C3-HNE assay

Abstract

The present invention relates to an immunoassay method for detecting HNE-producing fragments of the α1 chain of type III collagen in patient samples, and to the use thereof for detecting and / or monitoring inflammatory bowel disease (IBD) or a specific level of severity of said disease in patients. The present invention also relates to monoclonal antibodies and assay kits for use in said immunoassay method.

Description

Technical Field

[0001] The present invention relates to an immunoassay method for detecting the HNE-generated fragment of the α1 chain of type III collagen in a subject sample, and its use for detecting and / or monitoring inflammatory bowel disease (IBD) or a specific level of severity of the disease in a subject. The present invention also relates to monoclonal antibodies and assay kits for use in the immunoassay method.

Background Art

[0002] Inflammatory bowel disease (IBD) is a general term for chronic inflammatory diseases of the gastrointestinal (GI) tract characterized by damage to the epithelial barrier. The disease is classified into Crohn's disease (CD) or ulcerative colitis (UC) depending on the site and depth of the damage (Jostins et al., 2012, Graham et al., 2020). The chronic disease develops due to genetic susceptibility and activation of the immune response. When an abnormal immune response to the gut microbiota occurs, excessive intestinal damage and mucosal inflammation are induced (Qiu et al., 2022). At the same time, uncontrolled and high proteolytic activity is associated with IBD, and proteases have been secreted from various immune cells (neutrophils, lymphocytes, macrophages, etc.) (Curciarello et al., 2020, Kriaa et al., 2021).

[0003] The intestinal wall is an essential component for protecting the GI system, maintaining homeostasis through interactions between epithelial and stromal cells (Kong et al., 2018). Epithelial and stromal cells work together to generate and organize the intestinal extracellular matrix (ECM). Type III collagen is expressed throughout all layers of the intestinal mucosa, is the second most abundant collagen in the intestine (20%), and is primarily synthesized by fibroblasts (Graham 1988). ECM remodeling is a dynamic process that requires interactions between resident cells and ECM components, leading to the secretion of proteolytic enzymes. Healthy tissue is continuously and constantly remodeled; however, abnormal regulation can lead to an imbalance in ECM turnover, potentially inducing chronic inflammation (Kehlet et al., 2018). Inflammation is, Inflammation is a biological response to harmful stimuli, and can be acute or chronic (Signore et al., 2013, Pahwa et al., 2021). Acute inflammation is mediated by granulocytes such as neutrophils, but they play an essential role in chronic inflammation. Chronic inflammation of the intestinal mucosa causes granule release and neutrophil activation (NETosis), forming neutrophil extracellular traps (NETs). NETs consist of filamentous DNA interwoven with nucleoproteins such as histones, and cytotoxic granular components such as human neutrophil elastase (HNE) (Yuen et al., 2016, Menegazzi et al., 2012). HNE is a serine protease that cleaves ECM components such as elastin and collagen and upregulates several pro-inflammatory cytokines (Curciarello et al., 2020). Therefore, excessive neutrophil recruitment may induce tissue damage in inflamed mucosa (Saez et al., 2023). The presence and activity of neutrophils are increased in IBD patients, and plasma HNE levels in IBD patients are significantly higher than in healthy individuals (Hansberry et al., 2017, Fischbach et al., 1987). Elevated HNE levels were detected in both UC and CD, and the expression ratio between HNE and antiproteases (e.g., elaphin) was significantly shifted towards HNE in CD compared to UC (Schmid et al., 2007). Furthermore, HNE causes proteolysis of therapeutic antibodies in IBD patients, leading to treatment non-response (Curciarello et al., 2020). [Overview of the project]

[0004] A type III collagen fragment induced by the neutrophil-specific protease HNE was identified, which reflects neutrophil-induced mucosal damage and can be used as a direct measure of immune cell activity. A competitive enzyme-coupled immunosorbent assay (ELISA) targeting the HNE-producing neoepitope of the α1 chain of type III collagen was developed, demonstrating the usefulness of this HNE-producing neoepitope as a biomarker for inflammatory bowel disease (IBD) in general, and particularly for early-stage IBD.

[0005] Therefore, in a first aspect, the present invention provides an immunoassay method. This immunoassay method involves contacting a sample from a patient with a monoclonal antibody that specifically binds to an HNE-producing neoepitope of an HNE-producing fragment of the α1 chain of type III collagen, detecting the binding between the monoclonal antibody and a peptide in the sample, and determining the amount of binding, wherein the HNE-producing neoepitope consists of the N-terminal sequence of the HNE-producing fragment, which is located at the end of the HNE-producing fragment cleaved by HNE.

[0006] In a preferred embodiment, the immunoassay method comprises the following i) and ii); i) Contacting the patient's sample with a monoclonal antibody that specifically binds to the N-terminal amino acid sequence PGKNGERGGP (SEQ ID NO: 1) (hereinafter also referred to as "C3-HNE" or "C3-HNE target sequence"); and, ii) To detect the binding between the monoclonal antibody and the peptide in the sample and to determine the amount of binding.

[0007] In a preferred embodiment, the immunoassay is an immunoassay for detecting and / or monitoring inflammatory bowel disease or a specific level of severity of said disease in a patient, further comprising correlating the binding amount with a value associated with a normal healthy individual and / or a value associated with a known disease severity and / or a value obtained from the patient at a past point in time and / or a predetermined cutoff value. In a preferred embodiment, the inflammatory bowel disease (IBD) is Crohn's disease (CD) or ulcerative colitis (UC). In a preferred embodiment, the method is an immunoassay for detecting early-stage inflammatory bowel disease. In a preferred embodiment, the patient's sample is a human biological fluid sample. Preferably, the sample is a blood material sample such as blood (whole blood), plasma, or serum.

[0008] If the monoclonal antibody is a monoclonal antibody that specifically binds to the N-terminal amino acid sequence PGKNGERGGP (SEQ ID NO: 1), then preferably the monoclonal antibody does not specifically bind to a peptide having the N-terminal amino acid sequence APGKNGERGGP (SEQ ID NO: 2) (i.e., an elongated version of the C3-HNE target sequence extended by adding an alanine residue to the N-terminal), and / or does not specifically bind to a peptide having the N-terminal amino acid sequence GKNGERGGP (SEQ ID NO: 3) (i.e., a truncated version of the C3-HNE target sequence truncated by removing the first proline residue). Preferably, the ratio of the affinity of the antibody to the C3-HNE target sequence to the affinity of the antibody to the elongated version of the target sequence is at least 10:1, more preferably at least 20:1, or at least 30:1.

[0009] As used herein, “specifically binding” means that antibody binding is selective to an antigen and that this binding can be distinguished from unwanted or nonspecific interactions. The ability of a monoclonal antibody to bind to a specific epitope or peptide sequence can be measured by either the enzyme-linked immunosorbent assay (ELISA) described herein, or by other techniques well known to those skilled in the art, such as surface plasmon resonance (SPR) techniques (e.g., analyzed with BIAcore instruments) and conventional binding assays. The degree of binding of a monoclonal antibody to an unrelated protein is less than approximately 10% of the binding of a monoclonal antibody to an epitope or peptide as measured, for example, by ELISA. “Affinity” refers to the total strength of all non-covalent interactions between a single binding site of a molecule (e.g., the epitope-binding region of an antibody) and its binding partner (e.g., the epitope or antigen). Unless otherwise indicated herein, “binding affinity” refers to the intrinsic binding affinity that reflects the 1:1 interaction between the components of the binding pair (e.g., the antigen-binding portion and the antigen). The affinity of a molecule for its binding partner is generally expressed by the constants of the dissociation rate and the association rate (K, respectively). off , K on The dissociation constant (Kd) can be expressed as the ratio of the rate constants. Therefore, equivalent affinity may consist of different rate constants, as long as the ratio of the rate constants remains the same. The dissociation constant represents the concentration of the antigen when it occupies half of the binding sites on the antibody. A lower Kd indicates a higher binding affinity between the antibody and the antigen, while a higher Kd indicates a weaker binding. Several methods can be used to measure the Kd of an antibody, such as surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), and fluorescence emission principle assays. In certain embodiments, the dissociation constant (KD) of a monoclonal antibody bound to an epitope or peptide may be <1 pM, <100 nM, <10 nM, <1 nM, <0.1 nM, <0.01 nM, or <0.001 nM (e.g., 10 8 M or less, for example, 10 8 M to 10 13 Up to M, for example, 10 9M to 10 13 (Up to M)

