C4A3-HNE and C4A4-HNE assays

Abstract

Immunoassay methods for detecting HNE-generated fragments of the α3 or α4 chain of type IV collagen in a patient sample and their use for detecting and / or monitoring inflammatory bowel disease (IBD) or a particular level of severity of the disease in a patient are disclosed. Also disclosed are monoclonal antibodies and assay kits for use in the immunoassay methods.

Description

[Technical Field]

[0001] The present invention relates to an immunoassay method for detecting HNE-generated fragments of the α3 or α4 chain of type IV collagen in a patient sample, and its use for detecting and / or monitoring inflammatory bowel disease (IBD) or a particular level of severity of said disease in a patient. The present invention also relates to monoclonal antibodies and assay kits for use in the immunoassay method. [Background technology]

[0002] Inflammatory bowel disease (IBD) is a chronic, immune-mediated disease of the gastrointestinal (GI) system that encompasses Crohn's disease (CD) and ulcerative colitis (UC). The two diseases differ in the location and depth of their involvement; UC causes superficial inflammation of the colonic mucosa, whereas CD causes full-thickness ulcers in any part of the GI system. IBD results from an abnormal immune response to the intestinal microbiota and occurs in genetically susceptible individuals, where an excessive immune response induces persistent deterioration of the intestinal barrier. [1] Immune cells are thought to play a major role in the pathogenesis of IBD by expressing and secreting a plethora of different cytokines and proteases that initiate and sustain inflammation. [2] For these reasons, immune cells are important targets for biological therapy in IBD. Although disease control has improved substantially with the adoption of anti-tumor necrosis factor (TNF) antibodies, up to 40% of IBD patients suffer from non-response or impaired response after induction therapy. [3] Therefore, better patient profiling and monitoring are needed to provide the best possible treatment. Improved detection of asymptomatic ulceration is of paramount importance, as early-stage IBD offers a unique window of opportunity for intervention, as once deep inflammation is established, the disease becomes a self-perpetuating progressive process.

[0003] Neutrophils are the most abundant immune cells in the circulation. They are the first responding immune cells in the innate immune system and are well known for their rapid recruitment to inflammatory sites. Upon activation, neutrophils produce NETs (neutrophil extracellular traps), in which they release the antimicrobial protein calprotectin and proteases such as human neutrophil elastase (HNE) from granules stored within the neutrophil cytoplasm. [4] Continual activation and excessive recruitment of neutrophils are common features of IBD, and neutrophil infiltration into the intestinal tissue correlates with disease activity. [4] HNE is a serine protease and the most abundant protein in NETs. HNE levels are elevated in the plasma of IBD patients compared with healthy controls, HNE expression is upregulated in intestinal tissues of UC, and HNE levels correlate with histopathological disease scores in CD. [5-7] .

[0004] As previously mentioned, the breakdown of the intestinal barrier is a hallmark of IBD, leading to the exposure of intestinal bacteria and promoting early inflammation. Therefore, the extracellular matrix (ECM) is of high importance for the structural integrity of the intestinal tissue, especially the intestinal basement membrane (BM). The intestinal basement membrane (BM), which serves as the first line of defense, is a collagen-rich matrix, with type IV collagen as the major collagen component. [8] . Type IV collagen consists of six genetically distinct alpha chains, three of which form a single molecule. Three different alpha chain compositions are known for type IV collagen, which vary depending on the tissue. The composition α1α1α2 is thought to be present in almost all basement membranes, while the composition α5α5α6 is primarily localized in the epidermis. The third composition, α3α4α5, is thought to be present only in the kidney and lung alveoli (both organs require selective filtration through their BM). [9] However, immunostaining of all six alpha chains in the human large intestine showed that these chains are also present in the intestine, with α3 and α4 being expressed solely on the outermost mucosal surface facing the lumen of the GI tract. [10,11] . Summary of the Invention

[0005] Applicants have now developed and biologically evaluated two competitive enzyme-linked immunosorbent assays (ELISAs) targeting HNE-generated neo-epitopes in the α3 or α4 chain of type IV collagen, and demonstrated the utility of these HNE-generated neo-epitopes as biomarkers for inflammatory bowel disease (IBD) in general, and early-stage or mild IBD in particular.

[0006] Thus, in a first aspect, the present invention provides an immunoassay method, which comprises contacting a sample from a patient with a monoclonal antibody that specifically binds to an HNE-generated neo-epitope carried by an HNE-generated fragment of the α3 chain or α4 chain of type IV collagen, and detecting binding between the monoclonal antibody and a peptide in the sample to determine the amount of binding, wherein the HNE-generated neo-epitope consists of the N-terminal or C-terminal sequence of the HNE-generated fragment present at the end of the HNE-generated fragment cleaved by HNE.

[0007] In a preferred embodiment, the immunoassay method comprises the following steps i) and ii): i) contacting the patient's sample with a monoclonal antibody that specifically binds to the N-terminal amino acid sequence EGTRPGPPGP (SEQ ID NO: 1) (also referred to herein as "C4A3-HNE" or "C4A3-HNE target sequence") or that specifically binds to the N-terminal amino acid sequence TYPGRHGPPG (SEQ ID NO: 2) (also referred to herein as "C4A4-HNE" or "C4A4-HNE target sequence"); and ii) detecting binding between the monoclonal antibody and the peptide in the sample and determining the amount of binding.

[0008] In a preferred embodiment, the immunoassay method is for detecting and / or monitoring inflammatory bowel disease or a particular level of severity of the disease in a subject, and further comprises correlating the amount of binding with a value associated with normal healthy individuals and / or with a value associated with a known disease severity and / or with a value obtained from the subject at a previous time point and / or with a predetermined cut-off value.

[0009] 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 method for detecting mild or early stage inflammatory bowel disease. In a preferred embodiment, the patient sample is a human biological fluid sample. Preferably, the sample is a blood material sample such as blood (whole blood), plasma or serum.

