivity of the protein is essentially maintained and the autoantibodies as described herein bind in an analogous manner to the specific sequence provided.
  • all peptides, peptide fragments or structures comprising peptides gener- ated using the methods mentioned above - starting from the peptides of the invention - are peptides according to the invention, provided they accomplish the object of the invention and, in particular, interact with the pathogenic autoantibodies.
  • these autoantibodies can be agonistic autoantibodies activating receptors.
  • the CHI3L1 protein may also be described as an antigen, as it reacts with an antibody targeted to said CHI3L1 protein.
  • CHI3L1 may also be referred to as a protein or target.
  • a CHI3L1 antigen can be partially purified.
  • a CHI3L1 antigen also can be prepared recombinantly by expressing an encoding nucleic acid sequence as described herein using methods well known in the art (see, for example, Ausubel et al., Current Protocols in Molecular Biology John Wiley & Sons, Inc. New York (1999)).
  • diagnosis includes the use of the devices, methods, kits, and systems, of the present invention to determine the presence or absence of a medically relevant disorder in an individual.
  • the term also includes devices, methods, kits, and systems for assessing the level of disease activity in an individual.
  • statistical algorithms are used to diagnose a mild, moderate, severe, or fulminant form of the disorder based upon the criteria devel- oped by Truelove et al., Br. Med. J., 12:1041-1048 (1955).
  • statistical algorithms are used to diagnose a mild to moderate, moderate to severe, or severe to fulminant form of the IBD based upon the criteria developed by Hanauer et al., Am. J. Gastroenterol., 92:559-566 (1997).
  • prognosis relates to the prediction of an outcome or a specific risk for a subject to suffer from an autoimmune disease as described herein. This may also include an estimation of the chance of recovery or the chance of an adverse outcome for said subject.
  • risk assessment and “risk stratification”, may be used interchangeably, and relate to the grouping of subjects into different risk groups according to their further prognosis. Risk assessment also relates to stratification for applying preventive and/or therapeutic measures.
  • therapy control in the context of the present invention refers to the monitoring and/or adjustment of a therapeutic treatment of said subject.
  • therapy guidance refers to application of certain therapies or medical interventions based on the value of the outcome of the methods described herein.
  • the invention also encompasses use of the method for "disease monitoring", also known as monitoring the progression or regression of the autoimmune disease.
  • monitoring the progression or regression of the autoimmune disease includes the use of the devices, methods, kits and systems of the present invention to determine the disease state (e.g., presence or severity of the autoimmune disease) of an individual.
  • the results of a statistical algorithm e.g., a learning statistical classifier system
  • the devices, methods, kits and systems of the present invention can also be used to predict the progression of the autoimmune disease, e.g., by determining a likelihood for the autoimmune disease to progress either rapidly or slowly in an individual based on the presence or level of at least one marker in a sample.
  • the devices, methods, and systems of the present invention can also be used to predict the regression of the autoimmune disease, e.g., by determining a likelihood for the autoimmune disease to regress either rapidly or slowly in an individual based on the presence or level of at least one marker in a sample.
  • Therapy monitoring may also be conducted, whereby a subject is monitored for disease progression during the course of any given therapy.
  • the comparative analysis described herein between autoantibody binding to CHI3L1 in different patient populations, ie CD vs UC, is a preferred method of the present invention.
  • Direct comparison based on autoantibody binding as measured in the same experiment may be used.
  • the amount of CHI3L1 provided for the experiment should preferably be controlled carefully to enable direct comparative analysis.
  • control values or standards may be used that provide samples with autoantibodies or represent control amounts thereof, as have already been obtained from previous analytical tests. It is possible to use control values having been generated by the testing of cohorts or other large numbers of subjects suffering from any given disease or control group. Appropriate statistical means are known to those skilled in the art for analysis and comparison of such data sets. Control samples for positive controls (such as disease sufferers) or negative controls (such as from healthy sub- jects) may be used for reference values in either simultaneous of non-simultaneous comparison.
