Method for detecting inflammatory intestinal diseases

Abstract

The present invention is related to a method for determining or confirming inflammatory bowel disease (IBD) in a subject, the method comprising detecting the amount of keratin 18 (K18) and / or keratin 19 (K19) mRNA or protein in a biological sample obtained from said subject. The present method can also be used for differentiating microscopic colitis from IBD.

Description

Method for detecting inflammatory intestinal diseasesFIELD

[0001] The present invention relates generally to diagnostics methods and particularly to methods for detecting intestinal inflammation and identifying the disease based on disease-specific changes in intestinal keratin expression.BACKGROUND

[0002] The incidence of intestinal diseases and disorders has been growing for decades. While the severity of different gut disorders varies greatly from life threatening to transient, they share many similar symptoms, making diagnosis challenging. Inflammatory bowel disease (IBD) is among the most serious gut diseases and the number of IBD patients is increasing globally, currently affecting millions of patients. IBD also multiplies the risk for colorectal cancer - another serious gut disease. Less severe intestinal disorders and diseases, such as irritable bowel syndrome (IBS) and microscopic colitis have a significant prevalence as well. It is estimated that globally nearly 10% of the population have symptoms of IBS. Rapid and accurate non-invasive diagnostic methods are needed to differentiate between gut diseases, to select the most efficient treatments and to avoid unnecessary interventions.

[0003] Calprotectin concentration in stool has been used as a “golden standard” marker for IBD associated inflammation. However, calprotectin does not distinguish between the subtypes of IBD, or evaluate the severity or prediction of the disease (State et al. 2021, Freeman et al. 2019). The colonic epithelial barrier is often severely compromised in IBD immune cell flux (De Souza and Fiocchi, 2016), witnessed by erosion, edema and later regeneration and hyperplasia to reinstate cellular integrity and tissue homeostasis. Currently, there are no valid colon epithelial cell produced compounds utilized to indicate the local inflammatory responses, similar to, e.g., liver epithelia produced CRP.

[0004] Among the integrity maintaining epithelial cell components, intermediate filament keratins are key cytoskeletal proteins. There are 56 different keratins in humans and their expression profiles are tissue specific. Keratins 8, 18, 19 and 20 are the main keratins present in human colon and K23 can be found at minor levels. Typically, most tissue specific keratin patterns are stable in general. Keratin staining is utilized in cancer diagnostics to identify the tissue of origin of metastasis. To our knowledge keratin expression profiles of K18, K19, and K20 have not been used in the diagnostics of inflammatory intestinal diseases.

[0005] WO2023 156714 discloses a method for detecting and subtyping inflammatory intestinal diseases. In this method, an IBD diagnosis is based on keratin 7 expression.However, K7 is not present in healthy colon, thus, it differs from keratins 8, 18, 19, and 20 as a biomarker, since basal levels in colon of keratins 8, 18, 19, and 20 are clearly within a detectable area and therefore their upregulation and downregulation can be measured as well.SUMMARY OF THE INVENTION

[0006] The aim of this invention is to overcome the limitations in the diagnostic tools for inflammatory intestinal diseases. The invention is based on a novel finding that keratin 19 and 18 levels are altered in the colon of patients with inflammatory intestinal diseases, such as inflammatory bowel disease (IBD), compared to controls. Particularly, keratins 19 and 18 are upregulated or downregulated in said diseases and thus the level of keratins 19 and 18 is increased in colon of such patients. The present disclosure provides a method where changes in epithelial gene expression is used to diagnose inflammatory intestinal diseases. The present invention can also be utilized in predicting disease progression and designing personal medication for IBD patients.

[0007] According to the first aspect of the present invention, there is provided a method, preferably a non-invasive method, for determining or confirming an inflammatory intestinal disease or a risk thereof in a subject, the method comprising detecting the amount of keratin 19 (KI 9) and / or keratin 18 (KI 8) mRNA or protein in a biological sample obtained from said subject, and preferably comparing the detected amount to the baseline level of expression of keratin 19 (K19) and / or keratin 18 (K18) gene(s) in intestine, wherein an amount corresponding to upregulation or downregulation of keratin 19 (K19) and / or keratin 18 (KI 8) gene(s) compared to the baseline level of expression of said gene(s) in intestine indicates the presence or risk of said inflammatory intestinal disease in said subject.

[0008] According to the second aspect of the present invention, there is provided a method of treating inflammatory bowel disease (IBD) in a subject, wherein the method comprises steps of: a) determining or confirming inflammatory bowel disease (IBD) in said subject according to the method of the present disclosure; and b) administering an antiinflammatory drug, or an immune system suppressor treating inflammatory bowel disease (IBD) to said subject.

