Crosslinked type V collagen assay

Abstract

The present invention relates to a sandwich immunoassay for determining cross-linked collagen type V in a biological sample, and its use in identifying patients suffering from conditions associated with fibrosis, such as ankylosing spondylitis, inflammatory bowel disease, psoriasis and atopic dermatitis. The invention also relates to a kit for performing a sandwich immunoassay.

Description

Technical Field

[0001] The present invention relates to a sandwich immunoassay for detecting cross-linked type V collagen in a biological sample and its use in identifying patients with fibrosis-related disorders such as ankylosing spondylitis, inflammatory bowel disease, psoriasis and atopic dermatitis. The present invention also relates to a kit for performing the sandwich immunoassay. Background Art

[0002] Type V collagen, fibrous collagen

[0003] Fibrillar collagen is located in the interstitial matrix and is home to mesenchymal cells and fibroblasts, helping to maintain structural tissue integrity, tensile strength, and mechanical and signal transduction properties [1, 2]. Fibrillar collagen includes types I, II, III, V, XI, XXIV, and XXVII, of which types I, II, and III are considered the main fibrillar collagens and are the most abundant [2]. Type V collagen has three different α chains (α1, α2, and α3), of which the heterotypic type V collagen trimers (α12, α2) are the most abundant, and the homotrimers (α13) are less expressed [3]. Although collagen fibrils are mainly composed of major collagens, type V collagen forms the core to which type I and type III collagens are attached through initial formation, and the size of collagen fibrils is controlled by type V collagen non-cartilage tissue [4, 5]. After collagen maturation, type V collagen initiates the nucleation of collagen fibrils, to which type I and type III collagens attach, mediated by enzymatically formed cross-links.

[0004] The importance of type V collagen in collagen fibrillogenesis is evident in knockout mouse models, in which the absence of type V collagen results in deficient collagen fibrillogenesis despite normal secretion of type I collagen. Lack of type V collagen causes mice to die during the onset of organogenesis, indicating that type V collagen is required for normal fibrillogenesis [6]. The importance of type V collagen is also evident in genetic indications such as Ehlers-Danlos syndrome [7]. Patients exhibit fragile and soft tissues as well as impaired wound healing. In addition, several fibrotic diseases demonstrate significant upregulation of type V collagen [3].

[0005] Enzymatic cross-linking

[0006] The formation of intramolecular and intermolecular enzymatic cross-links of fibrillar collagen is catalyzed by the enzymatic action of lysyl hydroxylases (LH), prolyl 3-hydroxylase 3 (P3H3), lysyl oxidase (LOX), and lysyl oxidase-like (LOXL) enzymes [8]. Enzymatic cross-links are key post-translational modifications that connect fibrillar collagen and strengthen the extracellular matrix in organs [9], blood vessels

[10] , and the granulation matrix during wound healing [11, 12]. Under the catalysis of LH or P3H3, they are initiated by the hydroxylation of specific lysines within the collagen helical region and non-collagenous terminal peptides, followed by oxidation of the hydroxylated or non-hydroxylated lysines by LOXL [8]. In the extracellular environment, Cu 2+ LOXL catalyzes the oxidation of lysines in the telopeptides of fibrillar collagens, including type V collagen

[13] . However, conserved cross-linking lysines do not appear to be present in the C-terminal telopeptide of type V collagen and are likely present only in the N-terminal telopeptide and in the N- and C-terminal helical regions

[14] . Subsequently, hydroxylated and non-hydroxylated lysines in the helical and telopeptidyl regions react spontaneously to form mature trivalent cross-links

[15] . The functional and mechanical properties of collagen cross-links depend on the extent and biochemical nature of the cross-links

[16] . This is evident when comparing the extent of hydroxylated cross-link material between tissues, with higher levels of hydroxylation occurring in tough connective tissues compared to soft connective tissues

[16] . Differences in tissue expression of LH and LOXL, which control the extent of lysine hydroxylation and glycosylation, allow for tissue-specific collagen cross-linking rather than collagen-specific cross-linking.