  • the presence or level of anti-CHI3L1 antibodies or at least one marker is determined using an immunoassay or an immunohistochemical assay.
  • An immunoassay suitable for use in the method of the present invention includes an ELISA.
  • immunohistochemical assays suitable for use in the method of the present invention include, but are not limited to, immunofluorescence assays such as direct fluorescent antibody assays, IFA assays, anticomplement immunofluorescence assays, and avidin-biotin immunofluorescence assays. Other types of immunohistochemical assays include immunoperoxi- dase assays.
  • the immunoassay is used in the detection of antibodies, to which end binding of CHI3L1 antigen to a solid phase is envisaged.
  • the patient's antibody included therein binds to the CHI3L1 antigen.
  • the antibody which is obtained e.g. from the serum or stool of a patient and bound to CHI3L1 is subse- quently detected using a label, or labelled reagent and optionally quantified.
  • detection of the antibodies in this method is effected using labelled reagents according to the well-known ELISA (Enzyme-Linked Immunosorbent Assay) technology.
  • Labels according to the invention therefore comprise enzymes catalyzing a chemical reaction which can be determined by optical means, especially by means of chromogenic substrates, chemilu- minescent methods or fluorescent dyes.
  • the autoantibodies are detected by labelling with weakly radioactive substances in radioimmunoassays ( IA) wherein the resulting radioactivity is measured.
  • immunoassay techniques including competitive and non-competitive immunoassays, can be used to determine the presence or level of one or more markers in a sample (see, e.g., Self et al., Curr. Opin. Biotechnol., 7:60-65 (1996)).
  • immunoassay encompasses techniques including, without limitation, enzyme immunoassays (EIA) such as enzyme multiplied immunoassay technique (EMIT), enzyme-linked immunosorbent assay (ELISA), antigen capture ELISA, sandwich ELISA, IgM antibody capture ELISA (MAC ELISA), and microparticle enzyme immu- noassay (MEIA); capillary electrophoresis immunoassays (CEIA); radioimmunoassays (RIA); immunoradiometric assays (IRMA); fluorescence polarization immunoassays (FPIA); and chem- iluminescence assays (CL). If desired, such immunoassays can be automated.
  • EIA enzyme multiplied immunoassay technique
  • ELISA enzyme-linked immunosorbent assay
  • MAC ELISA enzyme-linked immunosorbent assay
  • MEIA microparticle enzyme immu- noassay
  • CEIA capillary electrophoresis immunoa
  • Immunoassays can also be used in conjunction with laser induced fluorescence (see, e.g., Schmalzing et al., Electrophoresis, 18:2184-2193 (1997); Bao, J. Chromatogr. B. Biomed. Sci., 699:463-480 (1997)).
  • Liposome immunoassays such as flow-injection liposome immunoassays and liposome immunosensors, are also suitable for use in the present invention (see, e.g., Rongen et al., J. Immunol. Methods, 204:105-133 (1997)).
  • nephelometry assays in which the formation of protein/antibody complexes results in increased light scatter that is converted to a peak rate signal as a function of the marker concentration, are suitable for use in the present invention.
  • Nephelometry assays are commercially available from Beckman Coulter (Brea, Calif.; Kit #449430) and can be performed using a Behring Nephelometer Analyzer (Fink et al., J. Clin. Chem. Clin. Biol. Chem., 27:261-276 (1989)).
  • the immunoassays described above are particularly useful for determining the presence or level of one or more markers in a sample (and may be considered examples of means for de- tecting a label), wherein a marker may be understood as an autoantibody targeted to CHI3L1.
  • a fixed neutrophil ELISA is useful for determining whether a sample is positive for ANCA or for determining ANCA levels in a sample.