[0010] If the monoclonal antibody is a monoclonal antibody that specifically binds to the N-terminal amino acid sequence PGKNGERGGP (SEQ ID NO: 1), The monoclonal antibody may be produced, for example, by immunizing a synthetic peptide having the N-terminal amino acid sequence PGKNGERGGP (SEQ ID NO: 1); for example, a monoclonal antibody may be produced by: (a) immunizing a rodent (or other suitable mammal) with a synthetic peptide containing the N-terminal amino acid sequence PGKNGERGGP (SEQ ID NO: 1), to which an immunogenic carrier protein (such as keyhole limpet hemocyanin) may optionally be linked to the N-terminal; (b) isolating and cloning a single antibody-producing cell; and (c) assaying the resulting monoclonal antibody to confirm that it has the desired specificity. Exemplary procedures for developing, producing, and characterizing suitable monoclonal antibodies are described in the Examples section below.

[0011] In certain exemplary embodiments, if the monoclonal antibody is a monoclonal antibody that specifically binds to the N-terminal amino acid sequence PGKNGERGGP (SEQ ID NO: 1), the monoclonal antibody may preferably include one or more complementarity-determining regions (CDRs) selected from the following: CDR-La1: SASSSVSYLN (Sequence ID 4) CDR-La2: STSNLAS (Sequence ID 5) CDR-La3: HQRSSYPPT (Sequence ID 6) CDR-Lb1: RSSQNIVHRDGNTYLE (Sequence ID 7) CDR-Lb2: RVSNRFS (Sequence ID 8) CDR-Lb3: FQGSHVPWT (Sequence ID 9) CDR-H1: TSGMGVG (Sequence ID 10) CDR-H2: HIYWDDDKYYNPSLKS (Sequence ID 11) CDR-H3: NLLPGGFAY (SEQ ID NO: 12) Preferably, the monoclonal antibody comprises at least two, three, four, five or six of the CDR sequences listed above.

[0012] Preferably, the monoclonal antibody has a light chain variable region comprising the following CDR sequences: CDR-La1: SASSSVSYLN (SEQ ID NO: 4) CDR-La2: STSNLAS (SEQ ID NO: 5) CDR-La3: HQRSSYPPT (SEQ ID NO: 6) Alternatively, preferably, the monoclonal antibody has a light chain variable region comprising the following CDR sequences: CDR-Lb1: RSSQNIVHRDGNTYLE (SEQ ID NO: 7) CDR-Lb2: RVSNRFS (SEQ ID NO: 8) CDR-Lb3: FQGSHVPWT (SEQ ID NO: 9) Preferably, the monoclonal antibody has a light chain comprising the framework sequences between the CDRs, and the framework sequences are substantially identical or substantially similar to the framework sequences between the CDRs in any of the following light chain sequences (CDRs are shown in bold and underlined, and framework sequences are shown in italics).

[0013]

Chemical Structure

[0014] Or,

[0015]

Chemical Structure

[0016] Preferably, the monoclonal antibody has a heavy chain variable region comprising the following CDR sequences.

[0017] CDR-H1: TSGMGVG (SEQ ID NO: 10) CDR-H2: HIYWDDDKYYNPSLKS (Sequence ID 11) CDR-H3: NLLPGGFAY (Sequence ID 12) Preferably, the monoclonal antibody has a heavy chain containing a framework sequence between CDRs, the framework sequence being substantially identical or substantially similar to the framework sequence between CDRs in the heavy chain sequence below (CDRs are shown in bold and underline, and framework sequences are shown in italics).

[0018] [ka]

[0019] Preferably, the monoclonal antibody has a light chain variable region sequence:

[0020] [ka]

[0021] Or,

[0022] [ka]

[0023] (CDR is in bold and underlined; framework array is in italics) and / or heavy chain variable region sequence:

[0024] [ka]

[0025] (CDR is in bold and underlined; framework array is in italics) Includes.

[0026] In this specification, the amino acid sequences of the framework present between the CDRs of an antibody are considered "substantially identical" or "substantially similar" to the amino acid sequences of the framework present between the CDRs of another antibody if they have at least 70%, 80%, 90%, or at least 95% similarity or identity with the amino acid sequences of the framework present between the CDRs of that other antibody. Similar or identical amino acids may be consecutive or discontinuous. A framework sequence may contain one or more amino acid substitutions, insertions, and / or deletions. Amino acid substitutions may be conservative substitutions, meaning that the substituted amino acid has similar chemical properties to the original amino acid. Those skilled in the art will understand which amino acids share similar chemical properties. For example, the following groups of amino acids share similar chemical properties in terms of size, charge, polarity, etc.: 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.

[0027] Programs like the CLUSTAL program can be used to compare amino acid sequences. This program compares amino acid sequences and finds the optimal alignment by appropriately inserting spaces within either sequence. For optimal alignment, it is possible to calculate amino acid identity or similarity (in addition to identity, conservation of amino acid types). Programs like BLASTx align the longest segments of similar sequences and assign values ​​to the matching sites. In this way, a comparison can be obtained, and several similar regions with different scores can be discovered. In this invention, it is conceivable to use these two types of analysis. Identity or similarity is preferably calculated over the entire length of the framework sequence.

[0028] In preferred embodiments, the immunoassay is a competitive assay or a sandwich assay. The immunoassay may be, for example, a radioimmunoassay or an enzyme-linked immunosorbent assay (ELISA). Such assays are known to those skilled in the art. Most preferably, the immunoassay is a competitive ELISA.

[0029] As used herein, the term “N-terminal” refers to the N-terminal peptide sequence at the tip of a polypeptide, i.e., the peptide sequence at the N-end of the polypeptide, and should not be interpreted as referring to its general direction. As used herein, the term “C-terminal” refers to the C-terminal peptide sequence at the tip of a polypeptide, i.e., the peptide sequence at the C-end of the polypeptide, and should not be interpreted as referring to its general direction. As used herein, the terms “peptide” and “polypeptide” are used synonymously.

[0030] As used herein, the term “monoclonal antibody” refers to both the whole antibody and its fragments that retain the binding specificity of the whole antibody, such as Fab fragments, F(ab')2 fragments, single-chain Fv fragments, or other such fragments known to those skilled in the art. As is well known, a whole antibody typically has a “Y-shaped” structure in which two identical polypeptide chain groups are paired, with each paired element consisting of one “light” chain and one “heavy” chain. The N-terminal regions of the light and heavy chains each contain a variable region, and the C-terminal portions of the heavy and light chains each constitute a constant region. The variable region contains 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 that retain binding specificity include at least the CDRs and a portion of the remainder of the variable region sufficient to retain binding specificity.

[0031] In the present invention, any monoclonal antibody containing any constant region known in the art can be used. In the case of mouse antibodies and human antibodies, the constant light chain is classified as either a kappa or lambda light chain. The constant heavy chain is classified as mu, delta, gamma, alpha, or epsilon, and the antibody isotype is defined as IgM, IgD, IgG, IgA, and IgE, respectively. In the case of humans, the IgG isotype has several subclasses, including IgG1, IgG2, IgG3, and IgG4, and in the case of mice, it has several subclasses, including IgG1, IgG2a, IgG2b, IgG2c, and IgG3, but is not limited thereto. The monoclonal antibody may preferably belong to an IgG isotype containing any one of the IgG subclasses.

