Immunoassay for inflammatory bowel disease

Abstract

The present invention relates to methods of immunoassay for detecting or monitoring inflammatory bowel disease or a severity thereof in a patient. In certain embodiments, the inflammatory bowel disease may be ulcerative colitis.

Description

[0001] Immunoassay for Inflammatory Bowel Disease Field of invention The present invention relates to methods of immunoassay for detecting or monitoring inflammatory bowel disease or a severity thereof in a patient. In certain embodiments, the inflammatory bowel disease may be ulcerative colitis. Background Inflammatory bowel disease (IBD) is a chronic immune-mediated disease of the gastrointestinal (GI) tract, encompassing Crohn’s disease (CD) and ulcerative colitis (UC). These two diseases differ by their location and depth of involvement; UC involves superficial inflammation of the colonic mucosa, while CD causes transmural ulceration of any portion of the GI tract. IBD develops in genetically susceptible individuals due to an abnormal immune response to the gut microbiota, where excessive inflammatory reactions lead to a continued deterioration of the intestinal barrier[1]. Immune cells are considered to be the main pathogenic factor in IBD, as they initiate and sustain inflammation by expressing and secreting a plethora of different cytokines and proteases[2]. For this reason, immune cells are the key targets of biologic treatments in IBD. Although disease management has substantially improved with the introduction of anti-tumor necrosis factor (TNF) antibodies, up to 40% of patients with IBD experience non-response or loss of response after induction therapy[3]. Better patient profiling and monitoring is therefore needed in order to provide optimal treatment. Improving the detection of subclinical ulceration is essential since early-stage IBD presents a unique window of opportunity for intervention, as the disease becomes a self-sustaining process once deep inflammation becomes established. As noted above, disruption of the intestinal barrier is a hallmark of IBD as it leads to the exposure of intestinal microbes, furthering the initial inflammation. Therefore, the extracellular matrix (ECM) is of high importance for structural integrity of the intestinal tissue – especially the intestinal basement membrane (BM). Acting as a front-line defense, the intestinal BM is a collagen rich matrix with type IV collagen being the major collagenous component[4]. Type VII collagen is the main component of the anchoring fibrils that connects the basement membrane to the underlying interstitial matrix. It consists of three identical alpha-1 chains with two non-collagenous (NC) domains and a central collagenous triple helical domain. It has been identified in the basement membranes of skin and mucous membranes[5]. Type VII collagen has mainly been investigated for its role in dystrophic epidermolysis bullosa, a severe skin disease. Mutations in the collagen type VII alpha-1 chain leads to the formation of abnormal, diminished or absent anchoring fibrils which causes separation of epidermis from dermis and thus skin blistering[5]. Type VII collagen has also been identified as the protein at fault in epidermolysis bullosa acquisita, an autoimmune disease-causing blistering of the skin and mucous membranes. It is caused by IgG autoantibodies directed at the collagen type VII NC1 domain[6]. Autoimmunity to collagen type VII has also been associated with inflammatory bowel disease and bullous systemic lupus erythematosus[7-8]. Sand et al have described the development of a competative enzyme-linked immunosorbent assay (ELISA) for detecting and quantifying the levels of a biomarker of type VII collagen turnover known as ”C7M”, and have shown that by using this assay to measure serological levels of C7M patients with chronic obstructive pulmonary disease (COPD) or systemic sclerosis SSc can be identified[9-10]. This C7M assay uses a monoclonal antibody that specifically binds to the C-terminus amino acid sequence GPPGPPGRLV (SEQ ID NO: 1) (i.e. “C7M”) in order to detect peptide fragments having this C-terminus amino acid sequence, which peptide fragments result from cleavage of collagen type VII between positions 1709 and 1710 of the alpha-1 chain. Nielsen et al have also described the use of the same C7M assay detect and determine the severity of hidradenitis suppurativa (HS)