[0009] According to the third aspect of the present invention, there is provided an antiinflammatory drug or an immune system suppressor for use in the treatment of inflammatory bowel disease (IBD) in a subject, wherein inflammatory bowel disease (IBD) is determined or confirmed in said subject according to the method of the present disclosure, wherein saidanti-inflammatory drug is preferably a corticosteroid, and said immune system suppressor is preferably a TNF alpha blocker or a JAK inhibitor.

[0010] The invention is defined by the features of the independent claims. Some specific embodiments are defined in the dependent claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1. Keratins 8, 18, 19, and 20 are significantly upregulated in ulcerative colitis (UC), and K18 is also significantly upregulated in Crohn’s disease (CD). K8 and K19 are downregulated in both microscopic colitis (MC) diseases, collagenous colitis (CC) and lymphocytic colitis (LC). The decrease of K8 is more prominent in LC and for K19 in CC. K20 is also downregulated in CC. N(tot)=56. Quantitation carried out using QuPathbioimageanalysis software and for each keratin intestinal disease patients are compared to control patients without intestinal disease (CTRL) or between diseases. Each dot represents single patient, whiskers represent min / max, the line in the middle median value.P: *<0.05; **<0.01; ***<0.001.

[0012] Figure 2. Representative HE and immunohistological DAB stainings for the keratins (K) 8, 18, 19 and 20 of colon tissue from patients with IBD (both Crohn’s Disease (CD) and Ulcerative colitis (UC)) and microscopic colitis (MC, including both lymphocytic (LC) and collagenous colitis (CC)). HE indicates sections stained for hematoxylin and eosin.

[0013] Figure 3. Relative amounts of KRT8 (K8), KRT18 (K18), KRT19 (K19), and KRT20 (K20) mRNA are increased in stool from patients with inflammatory bowel disease (IBD) compared to control patients without intestinal disease (healthy, H). Assays were carried out using Taqman qPCR assay. N(tot)=19. Each dot represents a single patient, whiskers represent min / max, the line in the middle median value. * P<0.05, ns = not statistically significant.EMBODIMENTS

[0014] As used herein, the term "antibody" encompasses naturally occurring and engineered antibodies, as well as full length antibodies, functional fragments, or analogs thereof that are capable of binding e.g., the target immune checkpoint or epitope (e.g., retaining the antigen-binding portion). The antibody may be from any origin including, without limitation, human, humanized, animal or chimeric, and may be of any isotype with a preference for an IgGl or IgG4 isotype, and further may be glycosylated or non-glycosylated. The term antibody also includes bispecific or multispecific antibodies so long as the antibody(s) exhibit the binding specificity herein described.

[0015] The term "binder” within the context of the present disclosure may be understood as referring to polypeptides and other molecules, such as antibodies and aptamers or fragments thereof, having a potential capability of specifically binding other compounds and / or structures, in particular epitopes, more in particular peptidic epitopes in other proteins such as keratin 18 (K18) or 19 (K19). The binder may also be an oligonucleotide primer or probe specifically binding to KI 8 or KI 9 mRNA or cDNA derived from said KI 8 or KI 9 mRNA.

[0016] As used herein, the term “fragment” includes native peptides (either degradation products, synthetically synthesized peptides or recombinant peptides) and modified peptides, which may have, for example, modifications rendering the peptides more stable or less immunogenic. Such modifications include, but are not limited to, cyclization, N-terminus modification, C-terminus modification, peptide bond modification, backbone modification and residue modification. The fragment may also comprise further elongations, deletions, substitutions or insertions.

[0017] As used herein, the term “polypeptide” refers herein to any chain of amino acid residues, regardless of its length or post-translational modification (e.g., glycosylation or phosphorylation).

[0018] As used herein, the terms “subject,” “individual,” “host,” and “patient,” are used interchangeably herein to refer to an animal being treated with one or more exemplary compounds as taught herein, including, but not limited to, simians, humans, avians, felines, canines, equines, rodents, bovines, porcines, ovines, caprines, mammalian farm animals, mammalian sport animals, and mammalian pets. A suitable subject for various embodiments can be any animal, including a human, that is suspected of having, has been diagnosed as having, or is at risk of developing a disease that can be ameliorated, treated or prevented by administration of one or more compounds known in the art to treat IBD. Preferably, said subject or patient is not known or suspected to suffer from cancer.

[0019] As used herein “keratin 7” and “K7” means keratin type II cytoskeletal 7 protein. Keratin 7 is also known by the names cytokeratin 7 and sarcolectin. Keratin 7 is encoded by the KRT7 gene. The amino acid sequence of human keratin 7 is found in Genbank under accession number NP 005547.3 and mRNA sequence under accession number NM_005556.4.

[0020] As used herein “keratin 8” and “K8” means keratin type II cytoskeletal 8 also known as cytokeratin 8. The amino acid sequence of human keratin 8 is found in Genbank under accession number NP 001243211.1 and mRNA sequence under accession number NM_001256282.2.