[0010] When the monoclonal antibody specifically binds to the N-terminal amino acid sequence EGTRPGPPGP (SEQ ID NO: 1), preferably the monoclonal antibody does not specifically bind to a peptide having the N-terminal amino acid sequence SEGTRPGPPGP (SEQ ID NO: 3) (i.e., an extension of the C4A3-HNE target sequence extended by adding a serine residue at its N-terminus) and / or does not specifically bind to a peptide having the N-terminal amino acid sequence GTRPGPPGP (SEQ ID NO: 4) (i.e., a truncation of the C4A3-HNE target sequence truncated by removing the first glutamic acid residue). Preferably, the ratio of the affinity of the antibody for the C4A3-HNE target sequence to the affinity of the antibody for extensions of the target sequence is at least 10:1, more preferably at least 20:1, or at least 30:1.

[0011] When the monoclonal antibody specifically binds to the N-terminal amino acid sequence TYPGRHGPPG (SEQ ID NO: 2), preferably the monoclonal antibody does not specifically bind to a peptide having the N-terminal amino acid sequence VTYPGRHGPPG (SEQ ID NO: 5) (i.e., an extension of the C4A4-HNE target sequence extended by adding a valine residue at its N-terminus) and / or does not specifically bind to a peptide having the N-terminal amino acid sequence YPGRHGPPG (SEQ ID NO: 6) (i.e., a truncation of the C4A4-HNE target sequence truncated by removing the first threonine acid residue). Preferably, the ratio of the affinity of the antibody for the C4A4-HNE target sequence to the affinity of the antibody for extensions of the target sequence is at least 10:1, more preferably at least 20:1, or at least 30:1.

[0012] When the monoclonal antibody specifically binds to the N-terminal amino acid sequence EGTRPGPPGP (SEQ ID NO: 1), the monoclonal antibody may be, for example, one raised against a synthetic peptide having the N-terminal amino acid sequence EGTRPGPPGP (SEQ ID NO: 1); and when the monoclonal antibody specifically binds to the N-terminal amino acid sequence TYPGRHGPPG (SEQ ID NO: 2), the monoclonal antibody may be, for example, one raised against a synthetic peptide having the N-terminal amino acid sequence TYPGRHGPPG (SEQ ID NO: 2). For example, monoclonal antibodies can be produced by: (a) immunizing a rodent (or other suitable mammal) with a synthetic peptide comprising the N-terminal amino acid sequence EGTRPGPPGP (SEQ ID NO: 1) or the N-terminal amino acid sequence TYPGRHGPPG (SEQ ID NO: 2), optionally linked to an immunogenic carrier protein (such as keyhole limpet hemocyanin) at its C-terminus; (b) isolating and cloning a single antibody-producing cell; and (c) assaying the resulting monoclonal antibodies to determine that they have the desired specificity.

[0013] In a preferred embodiment, 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 techniques known to those skilled in the art.

[0014] The term "N-terminus" as used herein refers to an N-terminal peptide sequence at the beginning of a polypeptide, i.e., a peptide sequence at the N-end of a polypeptide, and should not be construed as denoting its general orientation. The term "C-terminus" as used herein refers to a C-terminal peptide sequence at the beginning of a polypeptide, i.e., a peptide sequence at the C-end of a polypeptide, and should not be construed as denoting its general orientation. As used herein, the terms "peptide" and "polypeptide" are used interchangeably.

[0015] As used herein, the term "monoclonal antibody" refers to both whole antibodies and fragments thereof 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, whole antibodies typically have a "Y-shaped" structure consisting of two identical paired polypeptide chains, each of which is composed of one "light" chain and one "heavy" chain. The N-terminal regions of each of the light and heavy chains comprise the variable region, while the C-terminal portions of each of the heavy and light chains constitute the constant region. The variable regions contain three complementarity-determining regions (CDRs), which are primarily responsible for antigen recognition. The constant region enables the antibody to recruit cells and molecules of the immune system. Antibody fragments that retain binding specificity contain at least the CDRs and a sufficient portion of the remainder of the variable region to retain binding specificity.

[0016] The present invention can use monoclonal antibodies containing any constant region known in the art. In mouse and human antibodies, constant light chains are classified as kappa or lambda light chains. Constant heavy chains are classified as mu, delta, gamma, alpha, or epsilon, defining the antibody isotype as IgM, IgD, IgG, IgA, or IgE, respectively. The IgG isotype has several subclasses, including IgG1, IgG2, IgG3, and IgG4 in humans, and several subclasses, including IgG1, IgG2a, IgG2b, IgG2c, and IgG3 in mice, but is not limited to these. The monoclonal antibody preferably belongs to the IgG isotype, including any one of the IgG subclasses.

[0017] The CDRs of an antibody can be determined using methods known in the art, such as those 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 generated antibody can be determined using standard techniques. For example, RNA can be isolated from cells and used to generate cDNA by reverse transcription. The cDNA can then be subjected to PCR using primers that amplify the heavy and light chains of the antibody. For example, primers specific to the leader sequences of all VH (variable heavy) 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 a primer that binds to the 3' end of the kappa or lambda chain together with a primer that anneals to the leader sequence of Vkappa or Vlamda. Full-length heavy and light chains can be generated and sequenced.

[0018] As used herein, the term "amount bound" refers to the quantification of binding between an antibody and a peptide in a patient sample. This quantification may be determined, for example, by comparing the measured binding in the patient sample to a calibration curve generated using measured binding in standard samples containing known concentrations of the peptide to which the antibody specifically binds, to determine the amount of peptide in the patient sample to which the antibody specifically binds. Any analytical method suitable for measuring the amount of binding can be used. For example, an ELISA method can be used, which uses spectrophotometric analysis to measure both the amount of binding in the patient sample and the amount of binding when generating the calibration curve.

[0019] As used herein, the term "predetermined cutoff value" refers to a statistically determined amount of binding that indicates a high probability that a subject has a disease (e.g., IBD) or that the disease is of a particular severity, and in this sense, when a measured value of a target peptide in a subject's sample is equal to or greater than the statistical cutoff value, the measured value corresponds to at least a 70% probability, preferably at least a 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 the disease or the disease being of a particular severity.