[0032] The CDR of an antibody can be determined using methods known in the art, such as those described by Kabat et al. The antibody can be generated from B cell clones. The isotype of the antibody can be determined by ELISA specific to the IgM, IgG, or IgA isotype or subclass. The amino acid sequence of the generated antibody can be determined using standard methods. For example, RNA can be isolated from cells and used to generate cDNA by reverse transcription. The cDNA can then be PCR-treated using primers that amplify the heavy and light chains of the antibody. For example, primers specific to the leader sequence for all VH (variable heavy chain) sequences can be used together with primers that bind to sequences located in the constant region of a predetermined isotype. The light chain can be amplified using primers that bind to the 3' end of the kappa or lambda chain, together with primers that anneal to the V-kappa or V-lambda leader sequence. The full-length heavy and light chains can be generated and sequenced.

[0033] As used herein, the term “bound amount” refers to the quantification of binding between the antibody and the peptide in the patient sample. This quantification may be determined, for example, by comparing the measured binding amount in the patient sample with a calibration curve prepared using measured binding amounts in a standard sample containing the peptide to which the antibody specifically binds at a known concentration, in order to determine the amount of peptide to which the antibody specifically binds in the patient sample. Any analytical method suitable for measuring the bound amount can be used. For example, ELISA can be used, which utilizes spectrophotometric analysis to measure both the bound amount in the patient sample and the bound amount when creating the calibration curve.

[0034] As used herein, the term “predetermined cutoff value” means a statistically determined binding amount that indicates a high probability that a patient has a disease (e.g., IBD) or that the disease is of a particular severity, and in that sense, if the measured value of the target peptide in the patient sample is greater than or equal to the statistical cutoff value, then the measured value corresponds to a probability of at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95% that the disease is present or that the disease is of a particular severity.

[0035] As used herein, the term “value associated with normal healthy individuals” means the standardized binding amount determined by the method described above for samples obtained from subjects considered to be healthy, i.e., disease-free (i.e., not IBD); and the term “value associated with known disease severity” means the standardized binding amount determined by the method described above for samples obtained from patients known to have a disease of known severity (i.e., IBD).

[0036] In a second embodiment, the present invention provides a method for treating inflammatory bowel disease (IBD) in patients requiring treatment, comprising the following steps: (a) Perform an immunoassay on a sample obtained from a patient to detect whether it is IBD or whether its severity is at a specific level, according to the first aspect of the present invention; and (b) If it is determined in step (a) that the patient has IBD or that its severity is at a certain level, the patient is given therapy to treat IBD.

[0037] Preferred embodiments of the method according to the second embodiment are evident from the above discussion of preferred embodiments of the method according to the first embodiment. For example, IBD may include Crohn's disease (CD) or ulcerative colitis (UC); and / or, step (a) may include performing an immunoassay according to the invention of the first embodiment in order to detect mild or early-stage IBD, and if it is determined in step (a) that the patient has mild or early-stage IBD, the subsequent step (b) may include administering therapy to the patient to treat IBD. Step (a) may also include performing an immunoassay according to the invention of the first embodiment in order to detect patients who are relatively likely to respond to therapy, and if it is determined in step (a) that the patient is likely to respond to therapy, the subsequent step (b) may include administering therapy to the patient to treat IBD. The patient may have already been diagnosed with IBD, or the immunoassay may detect mild or early-stage IBD.

[0038] The aforementioned therapy may be any therapy suitable for treating the target inflammatory bowel disease. The therapy may include, for example, one or more medications, one or more lifestyle modifications, one or more surgeries, or a combination thereof, or consist of such therapies. The medication may be prescribed for topical or systemic administration. Topical medications may be prescribed for administration as, for example, a cream, foam, gel, lotion, or ointment. Systemic medications may be prescribed for administration as, for example, a gastrointestinal or parenteral administration. The surgery may be a curative surgery, a prophylactic surgery, a palliative surgery, and / or a reconstructive surgery.

[0039] For example, if inflammatory bowel disease is Crohn's disease, appropriate therapy may include one or more of the following therapies: lifestyle changes such as dietary adjustments, elemental diets, adequate hydration, and smoking cessation; antibiotics, aminosalicylic acid ester anti-inflammatory drugs, corticosteroids, 5-aminosalicylic acid (5-ASA), prednisone, azathioprine, 6-mercaptopurine, methotrexate, anti-TNF therapies (also referred to in prior art as TNF inhibitors) such as monoclonal antibodies that inhibit TNF activity, such as infliximab, adalimumab, and certolizumab; and other biological agents and antibodies such as vedolizumab, ustekinumab, and natalizumab; and combinations thereof.

[0040] If inflammatory bowel disease is ulcerative colitis, appropriate therapy may include one or more of the following: aminosalicylic acids such as mesalazine, sulfasalazine, valsalazide, and orsalazine; corticosteroids such as cortisone, prednisone, hydrocortisone, methylprednisolone, and budesonide; immunosuppressants such as mercaptopurine, azathioprine, and methotrexate; anti-TNF therapies (TNF inhibitors) such as infliximab, adalimumab, and golimumab; and other biological agents and antibodies such as tofacitinib and vedolizumab; surgical procedures such as partial or total colectomy; and combinations thereof.

[0041] In a third embodiment, the present invention provides a monoclonal antibody that specifically binds to the N-terminal amino acid sequence PGKNGERGGP (SEQ ID NO: 1) (i.e., the C3-HNE target sequence). The antibody according to the third embodiment of the invention is particularly suitable for use when carrying out the immunoassay method according to the first embodiment of the invention. Accordingly, preferred embodiments and features of the antibody according to the third embodiment are evident from the above discussion concerning preferred embodiments of the method according to the first embodiment.

[0042] In a fourth aspect, the present invention provides a monoclonal antibody according to a third aspect of the present invention, and an immunoassay kit comprising at least one of the following: - Streptoavidin-coated well plates - Biotinylated peptide PGKNGERGGP-L-biotin (SEQ ID NO: 1), where L is an optional linking group. - Second antibody used in sandwich immunoassays - Calibration protein containing the N-terminal amino acid sequence PGGNGERGGP (SEQ ID NO: 1) - Antibody biotinylation kit - Antibody HRP labeling kit - Antibody radiolabeling kit. The immunoassay kit according to the fourth embodiment of the invention is particularly suitable for use when carrying out the immunoassay method according to the first embodiment of the invention. Therefore, more preferred embodiments and features of the immunoassay kit according to the fourth embodiment are evident from the above discussion of preferred embodiments of the method according to the first embodiment. [Brief explanation of the drawing]

[0043] figure [Figure 1] Figure 1: Preliminary study: Acute DSS colitis rat SD model. Rats were divided into six groups: groups A, B, and C received DSS treatment, while groups D, E, and F received normal water intake. [Figure 2] Figure 2: Dose-dependent reactivity of the mAb was observed when incubated with the selected (standard) peptide, but no reactivity was observed when incubated with the elongated peptide or truncated peptide. [Figure 3]Figure 3: C3-HNE is elevated in both the CD (p=0.023) and UC (p=0.0361) samples compared to the healthy control. Data are shown as medians. Differences between groups were compared using the Kruskal-Wallis rank test with Dunn correction for multiple comparisons. Results are shown as box plots (showing all scores) and medians, along with interquartile ranges. Significance is indicated by p<0.05=* and p<0.01=**. [Figure 4] Figure 4: Serum levels of C3-HNE in Cohort I. C3-HNE formation in CD, UD samples and healthy donor samples within Cohort I. C3-HNE levels were elevated in both CD (p=0.0072) and UC (p=0.0437) samples compared to healthy donor samples. The Mann-Whitney U test was used to compare the differences between the two groups. Results are shown as box plots (showing all scores) and medians, along with interquartile ranges. Significance is indicated by p<0.05=* and p<0.01=**. [Figure 5] Figure 5: Serum levels of C3-HNE in Cohort III. C3-HNE levels were elevated in IBD (p=0.0034) samples compared to healthy donors. All quantified C3-HNE levels were higher than LLOQ levels. The Mann-Whitney U test was used to compare differences between groups. Results are shown as box plots (showing all scores) and medians, along with interquartile ranges. Significance is indicated by p<0.01=**.