[0021] As used herein “keratin 18” and “KI 8” means keratin type II cytoskeletal 18 also known as cytokeratin 18. The amino acid sequence of human keratin 18 is found in Genbank under accession number NP 000215.1 and mRNA sequence under accession number NM_199187.2.

[0022] As used herein “keratin 19” and “KI 9” means keratin type II cytoskeletal 19 also known as cytokeratin 19. The amino acid sequence of human keratin 19 is found in Genbank under accession number NP 002267.2 and mRNA sequence under accession number NM_002276.5.

[0023] As used herein “keratin 20” and “K20” means keratin type II cytoskeletal 20 also known as cytokeratin 20. The amino acid sequence of human keratin 20 is found in Genbank under accession number NP 061883.1 and mRNA sequence under accession number NM_019010.3.

[0024] The term "upregulation" as used herein in the context of upregulation of a gene refers to any process which results in an increase in the amount of a gene product, e.g., mRNA or protein in intestine (such as in bowel contents or stool) or intestinal tissue of a subject (such as in biopsy samples). The term "downregulation" as used herein in the context of downregulation of a gene refers to any process which results in a decrease in the amount of a gene product, e.g., mRNA or protein in intestine or intestinal tissue of a subject. The level of upregulation or downregulation of keratins can be measured by known procedures, some of which are described hereinafter, particularly in the Experimental Section.

[0025] The term “level of expression” refers herein to the amount of keratin mRNA or protein present in intestine (such as in bowel contents or stool) or intestinal tissue of a subject (such as in biopsy samples).

[0026] The term “baseline level of expression” refers herein to a pre-measured amount of keratin mRNA or protein (or a calculated average of pre-measured amounts) in intestine (such as in bowel contents or stool) or intestinal tissue of a subject (such as in biopsy samples) in a healthy population (i.e. a group of subjects without inflammatory intestinal diseases) or in any suitable population or group of subjects suffering from inflammatory intestinal diseases, such as microscopic colitis.

[0027] Intestinal diseases, such as inflammatory bowel disease, have been increasing globally. Rapid and accurate diagnosis methods are needed to estimate the severity of the disease so that the most efficient treatment can be selected. The present invention is directed to an ex vivo method for determining or confirming inflammatory intestinal disease or a risk thereof in a subject. The method is based on detecting the amount of keratin 18 and / or keratin 19 mRNA or protein in a biological sample obtained from a subject.

[0028] In some embodiments, the sample is selected from a stool sample or a gastrointestinal tract sample. Gastrointestinal tract sample may be, for example, a biopsy or a surgical removal. Preferably, the said gastrointestinal tract sample is a biopsy, more preferably a colon or small intestinal tissue biopsy. In an embodiment, a stool sample, as understood broadly in this disclosure, may also be any sample of bowel contents.

[0029] The term "inflammatory intestinal disease" as used herein refers to inflammation occurring in the intestines, and in a broad sense, may include all inflammatory diseases occurring in the intestines, such as infectious enteritis and ischemic bowel disease such as bacterial, viral, amoebic, or tuberculous enteritis, and the like; radiation enteritis, and the like. Examples of inflammatory intestinal disease include but are not limited to irritable bowel syndrome, inflammatory enteritis, microscopic colitis, such as collagenous colitis (CC) and lymphocytic colitis (LC), and inflammatory bowel disease and its subtypes.

[0030] In the present specification, the term "inflammatory bowel disease" (IBD) is used to mean a disease of multifactorial cause, wherein inflammation occurs in intestinal tissue and affects the surface layer of the alimentary canal mucosa of the large intestine, small intestine, etc., and part of the mucosa is thereby lost, and as a result, ulcers or erosions are developed. Inflammatory bowel disease may be chronic. Specific examples of inflammatory bowel disease may include ulcerative colitis (UC) and Crohn's disease (CD). Typical examples of the ulcerative colitis may include intractable ulcerative colitis, fulminant ulcerative colitis and drug-resistant ulcerative colitis.

[0031] In some embodiments, the said inflammatory intestinal disease is an inflammatory bowel disease (IBD) or its subtype. Preferably, the IBD is Crohn’s disease or ulcerative colitis, more preferably a drug-resistant ulcerative colitis. An increased presence (upregulation) of keratin 18 and / or keratin 19 protein or mRNA in the sample confirms that the sample is associated with IBD such as Crohn’s disease or ulcerative colitis.