[0011] . Summary of the Invention The present inventors have now determined and demonstrated that the levels of C7M in patient samples (and in particular patient serum samples) can be used identify patients with IBD in general, and UC in particular. Moreover, the inventors have now shown that the levels of C7M correlate with disease severity, such that the measured levels of C7M can be used to identify the severity of IBD in a patient. In this regard, the inventors have also now shown that in patients with IBD there is a correlation between baseline levels of C7M and the likelihood of the IBD going into remission in the future, such that measured levels of C7M can be used not just to determine the current level of severity of the disease but also its prognosis. Accordingly, in a first aspect, the present invention provides a method of immunoassay for detecting or monitoring inflammatory bowel disease (IBD) or a severity thereof in a patient, said method comprising: i) contacting a patient sample with a monoclonal antibody that specifically binds to the C-terminus amino acid sequence GPPGPPGRLV (SEQ ID NO: 1) (said sequence also being referred to herein as “C7M” or the “C7M target sequence”); ii) detecting and determining the amount of binding between said monoclonal antibody and peptides in the sample; and iii) correlating said amount of binding with values associated with normal healthy subjects and / or values associated with known disease severity and / or values obtained from said patient at a previous time point and / or with a predetermined cut-off value. In a preferred embodiment, the monoclonal antibody does not specifically bind to a peptide having the C-terminus amino acid sequence GPPGPPGRLVD (SEQ ID NO: 5) (i.e. an elongated version of the C7M target sequence extended at its C-terminus by the addition of an aspartic acid residue). Preferably, the ratio of the affinity of said antibody for the C7M target sequence to the affinity of said antibody for said elongated version of the target sequence is at least 10 to 1, and more preferably is at least 20 to 1, at least 30 to 1, at least 40 to 1, at least 50 to 1 or at least 100 to 1. In a preferred embodiment, the monoclonal antibody does not specifically bind to a peptide having the C-terminus amino acid sequence GPPGPPGRL (SEQ ID NO: 2) (i.e. a truncated version of the C7M target sequence truncated at its C-terminus by the removal of the last valine residue). Preferably, the ratio of the affinity of said antibody for the C7M target sequence to the affinity of said antibody for said truncated version of the target sequence is at least 10 to 1, and more preferably is at least 20 to 1, at least 30 to 1, at least 40 to 1, at least 50 to 1 or at least 100 to 1. In a preferred embodiment, the monoclonal antibody is raised against a synthetic peptide having the C-terminus amino acid sequence GPPGPPGRLV (SEQ ID NO: 1). For example, the monoclonal antibodies may be raised by: (a) immunizing a rodent (or other suitable mammal) with a synthetic peptide comprising the C-terminus amino acid sequence GPPGPPGRLV (SEQ ID NO: 1), which peptide may optionally be linked at its N- terminus to an immunogenic carrier protein (such as keyhole limpet hemocyanin (“KLH”)); (b) isolating and cloning a single antibody producing cell; and (c) assaying the resulting monoclonal antibodies to ensure that they have the desired specificity. An exemplary protocol of the development, production and characterization of suitable monoclonal antibodies is described in the Examples section, infra. As used herein the term “N-terminus” refers to an N-terminal peptide sequence at the extremity of a polypeptide, i.e. at the N-terminal end of the polypeptide, and is not to be construed as meaning in the general direction thereof. As used herein the term “C-terminus” refers to a C-terminal peptide sequence at the extremity of a polypeptide, i.e. at the C- terminal end of the polypeptide, and is not to be construed as meaning in the general direction thereof. As used herein, the terms “peptide” and “polypeptide” are used synonymously. As used herein the term “monoclonal antibody” refers to both whole antibodies and to fragments thereof that retain the binding specificity of the whole antibody, such as for example a Fab fragment, F(ab’)2 fragment, single chain Fv fragment, or other such fragments known to those skilled in the art. As is well known, whole antibodies typically have a "Y-shaped" structure of two identical pairs of polypeptide chains, each pair made up of one "light" and one "heavy" chain. The N- terminal regions of each light chain and heavy chain contain the variable region, while the C-terminal portions of each of the heavy and light chains make up the constant region. The variable region comprises three complementarity determining regions (CDRs), which are primarily responsible for antigen recognition. The constant region allows the antibody to recruit cells and molecules of the immune system. Antibody fragments retaining binding specificity comprise at least the CDRs and sufficient parts of the rest of the variable region to retain said binding specificity. In the present invention, a monoclonal antibody comprising any constant region known in the art can be used. In the case of mouse antibodies and human antibodies, the constant light chains are classified as either kappa or lambda light chains. Heavy constant chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. The