[0020] As used herein, the term "value associated with a normal healthy individual" refers to a normalized binding amount determined by the above method for a sample obtained from a subject who is considered to be healthy, i.e., disease-free (i.e., not IBD); and the term "value associated with known disease severity" refers to a normalized binding amount determined by the above method for a sample obtained from a subject who is known to have a disease of known severity (i.e., IBD).

[0021] 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 the steps of: (a) performing an immunoassay on a sample obtained from a patient to detect the presence or severity of IBD according to the first aspect of the invention; and (b) if it is determined in step (a) that the patient has IBD or that its severity is at a particular level, administering to the patient a therapy for treating IBD.

[0022] Preferred embodiments of the method according to the second aspect will be apparent from the above discussion of preferred embodiments of the method according to the first aspect. For example, IBD may particularly comprise Crohn's disease (CD) or ulcerative colitis (UC); and / or said step (a) may particularly comprise carrying out an immunoassay method according to the invention of the first aspect, for detecting mild or early stage IBD, and if in step (a) it is determined that the patient has mild or early stage IBD, said subsequent step (b) may comprise administering to the patient a therapy for treating IBD.

[0023] The therapy may be any therapy suitable for treating the inflammatory bowel disease of interest, and may include or consist of, for example, one or more medications, one or more lifestyle changes, one or more surgeries, or any combination thereof. The agent may be formulated for local or systemic administration. Topical agents may be formulated, for example, as creams, foams, gels, lotions, or ointments. Systemic agents may be formulated, for example, for gastrointestinal or parenteral administration. The surgery may be curative, preventative, palliative, and / or reconstructive surgery.

[0024] For example, if the inflammatory bowel disease is Crohn's disease, appropriate therapy may include one or more of the following therapies: lifestyle changes such as dietary modification, elemental nutrition, adequate hydration, and smoking cessation; anti-TNF therapies (also referred to as equivalents of TNF inhibitors) such as antibiotics, aminosalicylates, corticosteroids, 5-aminosalicylic acid (5-ASA), prednisone, azathioprine, 6-mercaptopurine, methotrexate, monoclonal antibodies that inhibit the action of TNF, such as infliximab, adalimumab, and certolizumab, and other biologics and antibodies such as vedolizumab, ustekinumab, and natalizumab; and combinations thereof.

[0025] Where the inflammatory bowel disease is ulcerative colitis, appropriate therapy may include one or more of the following therapies: aminosalicylates such as mesalazine, sulfasalazine, balsalazide, olsalazine, corticosteroids such as cortisone, prednisone, hydrocortisone, methylprednisolone, budesonide, immunosuppressants such as mercaptopurine, azathioprine, methotrexate; and anti-TNF therapy (TNF inhibitors) such as infliximab, adalimumab, and golimumab, and other biologics and antibodies such as tofacitinib and vedolizumab; surgical procedures such as partial or total colectomy; and combinations thereof.

[0026] In a third aspect, the present invention provides a monoclonal antibody that specifically binds to the N-terminal amino acid sequence EGTRPGPPGP (SEQ ID NO: 1) (i.e., the C4A3-HNE target sequence) or specifically binds to the N-terminal amino acid sequence TYPGRHGPPG (SEQ ID NO: 2) (i.e., the C4A4-HNE target sequence). The antibodies of the third aspect of the invention are particularly suitable for use in carrying out the immunoassay method of the first aspect of the invention. Preferred embodiments and features of the antibodies of the third aspect will therefore be apparent from the above discussion of preferred embodiments of the method of the first aspect.

[0027] In a fourth aspect, the present invention provides an immunoassay kit comprising a monoclonal antibody according to the third aspect of the invention and at least one of the following: - Streptavidin-coated well plates - the biotinylated peptide EGTRPGPPGP-L-biotin (SEQ ID NO: 7), where L is an optional linking group, or the biotinylated peptide TYPGRHGPPG-L-biotin (SEQ ID NO: 8), where L is an optional linking group - Secondary antibodies used in sandwich immunoassays a calibration protein containing the N-terminal amino acid sequence EGTRPGPPGP (SEQ ID NO: 1) or a calibration protein containing the N-terminal amino acid sequence TYPGRHGPPG (SEQ ID NO: 2); - Antibody Biotinylation Kit - Antibody HRP Labeling Kit - Antibody radiolabeling kit. The immunoassay kit according to the fourth aspect of the invention is particularly suitable for use in carrying out the immunoassay method according to the first aspect of the invention. Accordingly, further preferred embodiments and features of the immunoassay kit according to the fourth aspect will be apparent from the above discussion of preferred embodiments of the method according to the first aspect. [Brief explanation of the drawings]

[0028] figure [Figure 1] Figure 1: Analysis of dose-dependent reactivity of C4A3-HNE and C4A4-HNE monoclonal antibodies against selected, extended, and truncated peptides. [Figure 2]Figure 2: HNE digestion of synthetic peptides mimicking (A) type IV collagen alpha-3 chain and (B) type IV collagen alpha-4 chain. Peptides were incubated with active HNE for 4 hours. Negative controls were peptide (no protease) and protease (no peptide). HNE digestion of both peptides released neo-epitopes during incubation. Buffer was used as a background control. Values ​​shown are background-subtracted values. [Figure 3] Figure 3: Measurement of C4A3-HNE in CD and UC. C4A3-HNE is elevated in both CD and UC compared to healthy controls (p<0.05). Data are shown as median values.

[0029] [Figure 4] Figure 4: Measurement of C4A3-HNE in different stages of IBD. High levels of C4A3-HNE reflect early mucosal damage in IBD patients. [Figure 5] Figure 5: Measurement of FeCal and CRP in CD and UC. Inflammatory markers are lower in IBD patients with high levels of C4A3-HNE. [Figure 6] Figure 6: Measurement of C4A3-HNE and C4Ma3 in lung disease. The two immune cell-produced COL4 alpha-3 chain biomarkers do not reflect the same pathological process, as reflected by a weak correlation of 0.33.