[0007] Enzymatic cross-linking of fibrillar collagen is a key process under physiological conditions and is also a critical process during fibrosis. Fibrosis is caused by dysregulated wound healing resulting from the failure to resolve chronic inflammation induced by tissue injury and is responsible for 45% of deaths in developed countries

[17] . Continued wound healing leads to the release of multiple profibrotic and proinflammatory mediators, inducing fibroblast activation and differentiation into myofibroblasts, the primary mediators of tissue fibrosis

[18] . Activated myofibroblasts deposit excess fibrillar collagen into the surrounding tissue and enzymatically catalyze collagen cross-linking

[18] . Deposition of collagen cross-linking enzymes precedes collagen deposition, resulting in an increasingly rigid and non-compliant matrix that can ultimately inhibit tissue and even organ function

[19] . Animal models of liver [20, 21], heart

[22] , lung

[23] , and peritoneal fibrosis

[24] have demonstrated the potential effects of increased LOXL expression on matrix stiffness and the ensuing proinflammatory process. Extensive cross-linking can protect collagen from proteolysis by blocking the binding sites of proteases to collagen α-chains [25, 26] and promote fibrosis by stimulating myofibroblasts and recruiting cells to fibrotic sites [19, 27, 28]. Given the ability of highly cross-linked collagen to drive fibrosis independently of inflammation and the lack of approved drugs that can reverse fibrosis, there is increasing interest in the development and evaluation of pro-resolving therapies.

[0008] Resolution and assessment of type V collagen remodeling

[0009] Long considered irreversible, studies have demonstrated how timely and appropriate interventions, such as direct-acting antivirals used to treat hepatitis B or C, can lead to tissue regeneration and regression of inflammatory tissue. However, in more complex fibrotic diseases, a more direct approach targeting the proinflammatory extracellular matrix (ECM) may be required, as further supported by the lack of efficacy of anti-inflammatory treatments for fibrosis. Because established inflammatory ECM can reactivate fibrosis independently of inflammation, simply halting further collagen deposition may not be sufficient to treat patients with fibrosis. Therefore, additional options may include inducing regression of fibrosis through proteolytic clearance of cross-linked collagen. Achieving regression of inflammatory tissue requires the following: (1) cessation of tissue damage, (2) inactivation and phenotypic modulation of proinflammatory cells, such as phenotypic modulation of proinflammatory macrophages, (3) elimination or inactivation of myofibroblasts, and (4) degradation and clearance of extracellular matrix components. Pro-inflammatory resolution therapies aim to promote degradation and clearance of the established inflammatory ECM, disrupting the vicious self-activating feedback loop driven by a heavily cross-linked and non-compliant matrix. Two key processes in fibrosis resolution are inhibition of further enzymatic cross-linking and enhanced proteolytic degradation of collagen. LOXL inhibition using the nonspecific inhibitor β-aminopropionitrile or small molecule inhibitors has demonstrated the ability to reduce matrix stiffness, reduce collagen cross-linking, and reduce collagen deposition, suggesting that LOXL is an important therapeutic target.

[0010] In addition, enhancing collagen degrading proteases such as matrix metalloproteinases (MMPs) can help alleviate fibrosis. Reduction in collagen cross-linking can make the matrix more accessible to MMPs, leading to degradation and clearance of fibrillar collagens (including type V collagen), further promoting fibrosis regression. Multiple MMPs have been shown to be associated with fibrosis regression, among which specific MMPs vary between tissues and pathologies, indicating that the time and place of action of each MMP may depend on the cells that release the protease. Depending on the time of expression, MMPs can promote fibrosis. However, MMPs can also promote fibrosis regression, among which MMP-2, MMP-8, MMP-9, and MMP-13 have been shown to induce regression of liver fibrosis, MMP-1, MMP-3, and MMP-10 catalyze ECM degradation during wound healing in inflammatory bowel disease, MMP-7 can promote regression of pulmonary fibrosis, and MMP-2 can promote regression of renal fibrosis.

[0011] Important aspects in the study of anti-inflammatory and pro-inflammatory resolution therapies are sensitive and specific tools for assessing pharmacodynamics and achievement of clinical endpoints. The current gold standard typically involves endoscopy and tissue biopsy, which lacks direct measurement of collagen deposition and, more importantly, its resolution. The cited pro-inflammatory resolution studies utilize measures of collagen deposition and protease expression as markers of inflammatory resolution. While the histological reduction in type I and type III collagen deposition occurring concurrently with increased MMP expression supports the hypothesis of resolution and clearance of inflammatory ECM, objective and direct measurements are necessary. Collagen is the major protein component of tissues, and collagen remodeling and post-translational modifications (PTMs) are key aspects of many fibroinflammatory pathologies, where proteolytic activity releases fragments into the circulation. These collagen fragments can serve as objective, non-invasive biomarkers that reflect ongoing pathological tissue remodeling by quantifying fragments in blood samples

[318] .

[0012] Although few biomarkers related to type V collagen remodeling exist, at least two novel serological biomarkers exist. The PRO-C5 and C5M biomarkers developed by Nordic Bioscience A / S reflect type V collagen formation and MMP-catalyzed degradation, respectively. These biomarkers have demonstrated their use in patients with liver disease of various etiologies, patients with ankylosing spondylitis, and patients with IBD. The PRO-C5 biomarker assesses type V collagen formation by objectively assessing the C-terminal propeptide of type V collagen, a fragment proteolytically released during collagen maturation. Subsequently, during tissue inflammation, increased activity of MMPs releases the C5M biomarker from mature type V collagen. However, none of the current biomarkers for type V collagen assess proteolysis of cross-linked type V collagen. The use of such a biomarker would provide additional information, as this fragment would reflect proteolytic degradation as well as enzymatic cross-linking.