IgG isotype has several subclasses, including, but not limited to IgGl, IgG2, IgG3, and IgG4 in the case of humans and IgGl, IgG2a, IgG2b, IgG2c and IgG3 in the case of mice. The monoclonal antibody may preferably be of the IgG isotype, including any one of the IgG subclasses. The CDR of an antibody can be determined using methods known in the art such as that described by Kabat et al. Antibodies can be generated from B cell clones. The isotype of the antibody can be determined by ELISA specific for IgM, IgG or IgA isotype, or subclass. The amino acid sequence of the antibodies generated can be determined using standard techniques. For example, RNA can be isolated from the cells, and used to generate cDNA by reverse transcription. The cDNA is then subjected to PCR using primers which amplify the heavy and light chains of the antibody. For example, primers specific for the leader sequence for all VH (variable heavy chain) sequences can be used together with primers that bind to a sequence located in the constant region of the isotype which has been previously determined. The light chain can be amplified using primers which bind to the 3’ end of the Kappa or Lambda chain together with primers which anneal to the V kappa or V lambda leader sequence. The full length heavy and light chains can be generated and sequenced. In such exemplary embodiments, the monoclonal antibody may preferably comprise one or more complementarity-determining regions (CDRs) selected from: CDR-L1: RSSQSLLYSNGITYLY (SEQ ID NO: 6) CDR-L2: QMSNLAS (SEQ ID NO: 7) CDR-L3: AQNLELPWT (SEQ ID NO: 8) CDR-H1: ENTMN (SEQ ID NO: 9) CDR-H2: GINPKIGGSIYNQKFQD (SEQ ID NO: 10) CDR-H3: GIYDEGWHFDV (SEQ ID NO: 11) Preferably the monoclonal antibody comprises at least 2,3,4,5 or 6 of the above listed CDR sequences. Preferably the monoclonal antibody has a light chain variable region comprising the CDR sequences: CDR-L1: RSSQSLLYSNGITYLY (SEQ ID NO: 6) CDR-L2: QMSNLAS (SEQ ID NO: 7) and CDR-L3: AQNLELPWT (SEQ ID NO: 8). Preferably the monoclonal antibody has a light chain that comprises framework sequences between the CDRs, wherein said framework sequences are substantially identical or substantially similar to the framework sequences between the CDRs in the light chain sequence below (in which the CDRs are shown in bold and underlined, and the framework sequences are shown in italics) RSSQSLLYSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSSSGSGTDFTLRISRVEA EDVGVYYCAQNLELPWT (SEQ ID NO: 12) Preferably the monoclonal antibody has a heavy chain variable region comprising the CDR sequences: CDR-H1: ENTMN (SEQ ID NO: 9) CDR-H2: GINPKIGGSIYNQKFQD (SEQ ID NO: 10) and CDR-H3: GIYDEGWHFDV (SEQ ID NO: 11). Preferably the monoclonal antibody has a heavy chain that comprises framework sequences between the CDRs, wherein said framework sequences are substantially identical or substantially similar to the framework sequences between the CDRs in the heavy chain sequence below (in which the CDRs are shown in bold and underlined, and the framework sequences are shown in italics) ENTMNWVKQSHGKSLEWIGGINPKIGGSIYNQKFQDKATLTVDKSSSTAYMELRSLTSDDSA VYYCARGIYDEGWHFDV (SEQ ID NO: 13) Preferably, the monoclonal antibody comprises the light chain variable region sequence: DIVMTQAAFSNPVTLGTSASISCRSSQSLLYSNGITYLYWYLQKPGQSPQLLIYQMSNLASG (CDRs bold and underlined; Framework sequences in italics) and / or the heavy chain variable region sequence: EVQLQQSGPELVKPGASVKISCKTSGYTFIENTMNWVKQSHGKSLEWIGGINPKIGGSIYNQ KFQDKATLTVDKSSSTAYMELRSLTSDDSAVYYCARGIYDEGWHFDVWGAGTTVTVSS (SEQ ID NO: 15) (CDRs bold and underlined; Framework sequences in italics) As used herein, the framework amino acid sequences between the CDRs of an antibody are “substantially identical” or “substantially similar” to the framework amino acid sequences between the CDRs of another antibody if they have at least 70%, 80%, 90% or at least 95% similarity or identity. The similar or identical amino acids may be contiguous or non- contiguous. The framework sequences may contain one or more amino acid substitutions, insertions and / or deletions. Amino acid substitutions may be conservative, by which it is meant the substituted amino acid has similar chemical properties to the original amino acid. A skilled person would understand which amino acids share similar chemical properties. For example, the following groups of amino acids share similar chemical properties such as size, charge and polarity: Group 1 Ala, Ser, Thr, Pro, Gly; Group 2 Asp, Asn, Glu, Gln; Group 3 His, Arg, Lys; Group 4 Met, Leu, Ile, Val, Cys; Group 5 Phe Thy Trp. A program such as the CLUSTAL program to can be used to compare amino acid sequences. This program compares amino acid sequences and finds the optimal alignment by inserting spaces in either sequence as appropriate. It is possible to calculate amino acid identity or similarity (identity plus conservation of amino acid type) for an optimal alignment. A program like BLASTx will align the longest stretch of similar sequences and assign a value to the fit. It is thus possible to obtain a comparison where several regions of similarity are found, each having a different score. Both types of analysis are contemplated in the present invention. Identity or similarity is preferably calculated over the entire length of the framework sequences. In preferred embodiments, the patient sample is a human patient sample. In preferred embodiments, the patient