[0044] [Figure 6] Figure 6: C3-HNE rises before clinically apparent mucosal damage in the DSS-colitis rat model. A. Serum levels of C3-HNE (relative %) to baseline in rats treated with 5% DSS and standard water intake. Differences between groups were compared using a two-way ANOVA with multiple comparisons. Significance of the test is indicated by p<0.05=*. B. DAI scores were monitored daily in rats treated with 5% DSS and standard water intake. The induced DSS-colitis model is shown in red, and the control group is shown in blue. [Figure 7]Figure 7: Serum levels of C3-HNE after type III collagen cleavage. C3-HNE formation in the Scar-in-a-Jar (SIAJ) model when fibroblast-precipitated type III collagen is pre-cleaved with MMP8 and MMP13, and then final-cleaved with HNE. [Figure 8] Figure 8: C3-HNE formation in the culture supernatant of primary neutrophils inoculated into type III collagen-coated wells. Neutrophils were either inactive or activated for 6 hours (LPS 25 ng / ml or 100 ng / ml). IMDM was used as a control for inactive cells, and IMDM with LPS 25 ng / ml and IMDM with LPS 100 ng / ml were used as control for activated cells, respectively. [Figure 9] Figure 9: Serum levels of C3-HNE in IBD patients within cohorts II and III, stratified according to response to treatment. [Examples]

[0045] The embodiments disclosed herein are described in the following examples. These examples are provided to aid in understanding the disclosure and should not be construed in any way as limiting the scope of the disclosure specified in the claims below. The examples below are provided to a person skilled in the art to provide a complete disclosure and description of how to make and use the described embodiments and are not intended to limit the scope of the disclosure, nor are they intended to mean that the experiments described below are all or only experiments performed. Efforts have been made to ensure accuracy of the numerical values ​​used (e.g., quantity, temperature, etc.), but some experimental error and deviation should be taken into consideration. Unless otherwise indicated, parts are parts by weight, molecular weight is weight-average molecular weight, temperature is in Celsius, and pressure is atmospheric pressure or near atmospheric pressure.

[0046] Materials and methods Production of monoclonal antibodies Female Balb / C mice aged 6-7 weeks were immunized by subcutaneous injection of 200 μl of emulsified antigen and 100 μg of C3-HNE[PGKNGERGGP-GGC-KLH (SEQ ID NO: 19)] neoepitope peptide. The immunogen was prepared using the Sigma adjuvant system (Sigma(c), Cat No. S6322) and KHL-conjugated immunogen in a 1:1 ratio. Immunization was performed continuously at multiple locations on the abdomen at 2-week intervals for 11 weeks. At this stage, B cells differentiate into plasma B cells and memory B cells. Mice with the highest serum titer, i.e., highest antibody production, were selected for cell fusion and kept in recuperation for 1 month. Subsequently, these mice were boosted by intravenous administration of saline containing 50 μg / 100 μl of immunogenic peptide. Splenocytes and SP2 / 0 myeloma cells were fused in polyethylene glycol (PEG) as described by Gefter et al. (1977) to create immortalized hybridoma cells that produce monoclonal antibodies. The fused cells were selected for growth in hypoxanthine-aminopterin-thymidine (HAT) medium. Supernatants were collected for reactivity to neoepitopes using indirect ELISA with streptavidin-coated microtiter plates and the biotinylated peptide for C3-HNE [PGKNGERGGP-LYS-Biotin (SEQ ID NO: 20)]. Elongation peptide [ A The reactivity of clones to PGKNGERGGP (SEQ ID NO: 2) and the truncated peptide [_GKNGERGGP (SEQ ID NO: 3)] was tested, and the optimal clone for subsequent amplification was selected.

[0047] Antibody isotypes are determined using isotype-specific antisense primers or universal primers, and SMARTScribe TM The determination was made according to the technical manual for the Reverse Transcriptase Kit (Takara, Cat. No.: 639537). All procedures were carried out according to the manufacturer's instructions. The isotype, sequence, and CDR of this monoclonal antibody were determined. Two alternative sequences were detected in the light chain. While one might expect monoclonal antibodies to produce a single light chain sequence, it is common for approximately 30% of monoclonal antibody clones to produce additional sequences (Bradbury et al (2018)). The chain sequence is as follows (CDR is underlined and bold; N-terminal signal peptide and C-terminal constant region are italicized): Heavy chain sequence (mouse IgG2a isotype)

[0048] [ka]

[0049] Light chain sequence (mouse kappa isotype)

[0050] [ka]

[0051] Or,

[0052] [ka]

[0053] Antibody purification The clonal supernatant was purified using affinity chromatography with a 1 ml HiTrap Protein G column (GE Healthcare Life Science, Little Chalfront, Buckinghamshire, UK). Protein G is a recombinant protein derived from Escherichia coli that binds to immunoglobulin G (IgG) of eukaryotic species. Before starting the purification, the Protein G column was connected to a peristaltic pump, washed with 20% ethanol, and then washed with Mili Q water. Subsequently, a second column wash was performed at a flow rate of 1 ml / min with 10 columns of washing buffer (0.02 M sodium phosphate, pH 7) to remove preservatives. The procedure was divided into two days, with 250 ml of supernatant passed through the column each time. In this way, 250 ml of sterile filtered hybridoma supernatant was applied to the column, followed by one wash with 10 columns of washing buffer to remove nonspecifically bound proteins. Antibodies were collected by adding elution buffer (0.1 glycine, pH 2.7) to the column. Low pH elution buffer disrupts the binding between mAbs and protein G. The eluted mAbs were collected in four Eppendorf tubes containing 100 μl of neutralizing buffer (1M Tris-HCl, pH 9.0) to stabilize the antibody. The concentration of the eluted mAbs (IgG measurement) was measured using NanoDrop (NanoDrop 1000, Thermo Scientific). After measurement, all vials containing antibody concentrations greater than 1 mg / ml were combined into a single vial. The mAbs were desalted using a 5 ml spin column (Zeba spin desalting column 7k MWCO, Thermo Scientific). To replace the storage solution, the column was centrifuged and precipitated three times using Dulbecco's phosphate-buffered saline (DPBS, Thermo Scientific). The antibody was then passed through the column and collected in a clean tube. This procedure was repeated on day 2. The mAbs obtained from purification and desalting on both days were combined into one, their absorbance was measured using NanoDrop, and the mixture was divided into equal portions of 500 μl each in Eppendorf tubes and stored at -80°C.

[0054] Conditions for the C3-HNE assay The protocol for the C3 ELISA assay was established as follows: A white streptavidin-coated 96-well plate (biomat, LOT: SA2758) was coated with 100 μl of synthetic biotinylated peptide for C3-HNE [PGKNGERGGP-LYS-Biotin (SEQ ID NO: 20)] diluted to 0.5 ng / ml in assay buffer (50 mM PBS-BTB, 8 g NaCl, pH 7.4), and incubated on a 300 rpm shaker at 20°C for 30 minutes. After washing five times with wash buffer (25 mM Tris, 50 mM NaCl, 0.1% (v / v) Tween-20, pH 7.2), 1000 ng / ml of standard (selective) peptide (PGKNGERGGP (SEQ ID NO: 1)) was sequentially diluted 11 times (2-fold) and added to the plate at 20 μl / well. Then, the measurement was repeated twice, with 20 μl of sample, controls (CO1 and CO2), and quality controls (QC1 and QC2, serum / supernatant samples obtained from BioVit (Westerbury, NY, USA) or Nordic Bioscience). Simultaneously, 100 μl of 10 ng / ml horseradish peroxidase-conjugated monoclonal antibody was added, and the plate was incubated on a 300 rpm shaker at 4°C in the dark for 20 hours. ECL substrate was prepared 15-25 minutes before use according to the manufacturer's instructions (BM Chemiluminescence ELISA Substrate POD, Roche). The washing process was repeated, and 100 μl / well of equilibrium ECL substrate was added and incubated in the dark for 3 minutes. The plates were read using an ELISA reader (SpectraMax M5; Molecular Devices, UK) with Softmax Software under LUM mode (all wavelengths). Standard curves were plotted using a 4-parameter mathematical fitting model.