  • an ELISA using yeast cell wall phosphopeptidomannan is useful for determining whether a sample is positive for ASCA-lgA and/or ASCA-lgG, or for determining ASCA-lgA and/or ASCA-lgG levels in a sam- pie.
  • An ELISA using CHI3L1 protein or a fragment thereof is useful for determining whether a sample is positive for anti-CHI3L1 antibodies, or for determining anti-CHI3L1 antibody levels in a sample.
  • the autoantibodies are detected in an immunoassay, preferably with direct or indirect coupling of one reactant to a labelling substance.
  • an immunoassay preferably with direct or indirect coupling of one reactant to a labelling substance.
  • the autoimmune disease-specific antibodies are detected in an immunoassay wherein the antibodies are present dissolved in a liquid phase, preferably diluted in a con- ventional buffer solution well-known to those skilled in the art or in an undiluted body fluid.
  • detection can also be effected using stool samples.
  • soluble or solid phase-bound CHI3L1 molecules are used to bind the antibodies.
  • anti-human immunoglobulins are employed, preferably selected from the group comprising anti-human IgA, anti-human IgM and/or anti-human IgG antibodies, said anti-human immunoglobulins being detectably labelled conjugates of two components which can be conjugated with any conventional labelling enzymes, especially chromogenic and/or chemiluminescent substrates, preferably with horseradish peroxidase, alkaline phosphatase.
  • the advantage of this embodiment lies in the use of ELISA technology usually available in laboratory facilities so that detection according to the in- vention can be established in a cost-effective manner.
  • the antibody bound to CHI3L1 reacts with anti-human immunoglobulins, preferably selected from the group comprising anti-human IgA, anti-human IgM and/or anti-human IgG antibodies, detectably coupled to fluorescein isothiocyanate (FITC).
  • anti-human immunoglobulins preferably selected from the group comprising anti-human IgA, anti-human IgM and/or anti-human IgG antibodies
  • FITC fluorescein isothiocyanate
  • Specific immunological binding of the antibody to the marker of interest can be detected directly or indirectly via a label. Any given means for detecting these labels may be considered means for detecting the label according to the method of the invention.
  • Direct labels include fluorescent or luminescent tags, metals, dyes, radionuclides, and the like, attached to the antibody.
  • An anti- body labeled with iodine-125 (1251) can be used for determining the levels of one or more markers in a sample.
  • a chemiluminescence assay using a chemiluminescent antibody specific for the marker is suitable for sensitive, non-radioactive detection of marker levels.
  • An antibody labeled with fluorochrome is also suitable for determining the levels of one or more markers in a sample.
  • fluorochromes examples include, without limitation, DAPI, fluorescein, Hoechst 33258, R-phycocyanin, B-phycoerythrin, R-phycoerythrin, rhodamine, Texas red, and lissamine.
  • Secondary antibodies linked to fluorochromes can be obtained commercially, e.g., goat F(ab')2 anti-human IgG-FITC is available from Tago Immunologicals (Burlingame, Calif.).
  • Indirect labels include various enzymes well-known in the art, such as horseradish peroxidase (HRP), alkaline phosphatase (AP), ⁇ -galactosidase, urease, and the like.
  • HRP horseradish peroxidase
  • AP alkaline phosphatase
  • ⁇ -galactosidase urease, and the like.
  • a horseradish-peroxi- dase detection system can be used, for example, with the chromogenic substrate tetra- methylbenzidine (TMB), which yields a soluble product in the presence of hydrogen peroxide that is detectable at 450 nm.
  • TMB tetra- methylbenzidine
  • 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.
  • a ⁇ -galactosidase detection system can be used with the chromogenic substrate o-nitrophenyl- -D-galactopyranoside (ONPG), which yields a soluble product detectable at 410 nm.
  • ONPG o-nitrophenyl- -D-galactopyranoside
  • a signal from the direct or indirect label can be analyzed, for example, using a spectrophotometer to detect colour from a chromogenic substrate; a radiation counter to detect radiation such as a gamma counter for detection of 1251; or a fluorometer to detect fluorescence in the presence of light of a certain wavelength.