sample is a biofluid sample. Preferably, the sample is a blood-based sample, such as blood (whole blood), plasma or serum. In a particularly preferred embodiment, the sample is a serum sample. In preferred embodiments the immunoassay is a competition assay or a sandwich assay. The immunoassay may, for example, be a radio-immunoassay or an enzyme-linked immunosorbent assay (ELISA). Such assays are techniques known to the person skilled in the art. As used herein the term “amount of binding” refers to the quantification of binding between the antibody and peptides in the patient sample. Said quantification may for example be determined by comparing the measured values of binding in the patient sample against a calibration curve produced using measured values of binding in standard samples containing known concentrations of a peptide to which the antibody specifically binds, in order to determine the quantity of peptide to which the antibody specifically binds in the patient sample. Any suitable analytical method can be used for measuring the amount of binding. For example, an ELISA method can be used in which spectrophotometric analysis is used to measure the amount of binding both in the patient samples and when producing the calibration curve. The inflammatory bowel disease may for example be Crohn’s disease (CD) or ulcerative colitis (UC). In certain preferred embodiments, the inflammatory bowel disease is ulcerative colitis. In those embodiments where the method is a method for detecting or monitoring a severity of inflammatory bowel disease in a patient, the method may comprise detecting or monitoring a level of severity of inflammatory bowel disease and / or determining likely prognosis of the disease. For example, the method may comprise detecting or monitoring whether the patient has mild, moderate or severe CD or UC or a level of severity of CD or UC corresponding to a level of severity according to one of the standard classification systems for CD or UC (such as for example the Ulcerative Colitis Endoscopic Index of Severity (UCEIS), Mayo total score, Mayo Endoscopic Score (MES) or Montreal classification system). Alternatively or additionally, the method may comprise determining the likelihood of the inflammatory bowel disease going into remission within a set period of time. As used herein the term “predetermined cut-off value” means an amount of binding that is determined statistically to be indicative of a high likelihood of the disease in question (for example IBD in general, or CD or UC in particular) or a particular severity thereof in a patient, in that a measured value of the target peptide(s) in a patient sample that is at or above the statistical cut-off value corresponds to at least a 70% probability, preferably at least an 75% probability, more preferably at least an 80% probability, more preferably at least an 85% probability, more preferably at least a 90% probability, and most preferably at least a 95% probability of the presence of said disease or particular severity thereof. As used herein, the term “values associated with normal healthy subjects” means standardised quantities of binding determined by the methods described supra for samples from subjects considered to be healthy, i.e. without disease (e.g. without IBD); and the term “values associated with known disease severity” means standardised quantities of binding determined by the methods described supra for samples from patients known to have disease (e.g. IBD in general, or CD or UC in particular) of a known severity. In a second aspect, the present invention provides a method of treating inflammatory bowel disease (IBD) in a patient in need thereof, the method comprising: (a) carrying out a method of immunoassay in accordance with the first aspect of the present invention on a sample from a patient in order to detect whether said patient has IBD or a particular severity thereof; and (b) administering to the patient a therapy for the treatment of the disease if it is determined in step (a) that the patient has IBD or said severity thereof. For example, step (b) may comprise administering a therapy for the treatment of the disease if it is determined in step (a) that the patient has IBD, or has IBD of a particular level of severity, or has IBD that is unlikely (without therapeutic intervention) to go into remission within a set period of time. The therapy may be any therapy suitable for treating the inflammatory bowel disease in question. The therapy may for example comprise or consist of one or more surgeries, treatments, medicaments, or combinations thereof. Medicaments may be formulated for topical or systemic administration. Topical medicaments may for example be formulated as creams, foams, gels, lotions, or ointments for administration. Systemic medicaments may for example be formulated for enteral or parenteral administration. Surgeries may be curative surgeries, preventative surgeries, palliative surgeries and / or restorative surgeries. For example, where the inflammatory bowel disease is Crohn‘s disease suitable therapies may comprise one or more of: lifestyles changes such as dietary adjustments, elemental diet, proper hydration, smoking cessation; medicaments such as antibiotics, aminosalicylate anti-inflammatories, corticosteroids, 5-aminosalicylic acid (5-ASA), prednisone, azathioprine, 6-mercaptopurine, methotrexate, anti-TNF therapies (also referred to in the art as TNF