[0055] Technical Verification Various technical validation experiments were conducted to evaluate the robustness, precision, coherence, and stability of the assay. The lower limit of the buffer (LLOB) was determined by performing one run of 60 repetitions for the assay buffer. LLOB is defined as ±3xSD of the assay buffer that does not meet the acceptance criteria. The lower limit of quantification (LLOQ) is defined as the lowest concentration of the analyte in serum at which the CV% of the precision profile is 25%. The lower limit of quantification (LLOQ) was determined by performing five runs using four low-concentration samples (two below the evaluated LLOQ and two above the evaluated LLOQ).

[0056] Linearity of assay Evaluating the linearity of the assay determines its precision and simultaneously estimates the minimum reduction ratio (MRD) of the sample. Establishing the MRD early in assay validation is essential to check for matrix effects in the undiluted sample and to determine if the recovery rate of the diluted sample is acceptable. Therefore, to ensure assay linearity, at least three high-concentration samples (IC50+) were evaluated. Linearity is considered acceptable if the sample dilution curve follows the same pattern as the standard curve. The acceptable criterion for dilution recovery was 100±20%, equivalent to a number of concentrations calculated using the dilution factor [(concentration of diluted sample × dilution factor / concentration of undiluted sample) × 100]. Samples were diluted 2-fold, 4-fold, and 8-fold with assay buffer, and the protocol was repeated three times.

[0057] Assay accuracy The accuracy of the assay was determined by matrix-matrix spike recovery. This test evaluates whether the measured concentration in the sample is close to the actual concentration in the complex matrix. This test uses three sample sets consisting of serum samples spiked with high analyte concentrations (above IC50) and low analyte concentrations (around IC20) in different ratios. Each sample set had matrix-matrix spike-in samples with high-concentration and low-concentration samples in ratios of 100:0, 75:25, 50:50, 25:75, and 0:100. The recovery percentage was calculated based on the expected and measured concentrations. The acceptable recovery range was 100 ± 20%.

[0058] Precision and reproducibility of assays Assay precision is defined by repeated inter-sample variability, either within the same plate or between plates. Assay reproducibility must be established to ensure that quantitative data are equivalent and consistent. Inter-assay precision provides information about the robustness of the assay's standard curve and how precise repeated concentrations of individual samples are between individual runs, while intra-assay variability measures variability within a plate. Ten independent runs were performed using eight samples ranging from IC20 to IC80, covering the assay's measurement range. The upper limit of quantification (ULOQ) was calculated as the average of the highest points of the standard curve across all runs. Assay robustness was calculated as the coefficient of variation (CV%) of sample measurements. Acceptable CV%s for inter-assay variability and intra-assay variability were <15% and <10%, respectively. If the CV% for both parameters is low, the assay is considered robust.

[0059] Assay coherence Testing for interference is a necessary procedure to assess whether multiple different factors can affect the quantification of a sample and to avoid erroneous changes in analytical test results. Parameters that can affect the precision of the assay include how the serum sample was collected and stored, the temperature of the kit reagents, and the presence of endogenous or exogenous substances in the matrix. Therefore, it is impossible to completely standardize interference protocols. The endogenous analytes that most frequently cause interference in ELISA are hemoglobin, biotin, and lipids, and these are established as those that require minimal testing for interference. Three hemoglobin samples at high (5 ng / ml), medium (2.5 ng / ml), and low (0 ng / ml) concentrations, three lipid samples at high (5 ng / ml), medium (1.5 ng / ml), and low (0 ng / ml) concentrations, and eight biotin samples in the concentration range from 0 ng / mL to 100 ng / mL were spiked to three different samples (IC30-IC70). The interfering spike-in samples were prepared by adding 5 μL of each interfering substance to 95 μL of serum, thereby diluting hemoglobin, biotin, and lipids to a 1:20 ratio. The acceptable recovery rate was 100 ± 20%.

[0060] Assay stability The purpose of the stability test was to evaluate the stability of the endogenous analyte in both the freeze-thaw test of the sample and the assay stability test. The freeze-thaw procedure determined the stability of the analyte in the matrix by passing the target matrix through five freeze-thaw cycles. If all samples measured similarly after the cycle, there was virtually no change. Therefore, three samples (IC25-IC50) were subjected to freeze-thaw cycles at -20°C with 12-hour intervals. The thawing process was continued for at least 30 minutes. At the end of the freeze-thaw cycle, all partial samples were performed in a single ELISA plate, and the recovery percentage was calculated based on each non-cycled sample. Assay stability was evaluated to determine whether the measured values ​​of the sample were stable after incubation at room temperature for 24 hours. Three assay kits were included, and the same eight samples used in the assay precision and reproducibility tests were used. The acceptance criteria for both tests was 100 ± 20%.

[0061] In vitro neutrophil model To evaluate the C3-HNE assay, an in vitro neutrophil model was optimized. Primary neutrophil cells (100,000 cells / well in a 96-well plate) were inoculated into type III coated wells (78 μg / cm2). Activation medium (IMDM with LPS 25 or 100 ng / ml) was added to the wells, and the cells were incubated for 6 hours. Inactive neutrophils were included as a control. The culture supernatants of the control, as well as inactive and activated neutrophils inoculated into type III collagen coated wells, were analyzed using the C3-HNE assay.

[0062] Clinical evaluation Cohort I Cohort I originated from the Department of Gastroenterology and Hepatology at the Centre Zagreb clinical hospital in Croatia. Cohort I included patients with UC (n=40) and CD (n=85) as well as healthy donors (n=40), who were acquired from BioVIT (Westerbury, NY, USA). Blood and stool samples were collected on the day of hospital visit and stored at -70°C. Information on treatment, population statistics, and disease history was obtained through surveys and medical records. Anthropometric measurements were determined at the time of selection. The Mayo endoscopic score (MES, (0: ​​inactive disease; 1: mild activity; 2: moderate activity; 3: severe activity)), the complete Mayo score, and the partial Mayo score (pMayo, (<2: remission; 2-4: mild activity; 5-7: moderate activity; >7: severe activity)) were used to assess disease activity endoscopically in UC patients. A simplified endoscopic score for Crohn's disease (SES-CD, (0-2: remission; 3-6: mild disease activity; 7-15: moderate disease activity; >16: severe disease activity)) was used to determine disease activity endoscopically. The lesion site (L1=ileum, L2=colon, and L3=ileal-colon) and pathology (B1: inflammation, and B2: stricture) of Crohn's disease were classified according to the Montreal classification. Endoscopic examinations were performed within 3 months of blood collection. Endoscopic scores for disease activity were recorded and prospectively reviewed based on routine endoscopy and scoring. Both diseases were classified by experienced gastroenterologists. The study was approved by the ethics committee of the Centre Zagreb university hospital. Mann-Whitney U tests or Kruskal-Wallis tests (with Dunn correction) were performed to compare C3-HNE levels between groups. The discriminative utility of C3-HNE was evaluated using AUC with ROC curves.