  • a quantitative analysis of the amount of marker levels can be made using a spectrophotometer such as an EMAX Microplate Reader (Molecular Devices; Menlo Park, Calif.) in accordance with the manu- facturer's instructions.
  • the assays of the present invention can be automated or performed robotically, and the signal from multiple samples can be detected simultaneously.
  • the present invention provides methods of diagnosing the autoimmune disease or clinical subtypes thereof using CHI3L1.
  • inflammatory bowel disease (the autoimmune disease) markers such as biochemical markers, serological markers, genetic markers, or other clinical or echographic characteristics, are suitable for use and can be combined with statistical algorithms to classify a sample from an individual as an the autoimmune disease sample.
  • markers of the diseases include, but are not limited to, anti-neutrophil antibodies (e.g., ANCA, pANCA, cANCA, NSNA, SAPPA, etc.) or anti-Sac- charomyces cerevisiae antibodies (e.g., ASCA-lgA, ASCA-IgG, ASCA-lgM, etc.).
  • anti-neutrophil antibodies e.g., ANCA, pANCA, cANCA, NSNA, SAPPA, etc.
  • anti-Sac- charomyces cerevisiae antibodies e.g., ASCA-lgA, ASCA-IgG, ASCA-lgM, etc.
  • ANCA anti-neutrophil cytoplasmic antibody
  • ANCA activity can be divided into several broad categories based upon the ANCA staining pattern in neutrophils: (1 ) cytoplasmic neutrophil staining without perinuclear highlighting (cANCA); (2) perinuclear staining around the outside edge of the nucleus (pANCA); (3) perinuclear staining around the inside edge of the nucleus (NSNA); and (4) diffuse staining with speckling across the entire neutrophil (SAPPA).
  • ANCA levels in a sample from an individual can be determined, for example, using an immunoassay such as an enzyme-linked immunosorbent assay (ELISA) with alcohol-fixed neutrophils.
  • ELISA enzyme-linked immunosorbent assay
  • ASCA e.g., ASCA-lgA and/or ASCA-IgG
  • ASCA-lgA antibodies of the immunoglobulin A isotype that react specifically with S. cerevisiae.
  • ASCA-IgG antibodies of the immunoglobulin G isotype that react specifically with S. cerevisiae.
  • an antigen specific for ASCA can be any antigen or mixture of an- tigens that is bound specifically by ASCA-lgA and/or ASCA-IgG.
  • ASCA antibodies were initially characterized by their ability to bind S. cerevisiae, those of skill in the art will understand that an antigen that is bound specifically by ASCA can be obtained from S. cerevisiae or from a variety of other sources as long as the antigen is capable of binding specifically to ASCA antibodies.
  • the invention also relates to protein and nucleic acid molecules corresponding to the sequences described herein, for example proteins or nucleic acid molecules comprising or consisting of said sequences.
  • the determination of autoantibodies to the CHI3L1 or use thereof in an ELISA as a solid-phase antigen in serological diagnostics of the diseases described herein has neither been considered nor mentioned in the prior art.
  • the invention relates to a method wherein human IgA, IgM and/or IgG anti- body autoimmune diseases are detected.
  • CHI3L1 As used herein, the term "CHI3L1 ", "CHI3L1-isoform”, “CHI3L1-antigen”, “CHI3L1-molecule”, “CHI3L1-protein”, “CHI3L1-peptide” or “CHI3L1-autoantigen”, or other CHI3L1 -referencing phrase relates to the CHI3L1 protein or substantially similar or functionally analogous versions of the sequence SEQ ID NO 1 (and/or preferably SEQ ID NO 2, 3 and/or 4) as disclosed herein.
  • the CHI3L1 is of human, animal, recombinant or synthetic origin.