inhibitors) such as monoclonal antibodies that inhibit the effects of TNF such as infliximab, adalimumab and certolizumab, and other biologics and antibodies such as vedolizumab, ustekinumab, and natalizumab; and combinations thereof. Where the inflammatory bowel disease is ulcerative colitis suitable therapies may comprise one or more of: medicaments such as aminosalicylates such as Mesalazine, Sulfasalazine, Balsalazide, Olsalazine, corticosteroids such as Cortisone, Prednisone, Hydrocortisone, Methylprednisolone, Budesonide, immunosuppressive drugs such as Mercaptopurine, Azathioprine, Methotrexate; and anti-TNF therapies (TNF inhibitors) such as Infliximab, Adalimumab and Golimumab, and other biologics and antibodies such as Tofacitinib and Vedolizumab; surgical procedures such as a partial or total colectomy; and combinations thereof. Figures Figure 1: C7M assay and antibody selectivity. The C7M ELISA and mAb are reactive towards to the target sequence (standard peptide), whereas no reactivity is exhibited towards the elongated sequence (elongated peptide) or an unrelated sequence (nonsense peptide). Figure 2: C7M serum levels in ulcerative colitis (UC) vs. healthy subjects (HS). The data is represented with interquartile range (IQR) with 10-90 % percentile. Asterisks (*) depict statistically significant differences: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 Figure 3: Correlations of C7M serum levels to different disease scores for ulcerative colitis (UC), namely Ulcerative Colitis Endoscopic Index of Severity (UCEIS), Mayo total score, and Mayo Endoscopic Score (MES). Spearman rho correlations were applied. Figure 4: C7M serum levels are elevated in relation to Montreal extent of disease, where E1 = prochtitis, E2 = leftsided, E3 = pan-colitis. Asterisks (*) depict statistically significant differences: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 Figure 5: ROC-curve analysis displaying the discriminative power of baseline serum levels of C7M to predict UC patients achieving remission at week 6 based on the Mayo total score. Examples The presently disclosed embodiments are described in the following Examples, which are set forth to aid in the understanding of the disclosure, and should not be construed to limit in any way the scope of the disclosure as defined in the claims which follow thereafter. The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the described embodiments, and are not intended to limit the scope of the present disclosure nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric. Materials and methods C7M monoclonal antibody development and production The production of a monoclonal antibody (mAb) specific for the C-terminus neo-epitope amino acid sequence GPPGPPGRLV (i.e. C7M) has been described previously (see references[9-10]the contents of which are incorporated herein in their entirety). However, to briefly summarize: Four to six-week-old Balb / C mice were immunized subcutaneously with 200 µL emulsified antigen and 50 µg of synthetic peptide (KLH-CGG-GPPGPPGRLV (SEQ ID NO: 3)) using Freund’s incomplete adjuvant. Immunizations were performed every second week until stable sera titer levels were reached. The mouse with highest serum titer was selected for fusion. The mouse was rested for one month and then boosted intravenously with 50 µg synthetic peptide in 100 µL 0.9% sodium chloride solution three days before isolation of the spleen for cell fusion. Mouse spleen cells were fused with SP2 / 0 myeloma fusion partner cells. The resulting hybridoma cells were cloned using a semi-solid medium method, transferred into 96-well microtiter plates for further growth and incubated in a CO2 incubator. Standard limited dilution was used to promote monoclonal growth. The sequence and CDRs of the selected monoclonal antibody were determined. The sequence of the chains are as follows (CDRs underlined and in bold; N-terminus signal peptide and C- terminus Constant region in italics): Heavy Chain Sequence MGWSWIFLFLLSGTAGVLSEVQLQQSGPELVKPGASVKISCKTSGYTFIENTMNWVKQSHGK SLEWIGGINPKIGGSIYNQKFQDKATLTVDKSSSTAYMELRSLTSDDSAVYYCARGIYDEGW HFDVWGAGTTVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLS SGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCIC TVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREE QFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKE QMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWE AGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO: 16) Light Chain Sequence MRFSAQLLGLLVLWIPGSTADIVMTQAAFSNPVTLGTSASISCRSSQSLLYSNGITYLYWYL QKPGQSPQLLIYQMSNLASGVPDRFSSSGSGTDFTLRISRVEAEDVGVYYCAQNLELPWTFG GGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLN SWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO: 17) Supernatant from antibody-producing hybridoma cells was collected. Monoclonal antibody was purified using HiTrap affinity columns (GE Healthcare Life Science, Little Chalfont, Buckinghamshire, United Kingdom) and labelled with horseradish peroxidase (HRP) using Lightning-LinkHRP Conjugation Kit (Innova Biosciences, Babraham, Cambridge, United Kingdom), according to the manufacturer’s instructions. C7M ELISA The developed competitive ELISA procedure that uses the above described C7M mAb has also been described previously (see again references[9-10]the contents of which are incorporated herein in their entirety). However, to again briefly summarize: Streptavidin-coated plates were coated with 100 µL / well