[0032] In some embodiments, the method distinguishes inflammatory bowel disease subtypes, e.g., ulcerative Colitis and Crohn’s disease from collagenous colitis, microscopic colitis and from irritable bowel syndrome (IBS). An increased presence of K18 and / or K19protein and mRNA in the sample confirms that the sample is associated with inflammatory bowel disease subtypes. In other embodiments, the method comprises a step of differentiating microscopic colitis, preferably collagenous colitis or lymphocytic colitis, from inflammatory bowel disease, wherein the increased presence (i.e. upregulation) of KI 8 and / K19 protein or mRNA in the sample confirms that the sample is associated with inflammatory bowel disease. In other embodiments, a decreased presence (i.e. downregulation) of KI 8 and / KI 9 protein or mRNA in the sample confirms that the sample is associated with microscopic colitis, such as collagenous colitis (CC) and lymphocytic colitis (LC).

[0033] In some embodiments, the presence of KI 8 and / or KI 9 protein and mRNA in the biological sample is determined by immunohistochemistry or in situ hybridization, respectively. Preferably, the presence of K18 and / or K19 is determined by contacting said sample with a primary antibody specific to KI 8 and / or K19 and then the sample is visualized by further contacting said sample with a labelled secondary antibody binding to the primary antibody and a label-specific reagent. More preferably, the presence of the combination of the primary and secondary antibody is measured using an enzyme-linked immunosorbent assay (ELISA).

[0034] In some embodiments, the said ELISA is qualitative. In other embodiments, the said ELISA is quantitative. The method may comprise a step of diluting a stool sample in order to improve optical properties of the sample. In some embodiments, the sample is contacted with immobilized antibodies specific to the K18 and / or K19 protein to create a treated sample. Preferably, the treated sample is further contacted with enzyme-linked antibodies to create a readable sample. Most preferably, the optical density of said readable sample is determined at a suitable wavelength. In some embodiments, the method further comprises a step of generating a purified KI 8 and / or K19 protein standard curve. Preferably, optical density of the readable sample is compared to the standard curve to determine the concentration of the K18 and / or K19 protein in the sample, for example in a stool sample.

[0035] In other embodiments, the presence or expression of K18 and / or K19 is determined by preparing an RNA sample from the biological sample and detecting the presence and optionally the level of the K18 and / or K19 mRNA in said RNA sample. An increased presence of the KI 8 and / K19 mRNA indicates that said sample is associated with inflammatory bowel disease.

[0036] In some embodiments, the amount oftheK18 and orK19 protein orK18 and / or K19 mRNA that is detected in the biological sample is compared to the amount of the K18and / or KI 9 protein or KI 8 and / or KI 9 mRNA detected in corresponding samples taken from healthy population (i.e. population not suffering from intestinal diseases). Higher amount of the K18 and / or K19 protein or K18 and / or K19 mRNA detected in the biological sample than in the samples of healthy population confirms that the sample is associated with inflammatory bowel disease. In some embodiments, the amount of the K18 and / or K19 protein or KI 8 and or KI 9 mRNA that is detected in the biological sample is compared with a cut-off value provided by corresponding assays performed to a number of subjects from healthy population or patients suffering from microscopic colitis. A value above the cut-off is an indication that the subject has IBD or a risk for developing IBD.

[0037] The presence of K18 and / or K19 in the patient sample may be detected with any method suitable of protein detection. In some embodiments, the presence of keratin 18 or 19 (K18 / K19) protein in the biological sample is determined by flow cytometry, mass cytometry, nuclear magnetic resonance (NMR), lateral flow assay (see, e.g., WO2019215199) or by any immunofluorescence method.

[0038] In other embodiments, the method comprises an additional step, where the detection of K18 and / or K19 protein is combined with detection of a further biomarker in the samples. The further biomarker is selected from the group comprising keratin 7 (K7) protein, keratin 8 (K8) protein, keratin 16 (KI 6) protein, keratin 17 (KI 7) protein, keratin 20 (K20) protein, keratin 23 (K23) protein, keratin 24 (K24) protein, keratin 80 (K80) protein and calprotectin. More preferably, said further biomarker is K7, K8 and / or K20.

[0039] The present invention further relates to the use of a K18 and / or K19 -specific antibody for the detection of the presence of the KI 8 and / or KI 9 protein. In some embodiments, K18 and / or K19 is detected from a gastrointestinal tract sample or a stool sample with a K18 and / or K19 -specific antibody. Preferably, the said gastrointestinal tract sample is a colon tissue biopsy or a small intestinal tissue biopsy. In an embodiment said gastrointestinal tract sample is not a cancer sample or metastasis sample.

[0040] In other embodiments, the present disclosure is directed to a use of a KI 8 and / or K19 -specific binder for the detection of the presence of the K18 and / or K19 protein in a stool sample or a gastrointestinal tract sample such as a biopsy, said gastrointestinal tract sample not being a sample or biopsy of cancer or cancer metastasis, wherein said binder is preferably an antibody or aptamer. In a preferred embodiment, said gastrointestinal tract sample is a colon tissue biopsy or a small intestinal tissue biopsy. A further embodiment of the present disclosure is directed to a binder specific to K18 and / or K19 protein or K18 and / or K19 mRNA for use in a method of diagnosis of chronic inflammatory intestinaldisease or subtypes thereof, wherein said binder is preferably an antibody or aptamer for said protein or an oligonucleotide probe for said mRNA.