[0030] [Figure 7] Figure 7: Measurement of C4A4-HNE in CD and UC. C4A4-HNE is elevated in IBD compared to healthy controls. [Figure 8] Figure 8: Measurement of C4A4-HNE in CD and UC. C4A4-HNE is significantly elevated in both CD and UC compared to healthy donors (**** = p<0.0001, ** = p<0.01). [Figure 9]Figure 9: Measurement of C4A4-HNE in different stages of IBD. C4A4-HNE is higher in patients with mild endoscopic UC and decreases in a dose-dependent manner in patients with moderate and severe endoscopic UC. [Example]

[0031] The embodiments disclosed herein are described in the following examples. These examples are presented to aid in understanding the present disclosure and should not be construed in any way to limit the scope of the disclosure as defined in the claims that follow. The examples set forth below are presented 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 disclosure, nor are they intended to imply that the experiments described below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

[0032] Materials and Methods Assay Development Neo-epitope identification by in-silico cleavage The α3 and α4 chains of type IV collagen are expressed singly on the outermost surface of the lumen-facing mucosa of the GI tract [10,11] Based on the known expression patterns of these chains in the intestine, the applicant hypothesized that these chains are susceptible to all inflammatory disorders at an early stage, such as immune cell-secreted ECM-degrading proteases. The presence of HNE is also associated with an early-stage injury response, as neutrophils are rapidly recruited to newly formed inflammatory sites. Therefore, the applicant hypothesized that HNE-generated neo-epitopes of the α3 and α4 chains of type IV collagen may serve as biomarkers reflecting early tissue damage in IBD and aid in the implementation of prompt treatment.

[0033] Initial neo-epitope identification was performed by in silico cleavage of the alpha-3 and alpha-4 chains of type IV collagen based on the cleavage sites of HNE. The cleavage sites were C-terminal Ala, Val, and Ile. Based on in-house generated mass spectrometry data, HNE also appears to cleave C-terminal serine. The cleavage fragments were first generated manually and then analyzed using the Rapid Peptide Generator software.

[12] The fragments were validated using an in silico cleavage algorithm developed using RPG, Python v3.9. Fragments of <10 amino acids were avoided, as were proline-glycine repeats. In-house MS was probed to prevent fragments that may be generated naturally or as inactive by-products by other proteases. For the HNE-degraded alpha-3 chain, one neo-epitope fragment was selected that contains the N-terminal sequence EGTRPGPPGP (SEQ ID NO: 1) ("C4A3-HNE"), formed after cleavage of the intact alpha-3 chain past residue 1059, and for the HNE-degraded alpha-4 chain, another neo-epitope fragment was selected that contains the N-terminal sequence TYPGRHGPPG (SEQ ID NO: 2) ("C4A4-HNE"), formed after cleavage of the intact alpha-4 chain past residue 670.

[0034] Production of monoclonal antibodies, characterization of clones, and their specificity Female 6- to 7-week-old Balb / C mice were immunized by subcutaneous injection with 200 μl of emulsified antigen containing 100 μg of C4A3-HNE (EGTRPGPPGP-GGC-KLH (SEQ ID NO: 9)) or C4A4-HNE (TYPGRHGPPG-GGC-KLH (SEQ ID NO: 10)) antigenic peptide (KLH = keyhole limpet hemocyanin). Immunizations were performed consecutively every 2 weeks until a stable titer level was achieved. Mice with the highest antibody titers were selected for cell fusion and rested for one month. Subsequently, the mice were boosted with 50 μg of each immunogenic peptide in 100 μl of 0.9% NaCl solution intravenously 3 days before harvesting the spleen for cell fusion.

[0035] Hybridoma cells were prepared as described by Gefter et al.

[13] Hybridoma cells were generated by fusing mouse splenocytes with mouse SP2 / 0 myeloma cells as described by

[13] . Hybridoma cells were cloned using the semisolid medium method and then plated into 96-well microtiter plates for further expansion, using limiting dilution to promote monoclonal growth. The reactivity of the supernatants against each neoepitope was screened using streptavidin-coated microtiter plates and an indirect ELISA using a biotinylated peptide of C4A3-HNE (EGTRPGPPGP-K-Biotin (SEQ ID NO: 11)) or a biotinylated peptide of C4A4-HNE (TYPGRHGPPG-K-Biotin (SEQ ID NO: 12)).

[0036] The specificity of the clones was tested against the standard / selection peptide (EGTRPGPPGP (SEQ ID NO: 1) for C4A3-HNE or TYPGRHGPPG (SEQ ID NO: 2) for C4A4-HNE), the extended peptide (SEGTRPGPPGP (SEQ ID NO: 3) for C4A3-HNE or VTYPGRHGPPG (SEQ ID NO: 5) for C4A4-HNE), and the truncated peptide (GTRPGPPGP (SEQ ID NO: 4) for C4A3-HNE or YPGRHGPPG (SEQ ID NO: 6) for C4A4-HNE). Cross-reactivity between C4A3-HNE and C4A4-HNE was also tested. The supernatant was purified using a HiTrap affinity column (GE Healthcare Life Sciences, Little Chalfront, Buckinghamshire, UK) according to the manufacturer's instructions, and the antibody was further labeled with active horseradish peroxidase using a peroxidase labeling kit according to the kit's instructions (Roche Diagnostics, Mannheim, Germany, CAT no. 11829696001).

[0037] ELISA Protocol Preliminary experiments were performed to optimize the assay reagents, their concentrations and the length of incubation time. The final competitive ELISA procedure is as follows: A 96-well streptavidin plate was coated with synthetic biotinylated peptides (100 μl / well) diluted in assay buffer (25 mM PBS-BTB 6 g NaCl / L, pH 7.4 for C4A3-HNE and 25 mM PBS-BTB 8 g NaCl / L, pH 7.4 for C4A4-HNE) and incubated for 30 min at 20°C in the dark with shaking at 300 rpm. After each incubation step, the plates were washed with wash buffer (20 mM Tris, 50 mM NaCl, 0.1% (v / v) Tween-20®, pH 7.2) using a standardized ELISA plate washer (BioTek Instruments, Microplate washer, Elx405 Select CW). Standards and serum samples (20 μl / well) were appropriately diluted and added to the plate, followed by horseradish peroxidase-conjugated monoclonal antibodies (100 μl / well) diluted in assay buffer and incubated for 1 hour at 20°C in the dark with shaking at 300 rpm. Finally, BM chemiluminescent ELISA substrate (Merck, CAT no. 11582950001) was added to each well and incubated for 3 minutes, and the plate was then read using a fluorescent plate reader (Fluroskan FL, Thermo Fisher) with a read light exposure of 1,000 ms and no filter. Standard curves were plotted using a four-parameter mathematical fitting model.