of 2.5 ng / mL biotin-labelled coating peptide (Biotin-KK-GPPGPPGRLV (SEQ ID NO: 4) diluted in assay buffer (50mM TBS-BTB, 2g / L NaCl, pH 8.0) and incubated at 20oC, 300 rpm shaking for 30 minutes. Plates were washed five times in washing buffer (20 nM TRIS, 50 mM NaCl, pH 7.2). Sample or standard peptide (GPPGPPGRLV (SEQ ID NO: 1)) were added (20 µL / well) in double determinations and followed immediately by addition of 100 µL / well of 200 ng / mL HRP-labelled monoclonal antibody diluted in assay buffer and plates were incubated at 20oC, 300 rpm shaking for 3 hours. After incubation, plates were washed five times in washing buffer. A volume of 100 µL 3,3’,5,5’- tetramethylbenzidine (TMB) was added and incubated for 15 min at 20oC in the dark. To stop the enzyme reaction of TMB, 100 mL 0.1% sulphuric acid was added and the absorbance was measured at 450nm with 650nm as the reference using an ELISA reader. The standard peptide had a starting concentration of 125 ng / mL and was diluted 2-fold to create an 11 points calibration curve with the last point consisting of assay buffer only (c.f. Figure 1). The calibration curve was plotted using a 4- parametric mathematical fit model. Technical Validation As previously described[9-10], a twofold dilution of healthy human serum and plasma samples as well as rat and mouse serum was used to determine linearity, calculated as percentage of recovery of the undiluted sample. Antibody specificity was determined by percentage of signal inhibition of twofold diluted standard peptide (GPPGPPGRLV (SEQ ID NO: 1)), elongated peptide (GPPGPPGRLVD (SEQ ID NO: 5)), and nonsense peptide (GPKGGIGNRG) (SEQ ID NO: 18). Unspecific binding was tested by using a nonsense coating peptide (biotin-GPKGGIGNRG) (SEQ ID NO: 19). Lower limit of detection was calculated as the mean + 3 · standard deviation (SD) of the blank from 21 determinations of standard K (i.e., buffer). Upper limit of detection (ULOD) was determined as the mean–3 X SD of 10 measurements of Standard B. The intra- and inter-assay variation was determined by 10 independent measurements of eight QC samples and two kit control samples in double determination. The accuracy of the assay was measured in healthy human serum samples spiked with standard peptide or human serum, and was calculated as the percentage recovery of the theoretical amount of analyte in the sample. Analyte stability was determined in healthy human serum samples stressed by up to four freeze / thaw cycles or by storage at 4oC or 20oC for 2, 4, 24, or 48 h. Recovery was calculated with unstressed samples as reference. Interference was measured in healthy human serum spiked with lipids (low= 4.83mM, high = 10.98mM), biotin (low= 30 ng / mL, high = 90 ng / mL), or haemoglobin (low= 0.155mM, high = 0.310mM) and calculated as the percentage recovery of the analyte concentration in un- spiked serum. Clinical Evaluation in IBD Serum C7M levels were measured, using the above-described C7M ELISA, in serum samples from forty-nine UC patients with active disease and 50 healthy controls (HC). All had endoscopy and blood samples done at inclusion. Ulcerative Colitis Endoscopic Index of Severity (UCEIS) score, Total Mayo Score (TMS), Mayo Endoscopic Score (MES) and Montreal disease extent (E1-E3) scores were registered. Spearman’s correlations were applied for statistical analysis. MES and total Mayo response To evaluate C7M as a Pharmacodynamic (PD) biomarker the UC patients were classified as non-responder or responders based on the MES score and the total Mayo score. Of the 49 UC patients at baseline, 40 patients had a MES assessment and serum sample available for week 24. Response based on MES score was defined as having a MES decrease of 1 or more from baseline to week 24. As such 23 UC patients were classified as MES responders and 17 as MES non-responders. For the total Mayo score 33 UC patients had moderate and severe disease at baseline (based on the total Mayo score), and Mayo total response was defined as achieving remission at week 24. As such 21 UC patients were classified as responders and 12 UC patients were classified as non-responders based on the Mayo total score. C7M levels in CD patients undergoing surgery C7M was evaluated in human serum from 54 CD patients undergoing surgery for stenotic strictures, and serum samples were drawn 1 day after surgery and 1 month after the surgery for each patient. Results Technical Validation As previously described[9-10], the C7M ELISA and monoclonal antibody show high reactivity to the standard peptide (i.e., the synthetic peptide consisting of the C7M target sequence) which inhibited the signal in a dose-dependent manner (Fig. 1). Conversely, the antibody does not recognize the elongated peptide (i.e., a peptide with consisting of the C7M target sequence with an additional aspartic acid residue added to its C-terminus) or the nonsense peptide (Fig. 1). This shows that the C7M ELISA is neo-epitope specific and does not recognize intact type VII collagen. No reaction is seen either to the nonsense coating peptide, which shows that there is no non- specific binding in the system (Fig. 1). The measurement range of the C7M ELISA was determined to be 1.0 to 59.5 ng / mL, and intra- and inter-assay variation was acceptable with values of 6% and 10%, respectively. Linearity was evaluated for human serum and plasma as well as for rat and mouse serum. Human serum showed acceptable linearity