  • the CHI3L1 in accordance with one or more of the sequences disclosed herein is bound to a solid phase. Binding of CHI3L1 in accordance with one or more of the sequences disclosed herein to the solid phase can be effected via a spacer. All those chemical compounds having suitable structural and functional preconditions for spacer function can be used as spacers as long as they do not modify the binding behavior in such a way that binding of the CHI3L1 autoantibody in accordance with one or more of the sequences disclosed herein is adversely affected.
  • the molecule comprises a linker or spacer se- lected from the group of a-aminocarboxylic acids as well as homo- and heterooligomers thereof, ⁇ , ⁇ -aminocarboxylic acids and branched homo- or heterooligomers thereof, other amino acids, as well as linear and branched homo- or heterooligomers; amino-oligoalkoxyalkyl- amines; maleinimidocarboxylic acid derivatives; oligomers of alkylamines; 4-alkylphenyl derivatives; 4-oligoalkoxyphenyl or 4-oligoalkoxyphenoxy derivatives; 4-oligoalkylmercaptophenyl or 4-oligoalkylmercaptophenoxy derivatives; 4-oligoalkylaminophenyl or 4-oligoalkylaminophenoxy derivatives; (oligoalkylbenzyl)phenyl or 4-(oligoalkylbenzyl)phenoxy derivatives, as well as 4- (oligoalkylbenzyl)
  • the above-described detection method on a solid phase, for example by connection of the CHI3L1 molecule to the solid phase via a linker, in which case the storability of the peptide is advantageously increased as a result of the surprisingly stable linkage of the CHI3L1 antigen to the solid phase.
  • the CHI3L1 molecule is used as a soluble or solid phase-bound autoantigen for direct or indirect autoantibody detection or removal in stool and/or body fluids, especially blood and/or serum, in which case the use of the CHI3L1 molecule in accordance with one or more of the sequences as disclosed herein was found particu- larly advantageous.
  • sequences according to the present application, or the peptides which can be generated therefrom are immobilized. More specifically, the solid phase-bound CHI3L1 molecule in accordance with one or more of the sequences as disclosed herein is bound to organic, inorganic, synthetic and/or mixed polymers, preferably agarose, cellulose, silica gel, polyamides and/or polyvinyl alcohols.
  • immobilization is understood to involve various methods and techniques to fix the peptides on specific carriers, e.g. according to WO 99/56126 or WO 02/26292.
  • immobilization can serve to stabilize the peptides so that their activity would not be reduced or adversely modified by biological, chemical or physical exposure, especially during storage or in single- batch use.
  • Immobilization of the peptides allows repeated use under technical or clinical routine conditions; furthermore, a sample - preferably blood components - can be reacted with at least one of the peptides according to the invention in a continuous fashion.
  • this can be achieved by means of various immobilization techniques, with binding of the peptides to other peptides or molecules or to a carrier proceeding in such a way that the three-dimensional structure - particularly in the active center mediating the interaction with the autoantibodies - of the corresponding molecules, especially of said peptides, would not be changed.
  • there is no loss in specificity to the autoantibodies of patients as a result of such immobilization.
  • three basic methods can be used for immobilization:
  • crosslinking in crosslinking, the peptides are fixed to one another without adversely affecting their activity. Advantageously, they are no longer soluble as a result of such crosslinking.
  • binding to a carrier proceeds via adsorption, ionic binding or covalent binding, for example. Such binding may also take place inside microbial cells or liposomes or other membranous, closed or open structures.
  • the peptides are not adversely affected by such fixing. For example, multiple or continuous use of carrier-bound peptides is possible with advantage in clinical diagnosis or therapy.
  • inclusion in the meaning of the invention especially proceeds in a semipermeable membrane in the form of gels, fibrils or fibers.
  • encapsulated peptides are separated from the surrounding sample solution by a semipermeable membrane in such a way that interaction with the autoantibodies or fragments thereof still is possible.