[0041] A variety of medical options exist for the treatment of Crohn’s disease and ulcerative colitis including anti-inflammatory drugs, immune system suppressors, and biologic therapies (biologies). Unfortunately, these agents are also associated with adverse events ranging from mild nuisance symptoms to potentially life-threatening complications including infections and malignancies. Therefore, it is important to avoid unnecessary interventions with said drugs in patients having similar symptoms as patients with Crohn’s disease and ulcerative colitis but actually caused by milder intestinal conditions, such as IBS or microscopic colitis.

[0042] Said anti-inflammatory drugs can be selected from corticosteroids, such as cortisone and cortisol. In an embodiment, said anti-inflammatory drug is selected from a group consisting of: budenoside, prednisone, prednisolone, and methyprednosolone. Said anti-inflammatory drug can also be mirikizumab, risankinzumab, ustekinumab, or vedolizumab.

[0043] Said immune system suppressor can be a TNF alpha blocker, IL-22 / IL-23 inhibitor, or a Janus kinase (JAK) inhibitor. Preferably, the TNFalpha blocker is selected from the group consisting of adalimumab, infliximab, certolizumab pegol, golimumab, etanercept, and apremilast. Preferably, the JAK inhibitor is selected from the group consisting of tofacitinib, filgotinib and upadacitinib.

[0044] In an embodiment, the present invention is directed to a method of treating inflammatory bowel disease (IBD) in a subject, wherein the method comprises steps of: a) determining or confirming inflammatory bowel disease (IBD) in said subject according to the method of any one of claims 1-21; and b) administering an anti-inflammatory drug or an immune system suppressor treating inflammatory bowel disease (IBD) to said subject.

[0045] In a preferred embodiment, said method of treating comprises the steps of:- obtaining a stool sample from a patient;- quantifying the amount of K18 and / or K19 mRNA mRNA in said stool sample;- comparing the quantified mRNA amountto a corresponding baseline level(s) of K18 and / or K19 mRNA in a stool sample from a healthy subject, a subject with microscopic colitis and / or a subject with irritable bowel syndrome (IBS);- detecting upregulation of K18 and / or K19 mRNA mRNA in said stool sample; and- initiating said treatment with an anti-inflammatory drug or an immune system suppressor.

[0046] It is to be understood that the embodiments of the invention disclosed are notlimited to the particular structures, process steps, or materials disclosed herein, but are extended to equivalents thereof as would be recognized by those ordinarily skilled in the relevant arts. It should also be understood that terminology employed herein is used for the purpose of describing particular embodiments only and is not intended to be limiting.

[0047] Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment.

[0048] As used herein, a plurality of items, structural elements, compositional elements, and / or materials may be presented in a common list for convenience. However, these lists should be construed as though each member of the list is individually identified as a separate and unique member. Thus, no individual member of such list should be construed as a de facto equivalent of any other member of the same list solely based on their presentation in a common group without indications to the contrary. In addition, various embodiments and example of the present invention may be referred to herein along with alternatives for the various components thereof. It is understood that such embodiments, examples, and alternatives are not to be construed as de facto equivalents of one another, but are to be considered as separate and autonomous representations of the present invention.

[0049] Furthermore, the described features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. In the following description, numerous specific details are provided, such as examples of lengths, widths, shapes, etc., to provide a thorough understanding of embodiments of the invention. One skilled in the relevant art will recognize, however, that the invention can be practiced without one or more of the specific details, or with other methods, components, materials, etc. In other instances, well-known structures, materials, or operations are not shown or described in detail to avoid obscuring aspects of the invention.

[0050] While the forgoing examples are illustrative of the principles of the present invention in one or more particular applications, it will be apparent to those of ordinary skill in the art that numerous modifications in form, usage and details of implementation can be made without the exercise of inventive faculty, and without departing from the principles and concepts of the invention. Accordingly, it is not intended that the invention be limited,except as by the claims set forth below.

[0051] The verbs “to comprise” and “to include” are used in this document as open limitations that neither exclude nor require the existence of also un-recited features. The features recited in depending claims are mutually freely combinable unless otherwise explicitly stated. Furthermore, it is to be understood that the use of "a" or "an", i.e. a singular form, throughout this document does not exclude a plurality.EXPERIMENTAL SECTIONMaterial and methods

[0052] Patient material. Patient cohort histological samples were obtained from the Auria Biobank (Turku, Finland). Transport, handling and storage of the paraffin-embedded patient tissue samples were carried out in a standard way according to the biobank guidelines. The medical history of every patient, relevant to IBD, was filed and information was stored in encoded format and thus kept anonymous. The research project was authorized by the Auria Biobank’s Scientific Steering Committee (AB17-6901) and Hospital District of Southwest Finland (T05 / 032 / 19). Additional biopsy samples have been collected in Orebro University Hospital and Turku University Hospital. Patient stool samples have also been collected in Helsinki University Hospital.