[0063] Cohort II The study population for Cohort II consisted of IBD patients (n=149) treated with infliximab (IFX). Blood samples were taken at the start of the study and followed up twice. This study included the same healthy control sample (n=40) as Cohort I. The C3-HNE assay was performed blindly. The study was approved by the Danish Data Protection Agency and the Regional Ethics Committee of the Hovedstaden region, Denmark. Responders were defined as those with a score of 0, compliant with the Physician Global Assessment, at 3 / 3 of hospital visits. Statistical data for additional patient populations have not been determined. The Mann-Whitney U test was performed to compare C3-HNE levels between groups. The discriminative utility of C3-HNE was assessed by AUC using ROC curves.

[0064] Cohort III Cohort III was obtained from Herlev / Bispebjerg Hospital in Denmark and included Crohn's disease (CD) patients (n=49) and healthy donor (HD) individuals (n=44). Endoscopic and clinical disease activity was assessed using the Ulcerative Colitis Endoscopic Index (UCEIS) and the complete Mayo score. Serum C3-HNE levels were continuously measured from baseline to week 24 from 33 patients with active ulcerative colitis (UC) and classified according to their response to treatment (defined as a change in total Mayo score from severe / moderate disease at baseline to mild disease / remission at week 24). The Mann-Whitney U test was performed to compare C3-HNE levels between groups. The discriminative utility of C3-HNE was evaluated by AUC using ROC curves.

[0065] DSS-induced colitis model 12-week-old male Sprague Dolly (SD) rats (n=36) were divided into three groups (n=6) and treated with 5% (wt / vol) DSS (Sigma-Aldrich, Denmark) in their drinking water for 6 days to induce colitis (Groups A, B, and C). The DSS was then removed, and the rats drank ordinary water for 5 days (Wirtz et al., 2007). Control rats were also divided into three groups (n=6) and drank only ordinary water (Groups D, E, and F). Disease activity index (DAI) scores were monitored daily, and blood samples were collected according to each of the six groups. As shown in Figure 1, groups D and F were slaughtered on day 8, groups A and D on day 9, and groups B and E on day 10 for histological examination of the tissues.

[0066] Cleavage of type III collagen The purpose of the experimental cleavage was to evaluate whether HNE is capable of releasing desired fragments from fibroblast-deposited ECM. Because collagen helices are tightly packed structures, preliminary cleavage was performed using collagenases MMP-8 and -13 to unwind the tightly packed triple helix. The experiment was conducted on ice, and the buffers used were as follows: 1. HNE assay buffer for activated HNE (Abcam) (100 mM Tris-HCl, 500 mM NaCl, pH 7.5) 2. MMP assay buffer for MMP (50 mM Tris-HCl, 200 mM NaCl, 10 mM CaCl2, 100 μM ZnCl, pH 7.5)

[0067] Scar-in-a-Jar (SIAJ) is an in vitro model that stimulates fibrosis, in which fibroblasts (derived from the GI system) deposit extracellular matrix (ECM) in plastic wells. Human primary intestinal fibroblasts purchased from Cell Biologics (Chicago, Illinois, USA) (cat. no. H-6025) were grown until confluence. On day 2, 6-8 passages were inoculated at a concentration of 30,000 cells / well into Dulbecco's modified Eagle medium (DMEM) + Glutamax (cat. no. 31966, Gibco, Life Technologies, Carlsbad, California, USA) in a 48-well plate containing high-serum medium (10% fetal bovine serum (FBS)) (cat. no. F7524, Sigma-Aldrich, St. Louise, Missouri, USA). On day 1, the cells were deprived of serum in low-serum medium (0.4% FBS DMEM) to avoid interference with biomarker measurements. On day 0, during the induction of fibrillation, cells were cultured in a low-serum medium containing Ficol-70 (112.5 mg / mL, cat. no. F2878, Sigma-Aldrich, St. Louise, Missouri, USA) and Ficol-400 (75 mg / mL, cat. no. F4375, Sigma-Aldrich, St. Louise, Missouri, USA), with the addition of 1.0% L-ascorbic acid phosphate magnesium salt n-hydrate (cat. no. 013-19,641, Wako, Osaka, Japan), and stimulated with or without 20 ng / mL TGF-β1 (cat no. 100-B-010 / CF, R&D system, Minneapolis, Minnesota, USA). Cell culture was performed by incubating at 37°C in 95% O2 and 5% CO2 for 12 days, and replacing the culture medium with freshly prepared medium on days 0, 4, and 8. Wells were decellularized and incubated with HNE, or with matrix metalloproteinases - MMP8 (RnD systems, 904-MP, lot CLF0721041) and MMP13 (RnD systems, 511-MM, lot CJT1221111), or with MMP8 and MMP13 alone. The HNE reaction was incubated at 37°C for 72 hours and stopped by adding 2 μl of AEBSF (Thermo Fischer) inhibitor to a final concentration of 1 mM. Matrix metalloproteinases (MMPs) were activated at 37°C for 2 hours using MMP assay buffer containing 4-aminophenylmercury acetate (APMA) (1 mM per 100 μg / ml of proteinase) before incubation with type III collagen. The MMP reaction was then incubated at 37°C for 24 hours and stopped by adding EDTA to a final concentration of 1 mM. The control of uncleaved type III collagen was incubated at 37°C for 72 hours. The protease-to-protein ratio was set to 1:100, and the concentrations of each reagent can be found in Table 1.

[0068] [Table 1]

[0069] result This section describes the results obtained through assay development and evaluation of the technical performance of the C3-HNE biomarker assay. Subsequently, preliminary evaluation and biological relevance of serum from IBD (UC and CD) patients were investigated in one preclinical study and three clinical studies. Furthermore, as a proof of concept, fibroblast-precipitated type III collagen was cleaved.

[0070] The C3-HNE antibody is highly specific to the target fragment. The specificity of the neoepitope C3-HNE was evaluated by the reactivity of the mAb to a selective (standard) peptide (PGKNGERGGP (SEQ ID NO: 1)), an elongated peptide (APGKNGERGGP (SEQ ID NO: 2)), and a truncated peptide (GKNGERGGP (SEQ ID NO: 3)). The selective peptide representing the neoepitope dose-dependently inhibited the signal derived from the reaction between the coater peptide and the antibody. No reactivity was observed when the mAb was incubated with the elongated or truncated peptide. Thus, only the selective peptide competed for antibody binding. These results demonstrate that the mAb is highly specific to the neoepitope under study (Figure 2).

[0071] C3-HNE levels are elevated in IBD patients compared to healthy donors. Before performing technical validation of the assay, the biological relevance between commercially available IBD samples and healthy donor samples was evaluated. Samples were incubated for 1 hour at a coater concentration of 0.5 ng / ml and an mAb concentration of 15 ng / ml. C3-HNE levels were significantly elevated in both CD (n=16) and UC (n=14) samples compared to healthy donor samples (n=32) (38.2 ng / ml [32.94, 44.30] vs. 35.86 ng / ml [29.80, 47.16] vs. 30.85 ng / ml [25.10, 34.93]). (p=0.002 and p=0.036) (Figure 3).

[0072] C3-HNE is a technically robust ELISA assay. Measurement limits LLOB is the highest quantile value in the blank sample. The blank sample for the C3-HNE assay is the following assay buffer, also known as the background: 50 mM PBS-BTB, 8 g NaCl, pH 7.4. LLOB was determined by performing one run of 60 repetitions with the assay buffer. The arithmetic mean of the measurements was calculated to be 3.72 ng / ml. LLOQ is the lower limit at which the assay can yield a quantitative measurement. LLOQ was determined by performing five independent runs using four low-concentration human serum samples (2 measurements and 3 repetitions). LLOQ was established as 7.00 ng / ml.

[0073] Linearity of assay The minimum dilution ratio (MRD) and dilution recovery rate were determined by linearity verified during 2-fold dilution of human serum samples. For dilution recovery rate, four high-concentration human serum samples were diluted with assay buffer, and linearity was repeated three times, once daily. The acceptable standard for dilution recovery rate was 100 ± 20%, and the samples showed an acceptable dilution recovery rate for 4-fold dilution. At 8-fold dilution, more than 75% of the samples did not fall within the acceptable standard. When diluting in series, the concentration was reduced from 1+0 dilution to 1+2 dilution, but no matrix effect was observed. Therefore, the MRD was determined to be 1+0 (undiluted sample).