  • Various methods are available for immobilization, such as adsorption on an inert or electrically charged inorganic or organic carrier.
  • such carriers can be porous gels, aluminum oxide, bentonite, aga- rose, starch, nylon or polyacrylamide. Immobilization proceeds via physical binding forces, frequently involving hydrophobic interactions and ionic binding.
  • binding can be improved as a result of electrostatic binding forces between the charged groups of the peptides and the carrier, e.g. by using ion exchangers, particularly Se- phadex.
  • the carriers may have reactive groups forming homopolar bonds with amino acid side chains.
  • Suitable groups in peptides are carboxy, hydroxy and sulfide groups and especially the terminal amino groups of lysines.
  • Aromatic groups offer the possibility of diazo coupling.
  • the surface of microscopic porous glass particles can be activated by treatment with silanes and subsequently reacted with peptides.
  • hydroxy groups of natural polymers can be activated with bromocyanogen and subsequently coupled with peptides.
  • a large number of peptides can undergo direct covalent binding with polyacrylamide resins.
  • Inclusion in three-dimensional networks involves inclusion of the peptides in ionotropic gels or other structures well-known to those skilled in the art. More specifically, the pores of the matrix are such in nature that the peptides are retained, allowing interaction with the target molecules.
  • crosslinking the peptides are converted into polymer aggregates by crosslinking with bifunctional agents.
  • Such structures are gelatinous, easily deformable and, in particular, suitable for use in various reactors. By adding other inactive components such as gelatin in crosslinking, advantageous improvement of mechanical and binding properties is possible.
  • microencapsulation the reaction volume of the peptides is restricted by means of membranes. For example, microencapsulation can be carried out in the form of an interracial polymerization.
  • the peptides are made insoluble and thus reusable.
  • immobilized peptides are all those peptides being in a condition that allows reuse thereof. Restricting the mobility and solubility of the peptides by chemical, biological or physical means advantageously results in lower process cost, particularly when eliminating autoantibodies from blood components.
  • the invention also relates to a diagnostic kit for the determination of autoimmune diseases, comprising a CHI3L1 molecule in accordance with one or more of the sequences as disclosed herein.
  • the diagnostic kit optionally includes instructions concerning combining the contents of the kit and/or providing a formulation for the detection of inflammatory bowel diseases, particularly Crohn's disease, autoimmune disease of the liver and/or ulcerative colitis.
  • the instruction can be in the form of an instruction leaflet or other medium providing the user with information as to the type of method wherein the substances mentioned are to be used.
  • the information need not necessarily be in the form of an instruction leaflet, and the information may also be imparted via the Internet, for example.
  • one advantageous effect of such a kit is, for instance, that he or she, without directly addressing a physician, can determine the actual state of a disease even during a journey and optionally adapt diet and ac- tivities accordingly.
  • Enyzme-Linked Immunosorbent Assay for the detection of CHI3L1 antibodies.
  • the surface of the reaction vessel is coated with the CHI3L1 antigen (1 ).
  • unspecific binding sites are blocked with suitable components (here BSA) (2).
  • BSA suitable components
  • patient material In addition to the antibodies against CHI3L1 , the patient material (or sample) also contains many different other antibodies and proteins, but only CHI3L1-specific antibodies can interact with the bound protein (3).
  • a detection antibody is used which can bind the antigen-specific antibodies (4).
  • the detection antibody is conjugated with the horseradish peroxidase (HRPO), which is responsible for the reaction of an added substrate in the last step.
  • HRPO horseradish peroxidase
  • An embodiment of the invention is the Enyzme-Linked Immunosorbent Assay (ELISA) as shown in Fig. 1. This assay is performed essentially in 5 steps:
  • the antigen (CHI3L1 ) is applied to the surface of the reaction vessels and binds to it. Excess and unbound antigen is removed after the reaction by washing the reaction vessels.