[0038] Technical evaluation Proof of concept for disconnection Due to the complex structure of collagen molecules, their poor solubility, and their tendency to aggregate into fibrils, synthetic peptides mimicking the alpha-3 and alpha-4 chains of type IV collagen were designed with target sequences located in the middle (GenScript). Neo-epitopes were generated in vitro by incubating active neutrophil elastase purified from human cells by SDS-page (Abcam, cat. no. ab280938) with the synthetic peptides. Samples were incubated in 50 mM Tris buffer (pH 7.5) containing 500 mM NaCl in a reaction volume of 200 μl at 37°C for 4 h. The protein:protease ratio was 100:1. Proteolysis was inhibited by adding the serine protease inhibitor AEBSF to a final concentration of 1 mM. Serving as experimental controls were samples containing only human neutrophil elastase (HNE), synthetic peptides without HNE, or Tris buffer alone.

[0039] Technical Validation The assay was subjected to technical validation to evaluate its robustness, accuracy, precision, interference, and stability. The lower limit of blank (LLOB) was determined by 60 replicate measurements of the assay buffer. LLOB is defined as not meeting the acceptance criteria ±3xSD of the buffer (blank). The lower limit of quantitation (LLOQ) is defined as the lowest analyte concentration in serum at which the CV% of the precision profile is 25%. The LLOQ was determined by five independent runs of triplicate measurements of four low-level human serum samples. The upper limit of quantitation (ULOQ) is defined as the highest standard point of the assay standard curve with acceptable accuracy and precision. The ULOQ was determined by ten independent runs of the standard curve. The robustness of the standard curve and the half maximal inhibitory concentration (IC50) were also determined. The analytical working range is defined as the range between the LLOQ and ULOQ.

[0040] To assess linearity, 2-fold to 8-fold dilutions of four human serum samples were performed. Percent recovery from undiluted samples was calculated, with an acceptance criterion of 100% ± 25. The reportable measurement range is based on the accepted dilution recovery and is defined as the range between the LLOQ and ULOQ corrected for the highest dilution tested. Inter- and intra-assay variability was determined by performing 10 independent duplicate runs of eight human serum samples and two kit controls covering the quantifiable range of the assay. The acceptance criterion for inter- and intra-assay variability was a CV% <15.

[0041] To assess assay accuracy and any potential matrix effects, peptide spiking recovery and matrix-matrix spiking recovery were determined. Standard peptides serially diluted in buffer were spiked into three independent serum samples, and triplicate serum samples containing high and low analyte concentrations were spiked at different ratios relative to each other. Percent recovery was calculated based on predicted and measured concentrations, with an RE% criterion of ±25.

[0042] The stability of the analytes in serum was assessed by exposing three human serum samples to four freeze-thaw cycles. Percent recoveries were calculated using the respective non-cycled serum samples as references. To test whether components commonly found in blood interfere with the assay analytes, the interfering components biotin (0-100 ng / ml), hemoglobin (low = 2.5 mg / ml, high = 5 mg / ml), and lipids (low = 1.5 mg / ml, high = 5 mg / ml) were added to each of three human serum samples. The percent recovery of the analyte for each interfering sample was calculated using the respective control sample as a reference, with an acceptance criterion of 100% ± 20%.

[0043] Clinical evaluation Patient samples Serum samples from healthy individuals and patients with CD or UC were obtained from ProteoGenix. Patient serum samples were also obtained from Odense University Hospital (OUH), Denmark. The OUH study participants were enrolled in a prospective observational study (ClinicalTrials.gov ID: NCT02612103) and included patients with CD (n = 60) and UC (n = 101). Signed informed consent was collected from each subject prior to sample collection. The study was approved by the Regional Ethics Committee of Southern Denmark (journal number: S-20150107) and conducted in accordance with the Declaration of Helsinki. Disease activity was assessed based on the Harvey Bradshaw Index (HBI) for CD (0-4: remission; 5-7: mild; 8-16: moderate; >16: severe) and the Mayo score for UC (<2: remission; 2-4: mild; 5-7: moderate; >7: severe). All patients were classified according to the Montreal classification. Endoscopic scores for disease activity were recorded for some IBD patients. To assess endoscopic disease activity, the Simplified Endoscopic Score for CD (SES-CD) (0-2: remission; 3-6: mild; 7-15: moderate; >16: severe) was applied for CD, and the Mayo Endoscopic Subscore (0: inactive; 1: mild; 2: moderate; 3: severe) was applied for UC.

[0044] Additionally, additional human serum samples (Cohort 2) were obtained from the Clinical Hospital Sentre Zagreb (Department of Gastroenterology and Hepatology) containing serum samples from CD patients (N = 72) and UC patients (n = 28). The study was conducted in accordance with the Declaration of Helsinki. Patients with comorbidities or extraintestinal manifestations were excluded from the study. Demographic data and disease activity were obtained from electronic medical records and questionnaires. Endoscopy was performed within 3 months of blood sampling. Endoscopic disease activity for UC was graded using the Mayo Endoscopic Score. The endoscopic score was prospectively validated based on routine endoscopy and scoring by an experienced IBD endoscopist.

[0045] statistical analysis All data were considered nonparametric after visual assessment of normality using density plots. Differences in clinical data were compared using the Mann-Whitney U-test or the Kruskal-Wallis test with Dunn's correction for multiple comparisons. Receiver operating characteristic (ROC) statistics using the area under the curve (AUC) were used as the overall goodness of fit of the measurements to evaluate the discriminatory ability of the biomarkers. Sensitivity and specificity criteria were obtained by determining optimal cutoff values ​​using the Youden J statistic. AUC values ​​are given with the corresponding 95% confidence intervals (CI). Data visualization was performed using R studio (version 4.1.2; Rstudio, Boston, MA, United States) and GraphPad Prism 9.2.0 (GraphPad software, San Diego, CA, United States). A P value <0.05 was considered statistically significant.