[0053] Patient stool samples were collected in a controlled clinical trial including adolescents with suspected or diagnosed UC, CD or other disease. Samples were immediately frozen at home in -20 °C and transported to laboratory where they were stored at - 70 °C until processing.

[0054] Immunohistochemistry (IHC) and pathological evaluation. Prior IHC staining, pathologist inspected H&E stained slides of every biopsy to confirm the original diagnosis, the adequate presence of intestinal epithelial cells and orientation with the focus to view several full top to bottom crypts. The control tissues were collected from patients with the exclusion criteria of IBD and neoplastic intestinal diseases. UC and CD samples were harvested during colectomy or as needle biopsy samples during endoscopy. CC, LC and IBS control samples were collected as a needle biopsy during endoscopic examination. Keratin changes are presented in Figure 1 as separated subdiseases (UC, CD, LC and CC) as well as main diseases (IBD and MC).

[0055] The tissue samples were fixed in 4 % phosphate-buffered formaldehyde and embedded in paraffin according to standard procedures. K8, KI 8, KI 9, and K20 IHC stainings were carried out from 5 pm rehydrated sections with antibody to K8 (clone CAM 5.2, Becton Dickinson); KI 8 (clone DC 10, Abeam), KI 9 (clone A53-B / A2.26, Cell Marque) and K20 (clone SP33, Roche). The protein visualization was carried out using anti-mouse secondary antibody and 3,3'-Diaminobenzidine (DAB) as a chromogen and hematoxylin counter-stain (Figure 2).

[0056] Proteins and RNA were extracted from frozen stool sample using a modified calprotectin extraction buffer and a Stool RNA extraction kit, respectively. The presence of keratin proteins (K8, K18, K19) in fecal samples was quantified by image analysis. mRNA levels (Figure 3) were studied using RT-PCR or qPCR assay.

[0057] For grading of the severity of IBD, histology was used as the main reference standard. The inflammation activity in samples were graded into four classes: no activity (remission), mild activity (ad cryptitis), moderate activity (crypt abscesses) and severe (erosion / ulcers) according to ECCO guidelines (Magro et al., 2013).

[0058] Digital image analysis. The slides were scanned (Pannoramic 1000, 3D HISTECH, Budapest, Hungary) and pathological changes were evaluated. The quantification of K8, K18, K19, and K20 positive cells and the mean intensity of cellular K8, K18, K19, and K20 were measured using QuPath 0.5.1 bioimage analysis application (Bankhead et al., 2017). The epithelial cell layer region of interest (ROI) to be quantified was selected manually excluding immune, mesenchymal, endothelial and muscle cells from at least two distinct areas per samples, both including lumen and crypts, full crypts being prioritized when available. This epithelial ROI was chosen to contain over 1000 epithelial cells per sample, identified using the QuPath cell detection tool. Cellular K8, K18, K19, and K20 expression is based on mean intensity of cellular DAB staining.

[0059] Statistical analysis. The difference between more than two groups was measured using Kruskal-Wallis test, followed by Dunn’s multiple comparison. The difference between two factors was measured using Mann Whitney test. The linear correlation of two factors was studied using linear regression analysis.CITATION LISTPatent LiteratureWO2023156714WO2019215199Non-patent LiteratureDe Souza, H.S.P., and Fiocchi, C. (2016). Immunopathogenesis of IBD: Current state of the art. Nat. Rev. Gastroenterol. Hepatol. 13, 13-27.Freeman, K., Willis, B.H., Fraser, H., Taylor-Phillips, S., and Clarke, A. (2019). Faecal calprotectin to detect inflammatory bowel disease: A systematic review and exploratory meta-analysis of test accuracy. BMJ Open 9, 1-11.Magro, F., Langner, C., Driessen, A., Ensari, A., Geboes, K., Mantzaris, G.J., Villanacci, V., Becheanu, G., Nunes, P.B., Cathomas, G., et al. (2013). European consensus on the histopathology of inflammatory bowel disease. J. Crohn’s Colitis 7, 827-851.State, M., Negreanu, L., Voiosu, T., Voiosu, A., Balanescu, P., and Mateescu, R.B. (2021). Surrogate markers of mucosal healing in inflammatory bowel disease: A systematic review. World J. Gastroenterol. 27, 1828-1840.