  • Unspecific binding sites e.g. Free areas of the vessel floor are blocked with suitable sub- stances (e.g., bovine serum albumin (BSA), skimmed milk) so that in the next step only the antigen is available as a potential binding partner. Excess and unbound substances are removed by washing.
  • suitable sub- stances e.g., bovine serum albumin (BSA), skimmed milk
  • the patient material to be examined e.g., blood serum
  • the patient material to be examined e.g., blood serum
  • antibodies specifically directed against CHI3L1 can interact with it so that all other components can be removed. Excess and unbound components are removed by washing.
  • a secondary antibody which in turn is specific to certain antibody structures (e.g., heavy chain of IgG or IgA or secretory component of secretory IgA).
  • this antibody is conjugated with an enzyme, Horseradish Peroxidase (HRPO). Excess and unbound secondary antibodies are removed by washing.
  • HRPO Horseradish Peroxidase
  • the detection is performed.
  • a substrate is placed in the reaction vessel. This is typically 3, 3', 5, 5'-tetramethylbenzidine (TMB), which assumes a blue colour by activation with HRPO.
  • TMB 3, 3', 5, 5'-tetramethylbenzidine
  • HRPO 3', 5, 5'-tetramethylbenzidine
  • this reaction is stopped with sulfuric acid and a colour change from blue to yellow takes place.
  • the solution has an absorption maximum of 450 nm. At this wavelength, the optical density of the solution is determined. This varies depending on the amount of the antigen-specific antibody initially bound and is used for the subsequent evaluation of the results.
  • CD Crohn's disease
  • UC ulcerative colitis
  • AIH autoimmune hepatitis
  • PSC primary sclerosing cholangitis
  • reaction vessels were coated with CHI3L1 protein, unspecific binding was blocked, following incubation with diluted serum samples and HRPO-conjugated secondary detection antibody. After addition of TMB, the optical density (OD) was determined. Samples were measured in duplicates.
  • Table 1 Prevalence of positive and negative sera of patients with inflammatory bowel and autoimmune liver disorders as well as controls in the anti-
  • Table 2 Statistical analysis of anti-CHI3L1 (aCHI3L1 ) ELISA data.
  • MC Crohn's Disease
  • CU colitis ulcerosa
  • PSC primary sclerosing cholangitis
  • AIH auto-immune hepatitis
  • slgA secretory IgA
  • Johansen JS Jensen HS, Price PA. A new biochemical marker for joint injury. Analysis of YKL-40 in serum and synovial fluid. Br J Rheumatol. 1993 Nov;32(1 1 ):949-55.
  • Johansen JS Williamson MK
  • Rice JS Price PA. Identification of proteins secreted by human osteoblastic cells in culture. J Bone Miner Res Off J Am Soc Bone Miner Res. 1992 May;7(5):501-12.

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Abstract

L'invention concerne un procédé in vitro de diagnostic, de pronostic, d'évaluation de risque, de surveillance, de guidage thérapeutique et/ou de contrôle thérapeutique d'un trouble auto-immun du système digestif, tel qu'une maladie intestinale inflammatoire et une maladie hépatique auto-immune, dans lequel des auto-anticorps qui se lient à la protéine-1 de type chitinase-3 (CHI3L1) sont détectés en tant que marqueur d'une telle maladie. L'invention concerne donc l'utilisation de CHI3L1 en tant qu'auto-antigène dans des diagnostics auto-immuns dans des maladies du système digestif. L'invention concerne un kit et un système pour permettre de mettre en œuvre le procédé.
PCT/EP2018/058219 2017-07-20 2018-03-29 Utilisation de protéine-1 de type chitinase-3 (chi3l1) en tant qu'auto-antigène dans des troubles auto-immuns du système digestif Ceased WO2019015814A1 (fr)

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CN117368494A (zh) * 2022-12-09 2024-01-09 元典(天津)新技术有限公司 一种用于肝纤维化和肝硬化检测的试剂盒

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