[0074] Assay accuracy The accuracy of the assay was determined by matrix-matrix spike recovery. Three low-concentration samples (around IC20) were spiked into three high-concentration samples (around IC50). Each sample set had matrix-matrix spiked samples (spike-in) with high-concentration and low-concentration samples in ratios of 100:0, 75:25, 50:50, 25:75, and 0:100. The acceptable recovery rate was defined as 100 ± 20% for samples with a recovery rate of 75% or higher.

[0075] Precision and reproducibility of assays The robustness of the assay and the precision of concentrations measured within or between plates were determined by performing 10 independent runs within and between assays. The samples used were eight serum samples and two QCs covering the assay range. Assay robustness was calculated as the coefficient of variation (CV%) of sample measurements. The 11-point standard curve was robust, with 100% recovery for the first seven points, but the eighth point failed at 7.81 ng / ml (60% recovery). However, the curve fitting using the 11 points was the best and was therefore retained. Recovery rates for all parameters were less than 10%, particularly the mean IC50 of ~58 and the mean slope of ~1.07. Inter-assay variability ranged between 3-12%, and intra-assay variability ranged between 4-9%, both of which were within acceptable limits.

[0076] Assay stability Assay stability was tested by freeze-thaw and kit stability tests. In the freeze-thaw test, the analytes were subjected to five freeze-thaw cycles. The recovery percentage was within the acceptable range of 100 ± 20%. Kit stability was tested by performing three assay runs on the same eight samples used in the assay precision and reproducibility tests. The assay reagents from the three kits were left at room temperature for 24 hours. The results showed that the recovery percentage was within the acceptable range of 100 ± 20% for all samples. In all cases, the assays demonstrated good stability.

[0077] Assay interference Assay interference was tested by spiking hemoglobin, biotin, and lipids into three serum samples at different concentrations. Analysis showed no interference between all substances and serum samples. Recovery percentages were all acceptable within the range of 100 ± 20%.

[0078] Final assay parameters Technical validation results indicate that the C3-HNE assay is technically robust and can be used to test human serum samples. A summary of all parameters of the technical validation is presented in Table 2. These characteristics should be considered when identifying human serum samples using the C3-HNE assay.

[0079] [Table 2]

[0080] C3-HNE levels are elevated in IBD patients compared to healthy donors. The biological utility of C3-HNE was evaluated in serum samples from Cohort I. Cohort I included patients with CD (n=40) and UC (n=85), as well as healthy donors (n=40). C3-HNE levels were significantly higher in patients with CD and UC compared to healthy controls (37.44 ng / ml [27.07, 57.44] vs. 39.8 ng / ml [25.45, 57.25] vs. 31.2 ng / ml [24.48, 36.54]) (p=0.007 and p=0.043) (Figure 4). All quantile levels of C3-HNE were higher than LLOQ. These results suggest that C3-HNE fragment generation is associated with IBD. The biological relevance of C3-HNE was evaluated in serum samples from Cohort II. Cohort II included IBD patients treated with infliximab (n=149). This study included the same healthy control samples (n=40) as Cohort I. C3-HNE levels were significantly higher in IBD patients compared to healthy control donors (36.64 ng / ml [25.17, 50.71] vs. 31.2 ng / ml [24.48, 36.54]) (p=0.003) (Figure 5). All quantile levels of C3-HNE were higher than LLOQ. These results suggest that C3-HNE fragment formation is associated with IBD and demonstrate its potential as a biomarker.

[0081] C3-HNE has the potential to be a marker for the early stages of clinically evident mucosal injury. C3-HNE levels significantly increased during the DSS treatment period, while the control group showed a decrease in biomarker levels. During the period of colitis induction, C3-HNE levels rose earlier in time (day 1) than the increase in the disease activity index (DAI) (day 7). Upon discontinuation of DSS, the DAI score slowly increased to its maximum value by day 14, while C3-HNE levels rapidly decreased to the same level as the control group on day 14 (Figure 6). Therefore, these results suggest that the C3-HNE biomarker reflects an early stage of clinically evident mucosal damage during the experimental colitis period.

[0082] C3-HNE fragments are released from the fibroblast-deposited ECM upon HNE cleavage. C3-HNE levels measured in scar-in-a-jar (SIAJ) matrices cleaved with MMP-8, MMP-13, and HNE were elevated compared to negative controls (matrix + HNE, and matrix + MMP-8 + MMP-13) (Figure 7). All measured values ​​were lower than LLOQ (established for human serum).

[0083] Discrimination ability of commercially available samples, Cohort I, and Cohort II for C3-HNE Based on ROC analysis, the biomarker can significantly differentiate between healthy donors (HD) and Crohn's disease (CD) or ulcerative colitis (UC) in commercially available samples (P<0.001 and p=0.01), between HD and CD or UC in Cohort I (P<0.01 and p=0.01), and between HD and IBD in Cohort II (P<0.05) (Table 3).

[0084] [Table 3]

[0085] The C3-HNE fragment is released from primary neutrophil cells inoculated onto type III collagen when LPS is activated. C3-HNE levels were elevated in primary neutrophils activated with LPS for 6 hours. Although not statistically significant, this intriguing trend in LPS-activated neutrophils attracted attention. Levels of the C3-HNE biomarker increased with stimulation of 25 ng / ml LPS on type III collagen-coated wells compared to stimulation with 100 ng / ml LPS (Figure 8).

[0086] C3-HNE is a candidate with potential for monitoring treatment response in IBD patients. The percentile range of C3-HNE from visit 1 to visit 3 was higher in responders compared to non-responders, but no statistically significant difference was observed (Figure 9.A). For Cohort III, responders to 24 weeks of treatment showed an increase in C3-HNE levels compared to non-responders, but no statistically significant difference was observed between the groups (Figure 9.B, Table 4). In Cohort II, C3-HNE levels were elevated in non-responders compared to responders (ng / ml [IQR]: 54.47 [2642, 70.2] vs. 43.8 [23.2, 60.8], p=0.1) (Table 4). However, no significant differences were observed among patients grouped according to treatment. In Cohort III, when patients were stratified according to their response to treatment, C3-HNE was elevated in responders compared to non-responders (ng / ml [IQR]: 47.5 [30.8, 70.9] vs. 31.4 [23.2, 58.2], p=0.15), but the difference was not statistically significant (Table 4).

[0087] [Table 4]

[0088] In this specification, unless otherwise specified, the word "or" is used to mean an operator that returns true if one or both of the stated conditions are met, in contrast to the "exclusive OR" operator, which requires that only one of several conditions be met. The word "comprising" is used to mean "including, or consisting of." All prior teachings acknowledged above are incorporated into this specification by reference thereto. Any recognition of prior publications in this specification should not be taken as an admission or statement that the teachings in such publications were common knowledge in Australia or other countries at the time of this specification.

[0089] References Bradbury, ARM; Trinkrain, ND; Sie, H.; Wilkinson, IC; Tandon, AK; Anderson, S.; and Duvelp, S. (2018) When monoclonal antibodies are not single-specific: Hybridomas often express additional functional mutation regions. MAbs. 2018 May-Jun; 10(4): 539-546. doi: 10.1080 / 19420862.2018.1445456 Crisciarrello, R., Sobande, T., Jones, S., Giufrida, P., Di Sabatino, A., Dosena, G., ... and Coc, K. (2020) Human neutrophil elastase proteolytic activity in ulcerative colitis promotes the loss of function of therapeutic monoclonal antibodies. Journal of Inflammation Research, 13, 233. Fischbacher, W., Becker, W., Mossner, J., Olemuller, H., Kof, W., and Bohner, W. (1987) Leukocyte elastase in chronic inflammatory bowel disease: Is it a marker of inflammatory activity? Digestion, 37(2), 88-95. F, Z., Solpe, M., Akra, S., Chahar, G., and Hermann, LT (2018) Derived cleavage specificity of human neutrophil elastase, human proteinase 3, and their distantly related ortholog, Xenopus PR3 — three elastases with similar primary specificity but differing derivative specificity and stability. Frontiers in Immunology, 2387. Gefter, ML, Margulies, DH, and Schaaf, MD (1977) A simple method for polyethylene glycol-enhanced hybridization of mouse myeloma cells. Somatic cell genetics, 3(2), 231-236.