Claims

CLAIMS1. Method for determining or confirming an inflammatory intestinal disease or a risk thereof in a subject, the method comprising detecting the amount of keratin 19 (KI 9) and / or keratin 18 (KI 8) mRNA or protein in a biological sample obtained from said subject, and preferably comparing the detected amount to the baseline level of expression of keratin 19 (K19) and / or keratin 18 (KI 8) gene(s) in intestine, wherein an amount corresponding to upregulation or downregulation of keratin 19 (KI 9) and / or keratin 18 (KI 8) gene(s) in the sample compared to the baseline level of expression of said gene(s) in intestine indicates the presence or risk of said inflammatory intestinal disease in said subject.

2. The method according to claim 1, wherein said inflammatory intestinal disease is inflammatory bowel disease (IBD), wherein an amount corresponding to upregulation of keratin 19 (K19) and / or keratin 18 (K18) gene(s) in the sample compared to i) the baseline level of expression of said gene(s) in intestine and / or compared to ii) the level of expression of keratin 19 (K19) and / or keratin 18 (K18) gene(s) in intestine of patients with microscopic colitis indicates the presence or risk of IBD in said subject.

3. The method according to claim 2, wherein said inflammatory bowel disease (IBD) is Crohn’s disease or ulcerative colitis.

4. The method according to claim 1, wherein said inflammatory intestinal disease is microscopic colitis, wherein an amount corresponding to downregulation of keratin 19 (K19) and / or keratin 18 (KI 8) gene(s) in the sample compared to i) the baseline level of expression of said gene(s) in intestine and / or compared to ii) the level of expression of keratin 19 (K19) and / or keratin 18 (KI 8) gene(s) in intestine of patients with inflammatory bowel disease (IBD) indicates the presence or risk of microscopic colitis in said subject.

5. The method according to claim 4, wherein said microscopic colitis is collagenous colitis or lymphocytic colitis.

6. The method according to any one of claims 1-5, wherein the sample is a stool sample.

7. The method according to any one of claims 1-5, wherein the sample is a gastrointestinal tract sample, preferably a biopsy, more preferably a colon or small intestinal tissue biopsy.

8. The method according to claim 1, wherein said inflammatory intestinal disease is inflammatory bowel disease (IBD) such as Crohn’s disease or ulcerative colitis, wherein an increased amount of K19 and / or K18 protein or mRNA in the sample confirms that the sample is associated with IBD, wherein the amount of the K19 and / or K18 protein or K19 and / or KI 8 mRNA detected in said biological sample is compared with a cut-off value provided by corresponding assays performed to a number of subjects from healthy population, patients with microscopic colitis and / or patients with irritable bowel syndrome (IBS), wherein a value above the cut-off is an indication that the subject has IBD or a risk of developing IBD.

9. The method according to claim 8 comprising a further step of distinguishing inflammatory bowel disease (IBD) from microscopic colitis and from irritable bowel syndrome, wherein an increased amount of K19 and / or K18 protein or K19 and / or K18 mRNA in the sample confirms that the sample is associated with inflammatory bowel disease (IBD), wherein the amount of the K19 and / or KI 8 protein orK19 and / or KI 8 mRNA detected in said biological sample is compared with a cut-off value provided by corresponding assays performed to a number of subjects from patients with microscopic colitis and / or patients with irritable bowel syndrome (IBS), wherein a value above the cut-off is an indication that the subject has IBD instead of microscopic colitis or irritable bowel syndrome.

10. The method according to any one of claims 1-9, wherein the presence of K19 and / or K18 protein in the biological sample is determined by immunoassays such as ELISA, immunohistochemistry or immunofluorescence methods.

11. The method according to claim 10, wherein the presence of KI 9 and / or KI 8 protein in the biological sample is determined by contacting said sample with a primary antibody or other binder specific to K19 and / or K18 protein and then the sample is visualized by further contacting said sample with a labelled secondary antibody or other labelled binder binding to the primary antibody or other binder and a label-specific reagent.

12. The method according to any one of claims 1-11, wherein the amount of K19 and / or K18 protein in the biological sample is determined by preparing an RNA or cDNA sample from said biological sample and detecting the amount of the K19 and / or K18 mRNA or cDNA in said RNA or cDNA sample, respectively, wherein an increased presence of the K19 and / or KI 8 mRNA or cDNA indicates that said sample is associated with inflammatory bowel disease, or wherein decreased amount of the K19 and / or K18 protein or K19 and / or K18 mRNA detected in said biological sample than in the samples of healthy population confirms that the sample is associated with microscopic colitis.

13. The method according to claim 12, wherein increased amount of the KI 9 and / or KI 8 protein or K19 and / or K18 mRNA detected in said biological sample than in the samples of healthy population and / or patients with microscopic colitis confirms that the sample is associated with inflammatory bowel disease.