[0046] result The C4A3-HNE and C4A4-HNE ELISA assays are highly specific. The specificity of monoclonal antibodies raised against the C4A3-HNE (Figure 1A) and C4A4-HNE (Figure 1B) target sequences was evaluated. For both assays, select peptides representing the target sequences inhibited the signal in a dose-dependent manner. When mismatches were introduced by using extended and truncated peptides, signal inhibition was comparable to blanks (no reactivity). This data demonstrates that both antibodies and their ELISAs are highly specific for their respective targeted neo-epitopes.

[0047] The neo-epitopes represented by the C4A3-HNE and C4A4-HNE target sequences are detected by assays for the respective target sequences after they are released by HNE in vivo. The ability of HNE to release neo-epitopes was investigated in vivo by incubating synthetic peptides mimicking the alpha-3 and alpha-4 chains of type IV collagen with active HNE for 4 hours. The results showed that HNE was able to generate both neo-epitopes (Figures 2A and 2B). Furthermore, neo-epitopes were not detected in undigested samples (peptide only).

[0048] C4A3-HNE is a technically robust ELISA assay. The technical performance of the C4A3-HNE assay in serum was further evaluated through different technical validation steps and is summarized in Table 1. The intra-assay precision (CV%) was determined to be ≤13%, and the inter-assay precision (CV%) was determined to be ≤9%. The accuracy (RE%) was determined to range from -4% to 22%. Linearity studies showed that dilution recoveries up to 25% dilution were acceptable. Based on these data, the maximum dilution validated for human serum samples was 1 + 3 dilution. The lower limit of blank (LLOB) was determined to be 0.6 ng / ml. Furthermore, the C4A3-HNE analyte was stable to up to five freeze-thaw cycles in human serum. No interference was detected from serum containing low or high contents of biotin, lipids, or hemoglobin, with recoveries ranging from 82 to 108%. Taken together, these results demonstrate that the C4A3-HNE assay is a technically robust assay that can be used to test human serum samples.

[0049] [Table 1]

[0050] The technical performance of the C4A4-HNE assay in serum was evaluated through different technical validation steps and is summarized in Table 2. The intra-assay precision (CV%) was determined to be ≤7%, and the inter-assay precision (CV%) was determined to be ≤12%. The accuracy (RE%) was determined to range from -3% to 15%. Linearity studies showed that dilution recoveries up to 25% dilution were acceptable. Based on these data, the maximum dilution validated for human serum samples was 1 + 3 dilution. The lower limit of blank (LLOB) was determined to be 1.36 ng / ml. Furthermore, the C4A4-HNE analyte was stable to up to five freeze-thaw cycles in human serum. No interference was detected from serum containing low or high contents of biotin, lipids, or hemoglobin, with recoveries ranging from 82% to 117%. These results demonstrate that C4A4-HNE is a technically robust assay and can be used to test human serum samples.

[0051] [Table 2]

[0052] C4A3-HNE is elevated in IBD compared with healthy individuals. The biological relevance of C4A3-HNE was assessed by measuring the levels of C4A3-HNE in serum samples (obtained from Proteogenix) from IBD patients and healthy donors (obtained from Proteogenix) using a C4A3-HNE ELISA. As shown in Figure 3A, C4A3-HNE was elevated in both CD and UC compared with healthy donors (P = 0.001 and P < 0.0001, respectively) (AUC [95% CI]: 0.820 [0.669-0.971]; 0.875 [0.775-1.000]). These results were validated in serum samples from IBD patients in the OUH cohort (both p < 0.0001), as shown in Figure 3B.

[0053] C4A3-HNE is elevated in IBD patients with mild endoscopic activity. When examining ulcerative colitis patients stratified by endoscopic disease severity score, a pattern in C4A3-HNE levels was observed. Biomarker levels were highest in patients with endoscopically mild disease (71.77 ng / ml), with a dose-dependent decrease in levels for patients with endoscopically moderate (56.07 ng / ml) and severe (40.52 ng / ml) disease (Figure 4A). Patients were then divided into two groups based on whether their C4A3-HNE levels were above or below the median (62.75 ng / ml). The majority of patients with low biomarker levels had moderate (44%) and severe (22%) disease, whereas patients with high levels primarily had mild (52%) disease (Figure 4B). Taken together, C4A3-HNE decreased in a dose-dependent manner from patients with endoscopically mild disease to patients with endoscopically moderate / severe disease. Because not enough CD patients had registered endoscopic disease scores, we were unable to group them according to disease severity. When patients were grouped based on the median C4A3-HNE level (61.24 ng / ml), we found that patients with low C4A3-HNE levels had higher median endoscopic disease severity scores than patients with high levels of the marker (Figure 4C). These results reveal a similar pattern to that observed in patients with ulcerative colitis, suggesting that high levels of the C4A3-HNE biomarker reflect early mucosal damage in IBD patients.

[0054] Inflammatory markers are lower in IBD patients with high C4A3-HNE levels. CD and UC patients were again divided into groups according to high / low C4A3-HNE levels based on the median C4A3-HNE level in each group. Mean fecal calprotectin and C-reactive protein (CRP) levels were assessed between groups. Results showed that IBD patients with low C4A3-HNE levels had lower mean fecal calprotectin and C-reactive protein (CRP) levels compared with patients with high biomarker levels (FeCal (μg / g): UC - 840.0 vs. 491.6 / CD - 721.8 vs. 356.7) (CRP (ml / L): UC - 7.37 vs. 4.99 / CD - 8.34 vs. 6.45) (Figure 5). Taken together, these data indicate that high levels of C4A3-HNE are an indication of early disease.

[0055] C4A3-HNE does not reflect the same pathological process as the MMP-generated biomarkers of the same alpha-chain. To demonstrate the uniqueness of the C4A3-HNE marker, we compared it with another type IV alpha-3 collagen neo-epitope biomarker (C4Ma3). The biomarker of interest is generated by MMPs and therefore reflects lymphocyte activity and counters neutrophil activity. The alpha-3 chain has long been considered specific to the kidney and lung.