from undiluted to 1:8 dilution with a mean recovery of 102%. Citrate plasma had a recovery of 137% from undiluted to 1:4 dilution, but it showed acceptable linearity from undiluted to 1:2 dilution (109%). Only undiluted heparin plasma was within the measurement range of the C7M ELISA, and all measurements of EDTA plasma were below the measurement range. Rat serum showed a mean recovery of 149% for undiluted to 1:4 dilution, whereas only undiluted mouse serum was within the measurement range. Analyte stability in human serum samples after repeated freeze / thaw cycles or prolonged storage at 4oC or 20oC was high with a mean recovery of 102%, 104%, and 99%, respectively. Spiking of twofold dilutions of standard peptide or human serum in human serum resulted in mean recoveries of 107% and 131%, respectively. Lipid content in human serum samples did not interfere significantly with C7M measurements (mean recovery 108.5%), whereas high, but not low, concentrations of biotin or haemoglobin caused some degree of interference. Clinical Evaluation in IBD C7M serum levels were found to be significantly elevated in patients with active UC as compared to Healthy Controls (p<0.0001) (Figure 2). C7M serum levels correlated with TMS (p<0.0001), MES (p<0.001 and UCEIS (p<0.05) scores of UC severity (Figure 3). Serum levels of C7M were found to be significantly elevated in patients in the E3 subgroup (according to the Montreal disease extent classification system) as compared to patients in the E1 subgroup, demonstrating that C7M levels could differentiate between these subgroups (Difference ng / ml: 3.89 (SE of diff: 1.47), 13.15 (4.55), 10.08 (4.09), respectively, all p<0.05) (Figure 4). 18 / 49 of the UC patients in the study cohort achieved remission at 6 months after the study outset. C7M baseline levels (i.e. C7M serum levels as measured at the study outset) exhibited acceptable discriminative capabilities for distinguishing between patients likely to achieve remission (as defined using TMS scores) at 6 months and patients not likely to do so (AUC 0.72 (95% CI 0.58-0.87) p=0.01) (Figure 5). MES and total Mayo response C7M was significantly reduced at week 12 and week 24 in the UC patients responding to anti-TNF alpha treatment (based on the MES and total Mayo response classification), which demonstrates its potential to be PD biomarker for UC. (Figure 6). C7M levels in CD patients undergoing surgery C7M was significantly elevated one day after the surgery (POD) compared to baseline levels and C7M serum levels were normalized one month after surgery. (Figure 7) These data indicate that C7M is coming from the intestinal tissue, and therefore is a disease relevant biomarker for intestinal diseases such as Crohn’s disease and Ulcerative colitis. Conclusion The data presented herein demonstrates that C7M correlates with disease activity, and extent, and predicts future disease outcomes in UC. Measuring C7M thus provides a 'window' into transmural tissue remodelling and inflammatory and fibrotic burden. In this specification, unless expressly otherwise indicated, the word ‘or’ is used in the sense of an operator that returns a true value when either or both of the stated conditions is met, as opposed to the operator ‘exclusive or’ which requires that only one of the conditions is met. The word ‘comprising’ is used to mean ‘including or consisting of’. All prior teachings acknowledged above are hereby incorporated by reference. No acknowledgement of any prior published document herein should be taken to be an admission or representation that the teaching thereof was common general knowledge in Australia or elsewhere at the date hereof. References [1] C. McDowell, U. Farooq, M. Haseeb, Inflammatory Bowel Disease, 2022. http: / / www.ncbi.nlm.nih.gov / pubmed / 30137275. [2] S.H. Lee, J. eun Kwon, M. la Cho, Immunological pathogenesis of inflammatory bowel disease, Intest Res. 16 (2018) 26–42. https: / / doi.org / 10.5217 / ir.2018.16.1.26. [3] B.E. Sands, B.G. Feagan, P. Rutgeerts, J.F. Colombel, W.J. Sandborn, R. Sy, G. D’Haens, S. Ben-Horin, J. Xu, M. Rosario, I. Fox, A. Parikh, C. Milch, S. Hanauer, Effects of vedolizumab induction therapy for patients with Crohn’s disease in whom tumor necrosis factor antagonist treatment failed, Gastroenterology. 147 (2014) 618- 627.e3. https: / / doi.org / 10.1053 / j.gastro.2014.05.008. [4] J.H. Mortensen, M. Lindholm, L.L. Langholm, J. Kjeldsen, A.C. Bay-Jensen, M.A. Karsdal, T. Manon-Jensen, The intestinal tissue homeostasis–the role of extracellular matrix remodeling in inflammatory bowel disease, Expert Rev Gastroenterol Hepatol. 13 (2019) 977–993. https: / / doi.org / 10.1080 / 17474124.2019.1673729. [5] Chung, H. J. and J. Uitto. 2010. Type VII collagen: the anchoring fibril protein at fault in dystrophic epidermolysis bullosa. Dermatol.Clin. 28:93-105. [6] Chen, M., G. H. Kim, L. Prakash, and D. T. Woodley. 2012. Epidermolysis bullosa acquisita: autoimmunity to anchoring fibril collagen. Autoimmunity 45:91-101. [7] Hundorfean, G., M. F. Neurath, and C. Sitaru. 2010. Autoimmunity against type VII collagen in inflammatory bowel disease. J.Cell Mol.Med. 14:2393-2403. [8] Gammon, W. R., D. T. Woodley, K. C. Dole, and R. A. Briggaman. 1985. Evidence that anti-basement membrane zone antibodies in bullous eruption of systemic lupus erythematosus recognize epidermolysis bullosa acquisita autoantigen. J.Invest Dermatol. 84:472-476. [9] WO 2017 / 129480 A1