14. The method according to any one of claims 1-11, wherein the presence of K19 and / or KI 8 DNA in the biological sample is determined by preparing a DNA sample from said biological sample and detecting the amount of the KI 9 and / or KI 8 DNA in said sample, wherein increased amount of the K19 and / or K18 DNA detected in said biological sample than in the samples of healthy population and / or patients with microscopic colitis confirms that the sample is associated with inflammatory bowel disease or wherein decreased amount of the KI 9 and / or KI 8 DNA detected in said biological sample than in the samples of healthy population confirms that the sample is associated with microscopic colitis.

15. The method according to claim 11, wherein the presence of the combination of the primary and secondary antibody or other binder is measured using an enzyme-linked immunosorbent assay.

16. The method according to claim 15, wherein the presence of the K19 and / or K18 protein is / are measured quantitatively.

17. The method according to claim 6 further comprising a step of diluting or mixing a stool sample with reagents or a buffer in order to improve sample properties such as optical properties.

18. The method according to claim 15 further comprising a step of contacting the sample with immobilized antibodies or other binders specific to the KI 9 and / or KI 8 protein to create a treated sample.

19. The method according to claim 18 further comprising a step of contacting said treated sample with enzyme-linked antibodies or other binders to create a readable sample.

20. The method according to claim 19 further comprising a step of determining the optical density of said readable sample at suitable wavelength.

21. The method according to claim 16 further comprising a step of generating a purified KI 9 and / or KI 8 protein standard curve.

22. The method according to claim 17 further comprising a step of comparing said optical density of said readable sample to said standard curve to determine the concentration of the K19 and / or K18 protein in said stool sample.

23. The method according to any one of claims 1-14, wherein the presence of K19 and / or KI 8 protein in the biological sample is determined by flow cytometry or mass cytometry.

24. The method according to any one of claims 1-14, wherein the presence of K19 and / or KI 8 protein in the biological sample is determined by lateral flow assay, or other point of care assay, or nuclear magnetic resonance (NMR).

25. The method according to any one of claims 1-24, wherein the method comprises a step of detecting the presence or amount of a further biomarker selected from the group consisting of: keratin 7 (K7) protein, keratin 8 (K8) protein, keratin 16 (KI 6) protein, keratin 17 (KI 7) protein, keratin 20 (K20) protein, keratin 23 (K23) protein, keratin 24 (K24) protein, keratin 80 (K80) protein and calprotectin or a mRNA thereof; in said biological sample.

26. The method according to any one of claims 6-25 comprising:- obtaining a stool sample from a patient;- quantifying the amount of K19 and / or K18 mRNA in said stool sample;- comparing the quantified mRNA amount to a corresponding baseline level(s) of K19 and / or K18 mRNA in a stool sample from a healthy subject, a subject with microscopic colitis and / or a subject with irritable bowel syndrome (IBS).

27. Method of treating inflammatory bowel disease (IBD) in a subject, wherein the method comprises steps of a) determining or confirming inflammatory bowel disease (IBD) in said subject according to the method of any one of claims 1-26; and b) administering an anti-inflammatory drug or an immune system suppressor treating inflammatory bowel disease (IBD) to said subject.

28. The method according to claim 27, wherein the method comprises- obtaining a stool sample from a patient;- quantifying the amount of K19 and / or K18 mRNA in said stool sample;- comparing the quantified mRNA amountto a corresponding baseline level(s) of K19 and / or K18 mRNA in a stool sample from a healthy subject, a subject with microscopic colitis and / or a subject with irritable bowel syndrome (IBS);- detecting upregulation of K19 and / or K18 mRNA in said stool sample; and- initiating said treatment with an anti-inflammatory drug or an immune system suppressor.

29. The method according to claim 27 or 28, wherein said anti-inflammatory drug is a corticosteroid, preferably cortisone or cortisol, or said drug is selected from the group consisting of budenoside, prednisone, prednisolone, and methyprednosolone.

30. The method according to claim 27 or 28, wherein said immune system suppressor is a TNF alpha blocker, an IL-22 / IL-23 inhibitor, or a Janus kinase (JAK) inhibitor.

31. The method according to claim 30, wherein the TNF alpha blocker is selected from the group consisting of adalimumab, infliximab, certolizumab pegol, golimumab, etanercept, and apremilast.

32. The method according to claim 30, wherein the JAK inhibitor is selected from the group consisting of tofacitinib, filgotinib and upadacitinib.

33. The method according to claim 27 or 28, wherein said anti-inflammatory drug is mirikizumab, risankinzumab, ustekinumab, or vedolizumab.

34. An anti-inflammatory drug or an immune system suppressor for use in the treatment of inflammatory bowel disease (IBD) in a subject, wherein inflammatory bowel disease (IBD) is determined or confirmed in said subject according to the method of any one of claims 1- 26, wherein said anti-inflammatory drug is preferably a corticosteroid, and said immune system suppressor is preferably biologies such as a TNF alpha blocker, an IL-22 / IL-23 inhibitor, or a JAK inhibitor.

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