[14] This biomarker was primarily developed to assess fibrosis in lung diseases. To compare these two biomarkers, we measured them in cohorts of patients with idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease (COPD). Both biomarkers were found to be elevated compared to healthy controls, but Spearman correlation analysis revealed only a weak correlation (Figure 6). In other words, C4A3-HNE does not reflect the same pathological process as C4Ma3; they differ by up to 70% (Figure 6).

[0056] C4A4-HNE is also elevated in IBD compared with healthy individuals. The biological relevance of C4A4-HNE was also assessed by measuring the levels of C4A4-HNE in serum samples (obtained from Proteogenix) from IBD patients and healthy subjects using a C4A4-HNE ELISA. As shown in Figure 7, C4A4-HNE is elevated in IBD patients compared with healthy controls (p<0.01). We further validated the biological relevance of C4A4-HNE in another cohort of IBD patients (Cohort 2). As shown in Figure 8, C4A4-HNE was elevated in both CD and UC compared with healthy controls (HD vs. CD vs. UC [IQR]: 16.68 ng / ml [11.93, 19.55] vs. 26.33 ng / ml [21.05, 30.30] vs. 24.66 ng / ml [19.24, 28.93], p<0.0001 and <0.01, respectively) (AUC [90% CI]: 0.91 [0.70-0.99]; 1.00 [0.87-1.00]) (Figure 8).

[0057] C4A4-HNE decreases dose-dependently from patients with endoscopically mild to severe disease When UC patients in Cohort 2 were grouped according to endoscopic disease severity score, a similar pattern was observed for C4A4-HNE as for C4A3-HNE levels. As shown in Figure 9, patients with endoscopically mild disease had the highest C4A4-HNE levels (ng / ml [IQR]: 28.2 [21.5, 28.2]), which decreased dose-dependently across severity groups (ng / ml [IQR]: 24.6 [21.9, 24.7]; 23.3 [14.6, 28.8]).

[0058] In this specification, unless otherwise specified, the word "or" is used to mean an operator that returns a true value if one or both of the stated conditions are met, as opposed 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 herein by reference. Any acknowledgment in this specification of a prior-published document should not be taken as an admission or representation that the teaching of that document was common general knowledge in Australia or elsewhere at the time of this specification.

[0059] [References] [1] C. McDowell, U. Farook, M. Haseeb, inflammatory bowel disease, 2022. http: / / www.ncbi.nlm.nih.gov / pubmed / 30137275. [2] SH Lee, J. Ieun Kwon, M. Ra Cho, Pathology 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. Fairgan, P. Lutzgerts, J.F. Colombel, W.J. Sandborn, R. Sai, G. Dee'Haens, S. Ben-Holin, J. Kuss, M. Rosario, I. Fokus, A. Perik, C. Milk, S. Hanauer Efficacy of vedolizumab induction therapy for patients with Crohn's disease who are refractory to tumor necrosis factor inhibitor therapy Gastroenterology. 147 (2014) 618-627.e3. https: / / doi.org / 10.1053 / j.gastro.2014.05.008. [4] B. Drury, G. Hardisty, R.D. Gray, G. Tsar Ho, Neutrophil extracellular traps in inflammatory bowel disease: pathogenetic mechanisms and clinical interpretation Cmgh. 12 (2021) 321-333. https: / / doi.org / 10.1016 / j.jcmgh.2021.03.002. [5] W. Fischbach, W. Becker, J. Moessner, H. Ohl-Muehler, W. Koch, W. Boerner, Leukocyte elastase in chronic inflammatory bowel disease: a marker of inflammatory activity? Digestion. 37 (1987) 88-95. https: / / doi.org / 10.1159 / 000199473.

[0060] [6] S. Kirov, A. Sasson, C. Zhang, S. Chasarou, A. Dongre, H. Steen, A. Stensbarre, V. Andersen, S. Berkland, T.B. Bennicke, Extracellular matrix degradation is part of the pathology of ulcerative colitis. Mol Omics. 15 (2019) 67-76. https: / / doi.org / 10.1039 / c8mo00239h. [7] S.K. Jorhat, P. Cubes, The newly discovered role of neutrophil extracellular traps in non-infectious diseases, 23 (2017) 279-287. https: / / doi.org / 10.1038 / nm.4294. [8] J.H. Mortensen, M. Lindholm, LL Langholm, J. Kjeldsen, A.C. By-Jensen, M.A. Kalsdal, and T. Manon-Jensen. Intestinal tissue homeostasis - 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. [9] HP Baechinger, K. Mizuno, JA Vranka, SP Boudko, Collagen formation and structure, Comprehensive Natural Products II: Chemistry and Biology. 5 (2010) 469-530. https: / / doi.org / 10.1016 / b978-008045382-8.00698-5.

[10] Y. Oka, I. Knight, K. Manabe, Y. Sado, H. Matsushima, Y. Ninomiya, M. Mizuno, T. Tsuji, Immunofluorescence staining using chain-specific epitope-defined monoclonal antibodies Distribution of type IV collagen α1-6 chains in normal human colorectum and colorectal cancer, as demonstrated by Journal of Gastroenterology and Hepatology (Australia). 17 (2002) 980-986. https: / / doi.org / 10.1046 / j.1440-1746.2002.02789.x.

[0061]

[11] H. Sato, I. Knight, R. Momota, Y. Naomoto, T. Yamatsuji, Y. Sado, Y. Ninomiya, A. Otsuka, Differential distribution of type IV collagen α chains in the epithelial basement membrane of the human gastrointestinal tract Arch Histol Cytol. 70 (2007) 313-323. https: / / doi.org / 10.1679 / aohc.70.313.

[12] N. Maillet, Rapid Peptide Generator: Fast and efficient in silico protein digestion, NAR Genom Bioinform. 2 (2020) lqz004. https: / / doi.org / 10.1093 / nargab / lqz004.