[0010] Sand JMB, Lamy P, Juhl P, Siebuhr AS, Iversen LV, Nawrocki A, Larsen MR, Domsic RT, Franchimont N, Chavez J, Karsdal MA, Leeming DJ. Development of a Neo-Epitope Specific Assay for Serological Assessment of Type VII Collagen Turnover and Its Relevance in Fibroproliferative Disorders. Assay Drug Dev Technol. 2018 Feb / Mar;16(2):123-131. doi: 10.1089 / adt.2017.820. Epub 2018 Mar 1. PMID: 29493258.

[0011] Holm Nielsen S, Groen SS, Yao Y, Jørgensen AR, Nielsen VW, Karsdal M, Gehring K, Bay-Jensen AC, Thomsen SF. Biomarkers of Tissue Turnover and Systemic Inflammation Are Associated with Disease Severity and Activity in Patients with Hidradenitis Suppurativa. J Invest Dermatol. 2023 Feb;143(2):328-331.e3. doi: 10.1016 / j.jid.2022.08.049. Epub 2022 Sep 13. PMID: 36108804.

Claims

Claims 1. A method of immunoassay for detecting or monitoring inflammatory bowel disease or a severity thereof in a patient, said method comprising: i) contacting a patient sample with a monoclonal antibody that specifically binds to the C-terminus amino acid sequence GPPGPPGRLV(SEQ ID NO: 1); ii) detecting and determining the amount of binding between said monoclonal antibody and peptides in the sample; and iii) correlating said amount of binding with values associated with normal healthy subjects and / or values associated with known disease severity and / or values obtained from said patient at a previous time point and / or with a predetermined cut-off value.

2. A method as claimed in claim 1, wherein the monoclonal antibody does not specifically bind to a peptide having the C-terminus amino acid sequence GPPGPPGRLVD (SEQ ID NO: 5).

3. A method as claimed in claim 1 or 2, wherein the monoclonal antibody is raised against a synthetic peptide having the C-terminus amino acid sequence GPPGPPGRLV (SEQ ID NO: 1).

4. A method as claimed in any preceding claim, wherein the patient sample is a biofluid sample.

5. A method as claimed in any preceding claim, wherein the biofluid sample is selected from blood, plasma or serum.

6. A method as claimed in any preceding claim, wherein the immunoassay is a competition assay or a sandwich assay.

7. A method as claimed in any preceding claim, wherein the immunoassay is a radio-immunoassay or an enzyme-linked immunosorbent assay.

8. A method as claimed in any preceding claim, wherein the inflammatory bowel disease is ulcerative colitis.

9. A method as claimed in any preceding claim, wherein the method is a method for detecting or monitoring a level of severity of inflammatory bowel disease or in a patient. 10 A method as claimed in any preceding claim, wherein the method is a method for determining the likelihood of the inflammatory bowel disease going into remission.

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