[0090] Graham, DB, and Xavier, RJ (2020) Pathway paradigms revealed by the genetic characteristics of inflammatory bowel disease Nature, 578(7796), 527-539. Graham, MF; Dee Gelman, RF; Elson, CO; Lindblad, WJ; Gottcharuk, N.; Guy, S.; and Guy, R. (1988) Collagen quantity and type in intestinal strictures in Crohn's disease Gastroenterology, 94(2), 257-265. Hansbury, D., Shah, K., Agarwal, P., and Agarwal, N. (2017) Fecal myeloperoxidase as a biomarker for inflammatory bowel disease Cureus, 9(1). Jostins, L., Lipke, S., Wielsma, RK, Duel, RH, McGovern, DP, Huey, KY, ... and Cho, JH (2012) Host-microbe interactions shaped the genetic structure of inflammatory bowel disease. Nature, 491(7422), 119-124. Kellett, S., Wilmssen, N., Armbrecht, G., Dietzel, R., Brix, S., Henriksen, K., and Karsdal, MA (2018) Age-related collagen turnover in the intestinal matrix and basement membrane: Close relationship between age- and sex-dependent extracellular matrix remodeling. PLoS One, 13(3), e0194458.

[0091] Kon, S., Chang, YH, and Chang, W. (2018) Regulation of the characteristics and function of intestinal epithelial cells by amino acids BioMed research international, 2018. Clear A., ​​Yaburaoui A., Leemi S., Sousou S., Mukaouaru H., Mariaure V., ... and Leemi M. (2021) SP-1, a serine protease derived from the gut microbiota, affects colitis and causes intestinal dysbiosis in mice. Cells, 10(10), 2658. Mylet, N., (2020) Rapid peptide generator: Fast and efficient in silico protein digestion NAR Genomics and Bioinformatics, 2(1), lqz004. Menegazzi, R., Declever, E., and Dori, P. (2012) Killing by neutrophil extracellular traps: Fact or legend? Blood, The Journal of the American Society of Hematology, 119(5), 1214-1216. Mimura, Y., Church, S., Zealland, R., Ashton PR, Don, S., Goodall, M., ... and Jefferys, R. (2000) Effect of glycosylation on temperature stability and effector function expression of human IgG1-Fc: Properties of a series of truncated glycoforms Molecular immunology, 37(12-13), 697-706.

[0092] Pawar, R., Goyal, A., and Diaral, I. (2021) chronic inflammation StatPearls [Internet]. Kiu, P., Ishimoto, T., Fu, L., Chang, J., Chang, Z., and Liu, Y. (2022) Gut microbiota in inflammatory bowel disease Frontiers in Cellular and Infection Microbiology, 102. Saez, A., Herleo Fernandez, B., Gomez-Bliss, R., Sanchez-Martinez, H., and Gonzalez-Granado, JM (2023) Pathophysiology of inflammatory bowel disease: Innate immune system International Journal of Molecular Sciences, 24(2), 1526. Schmid, M., Ferremann, K., Fritz, P., Wiedow, O., Stanji, E., and Wekamp, ​​J. (2007) Inducible attenuation of elaphin, an epithelial and leukocyte serine antiprotease, and secretory leukocyte protease inhibitors in Crohn's disease. Journal of leukocyte biology, 81(4), 907-915. Signoir, A., (2013) About inflammation and infection EJNMMI research, 3(1), 8. Wiltz, S., Neufeld, C., Weigmann, B., and Neuras, MF (2007) A mouse model of chemically induced enteritis Nature protocols, 2(3), 541-546. Yuen, J., Purcelo, F.G., Duda, DN., Liedol, M., Cherry, A., Ulanova, M., ... and Licht, C. (2016) Neutrophils forming a NET activate the NET itself or bacterial complement via alternative or non-alternative pathways. Frontiers in immunology, 7, 137.

Claims

1. Immunoassay methods comprising the following i) and ii); i) Contacting the patient's sample with a monoclonal antibody that specifically binds to the N-terminal amino acid sequence PGKNGERGGP (SEQ ID NO: 1); and, ii) To detect the binding between the monoclonal antibody and the peptide in the sample and to determine the amount of binding.

2. The method is an immunoassay method for detecting and / or monitoring inflammatory bowel disease or a specific level of severity of said disease in a patient, further comprising iii) below, according to claim 1; iii) Correlating the binding amount with a value associated with a normal healthy person, and / or a value associated with a known disease severity, and / or a value obtained from the patient at a past point in time, and / or a predetermined cutoff value.

3. The immunoassay method according to claim 2, wherein the inflammatory bowel disease is Crohn's disease or ulcerative colitis.

4. The immunoassay method according to any one of the claims, wherein the sample of the patient is selected from blood, plasma, or serum.

5. The immunoassay method according to any one of the claims, wherein the monoclonal antibody specifically binds to the N-terminal amino acid sequence PGKNGERGGP (SEQ ID NO: 1) and does not specifically bind to the peptide having the N-terminal amino acid sequence APGKNGERGGP (SEQ ID NO: 2).

6. The immunoassay method according to any one of the claims, wherein the monoclonal antibody specifically binds to the N-terminal amino acid sequence PGKNGERGGP (SEQ ID NO: 1) and does not specifically bind to the peptide having the N-terminal amino acid sequence GKNGERGGP (SEQ ID NO: 3).

7. The immunoassay method according to any one of the claims, wherein the monoclonal antibody is produced in opposition to a synthetic peptide having the N-terminal amino acid sequence PGKNGERGGP (SEQ ID NO: 1).

8. The immunoassay method according to any one of the claims, wherein the immunoassay is a competitive assay or a sandwich assay.

9. The immunoassay method according to any one of the claims, wherein the immunoassay is a radioimmunoassay or an enzyme-linked immunosorbent assay.

10. A monoclonal antibody that specifically recognizes the N-terminal amino acid sequence PGKNGERGGP (SEQ ID NO: 1).

11. The monoclonal antibody according to claim 13, wherein the monoclonal antibody specifically binds to the N-terminal amino acid sequence PGKNGERGGP (SEQ ID NO: 1) and does not specifically bind to the peptide having the N-terminal amino acid sequence APGKNGERGGP (SEQ ID NO: 2).

12. The monoclonal antibody according to claim 13 or claim 14, wherein the monoclonal antibody specifically binds to the N-terminal amino acid sequence PGKNGERGGP (SEQ ID NO: 1) and does not specifically bind to the peptide having the N-terminal amino acid sequence GKNGERGGP (SEQ ID NO: 3).

13. The monoclonal antibody according to any one of claims 13 to 15, wherein the monoclonal antibody is produced in opposition to a synthetic peptide having the N-terminal amino acid sequence PGKNGERGGP (SEQ ID NO: 1).

14. An immunoassay kit comprising a monoclonal antibody according to any one of claims 13 to 19, and at least one of the following: - Streptoavidin-coated well plates - Biotinylated peptide PGKNGERGGP-L-biotin (SEQ ID NO: 20), where L is an optional linking group. - Second antibody used in sandwich immunoassays - Calibration protein containing N-terminal amino acid sequence PGKNGERGGP (SEQ ID NO: 1) - Antibody biotinylation kit - Antibody HRP labeling kit - Antibody radiolabeling kit.

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