[13] ML Gefter, DH Margulies, MD Scharf, A simple method for polyethylene glycol-promoted fusion of mouse myeloma cells. Somatic Cell Genet. 3 (1977) 231-6. https: / / doi.org / 10.1007 / BF01551818.

[14] J.M. Sand, L. Larsen, C. Hogaboam, F. Martinez, M.L. Han, M.R. Larsen, A. Nawrocki, Q. Zeng, M.A. Kalsdal, D.J. Leeming, MMP-mediated degradation of type IV collagen alpha 1 and alpha 3 chains reflects basement membrane remodeling in experimental and clinical fibrosis - Validation of two novel biomarker assays PLoS One. 8 (2013) 1-12. https: / / doi.org / 10.1371 / journal.pone.0084934.

Claims

1. An immunoassay method, which is a method comprising the following i) and ii): i) contacting a sample from a patient with a monoclonal antibody that specifically binds to the N-terminal amino acid sequence EGTRPGPPGP (SEQ ID NO: 1) or that specifically binds to the N-terminal amino acid sequence TYPGRHGPPG (SEQ ID NO: 2); and ii) detecting binding between the monoclonal antibody and the peptide in the sample and determining the amount of binding.

2. The method is an immunoassay method for detecting and / or monitoring inflammatory bowel disease or a particular level of severity of the disease in a subject, further comprising: iii) Correlating the amount of binding with values ​​associated with normal healthy individuals and / or with values ​​associated with known disease severity and / or with values ​​obtained from the subject at previous time points and / or with predetermined cut-off values.

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

4. 10. The immunoassay method according to any preceding claim, wherein the patient sample is selected from blood, plasma or serum.

5. An immunoassay method described in any of the preceding claims, wherein the monoclonal antibody specifically binds to the N-terminal amino acid sequence EGTRPGPPGP (sequence number 1) and does not specifically bind to a peptide having the N-terminal amino acid sequence SEGTRPGPPGP (sequence number 3).

6. An immunoassay method described in any of the preceding claims, wherein the monoclonal antibody specifically binds to the N-terminal amino acid sequence EGTRPGPPGP (sequence number 1) and does not specifically bind to a peptide having the N-terminal amino acid sequence GTRPGPPGP (sequence number 4).

7. 10. An immunoassay method according to any preceding claim, wherein the monoclonal antibody is raised against a synthetic peptide having the N-terminal amino acid sequence EGTRPGPPGP (SEQ ID NO: 1).

8. The immunoassay method according to any one of claims 1 to 4, wherein the monoclonal antibody specifically binds to the N-terminal amino acid sequence TYPGRHGPPG (SEQ ID NO: 2) and does not specifically bind to a peptide having the N-terminal amino acid sequence VTYPGRHGPPG (SEQ ID NO: 5).

9. The immunoassay method according to any one of claims 1 to 4 or 8, wherein the monoclonal antibody specifically binds to the N-terminal amino acid sequence TYPGRHGPPG (SEQ ID NO: 2) and does not specifically bind to a peptide having the N-terminal amino acid sequence YPGRHGPPG (SEQ ID NO: 6).

10. 10. The immunoassay method of any one of claims 1 to 4, 8 or 9, wherein the monoclonal antibody is raised against a synthetic peptide having the N-terminal amino acid sequence TYPGRHGPPG (SEQ ID NO: 2).

11. 10. The immunoassay method according to any preceding claim, wherein the immunoassay is a competitive assay or a sandwich assay.

12. 10. The immunoassay method according to any preceding claim, wherein the immunoassay is a radioimmunoassay or an enzyme-linked immunosorbent assay.

13. A monoclonal antibody which specifically recognizes the N-terminal amino acid sequence EGTRPGPPGP (SEQ ID NO: 1) or specifically recognizes and binds to the N-terminal amino acid sequence TYPGRHGPPG (SEQ ID NO: 2).

14. The monoclonal antibody described in claim 13, which specifically binds to the N-terminal amino acid sequence EGTRPGPPGP (sequence number 1) and does not specifically bind to a peptide having the N-terminal amino acid sequence SEGTRPGPPGP (sequence number 3).

15. The monoclonal antibody described in claim 13 or 14, which specifically binds to the N-terminal amino acid sequence EGTRPGPPGP (sequence number 1) and does not specifically bind to a peptide having the N-terminal amino acid sequence GTRPGPPGP (sequence number 4).

16. A monoclonal antibody according to any one of claims 13 to 15, wherein the monoclonal antibody is raised against a synthetic peptide having the N-terminal amino acid sequence EGTRPGPPGP (SEQ ID NO: 1).

17. The monoclonal antibody described in claim 13, which specifically binds to the N-terminal amino acid sequence TYPGRHGPPG (sequence number 2) and does not specifically bind to a peptide having the N-terminal amino acid sequence VTYPGRHGPPG (sequence number 5).

18. The monoclonal antibody described in claim 13 or 17, which specifically binds to the N-terminal amino acid sequence TYPGRHGPPG (sequence number 2) and does not specifically bind to a peptide having the N-terminal amino acid sequence YPGRHGPPG (sequence number 6).

19. 19. The monoclonal antibody of claim 13, claim 17 or claim 18, wherein the monoclonal antibody is raised against a synthetic peptide having the N-terminal amino acid sequence TYPGRHGPPG (SEQ ID NO: 2).

20. 20. An immunoassay kit comprising the monoclonal antibody of any one of claims 13 to 19 and at least one of the following: - Streptavidin-coated well plates - the biotinylated peptide EGTRPGPPGP-L-biotin (SEQ ID NO: 7), where L is an optional linking group, or the biotinylated peptide TYPGRHGPPG-L-biotin (SEQ ID NO: 8), where L is an optional linking group. - Secondary antibodies used in sandwich immunoassays a calibration protein containing the N-terminal amino acid sequence EGTRPGPPGP (SEQ ID NO: 1) or a calibration protein containing the N-terminal amino acid sequence TYPGRHGPPG (SEQ ID NO: 2); - Antibody Biotinylation Kit - Antibody HRP labeling kit - Antibody radiolabeling kit.

Images