[0013] "Fibrotic disease" refers to any disease that causes fibrosis, whether fibrosis is a primary or secondary symptom. Fibrosis is the end result of a chronic inflammatory response triggered by a variety of stimuli, including persistent infection, autoimmune reactions, allergic reactions, chemical injury, radiation, and tissue damage. Fibrotic diseases include those listed in Table A, such as inflammatory bowel disease, psoriasis, cirrhosis, and ankylosing spondylitis. Crohn's disease (CD) and ulcerative colitis (UC) are two major gastrointestinal diseases of inflammatory bowel disease (IBD) with similar symptoms, such as increased inflammation and structural damage to the intestine. CD can affect the entire gastrointestinal tract, while UC is primarily limited to the colonic mucosa [1]. The cause of IBD is not fully understood, but it is believed to have a genetic basis and an abnormal response of the immune system to environmental factors [2].

[0014] Applicants have designed a specific sandwich immunoassay that utilizes a neo-epitope in the C-helical region generated by protease cleavage of intact type V collagen to detect cross-linked type V collagen. The level of cross-linked, degraded type V collagen can be used to assess patients with fibrotic diseases such as inflammatory bowel disease (IBD), ankylosing spondylitis, psoriasis, and atopic dermatitis. Summary of the invention

[0015] The present invention relates to a monoclonal antibody that specifically recognizes and binds to a peptide that is a new epitope of the C helical region generated by protease cleavage of intact type V collagen. Preferably, the new epitope is contained in the amino acid sequence PKGEKGHPGL-COOH (SEQ ID NO: 1).

[0016] The present invention also provides a sandwich immunoassay for detecting cross-linked type V collagen (CTX-V) in a biological sample, wherein the cross-linked type V collagen comprises at least two chains of the C helical region of type V collagen linked together by interchain crosslinking. The method comprises contacting a biological sample comprising cross-linked type V collagen with a first monoclonal antibody bound to a surface, wherein each chain of type V collagen contained in the cross-linked type V collagen has a new epitope of the C helical region generated by protease cleavage of intact type V collagen, and adding a second monoclonal antibody. Both monoclonal antibodies specifically react with the new epitope of the C helical region generated by protease cleavage of intact type V collagen. Preferably, the new epitope is contained in the amino acid sequence PKGEKGHPGL-COOH (SEQ ID NO: 1). The method also comprises determining the binding amount of the second monoclonal antibody.

[0017] The present invention also relates to a kit for sandwich immunoassay as described herein, which comprises a solid support bound to the first monoclonal antibody as described above, and a labeled second monoclonal antibody as described herein.

[0018] The present invention also relates to a method for diagnosing a fibrotic disease, comprising detecting cross-linked type V collagen (CTX-V) in a biological sample from a patient using the immunoassay of the present invention. The method comprises contacting a biological sample containing cross-linked type V collagen with a first monoclonal antibody bound to a surface, wherein each chain of type V collagen contained in the cross-linked type V collagen has a new epitope in the C-helical region generated by protease cleavage of intact type V collagen, and adding a second monoclonal antibody. Both monoclonal antibodies specifically react with the new epitope in the C-helical region generated by protease cleavage of intact type V collagen. Preferably, the new epitope is contained in the amino acid sequence PKGEKGHPGL-COOH (SEQ ID NO: 1). The method also includes determining the binding amount of the second monoclonal antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 : Evaluation of monoclonal antibody specificity. The specificity of the CTX-V monoclonal antibody was tested by its reactivity to the selected peptide, extended peptide, truncated peptide and buffer in an indirect competitive ELISA.

[0020] Figure 2 : Correlation of CTX-V biomarker with IBD patients. Plasma CTX-V levels in healthy subjects (HS), ulcerative colitis (UC), and Crohn's disease (CD) patients were plotted and presented as Tukey plots. Statistical differences between plasma levels in HS and those in UC and CD patients were performed by Kruskal-Wallis test with Dunn correction for multiple comparisons. Significance is indicated as ****p<.0001.

[0021] Figure 3 : Proteolysis of cross-linked type V collagen in an in vitro setting. A) CTX-V levels measured in the supernatants of cells stimulated with or without TGF-β1 at days 4, 8, and 12 are plotted. The LLOQ line on the y-axis indicates the lower limit of quantification of the assay. B) Fold changes of CTX-V cleaved by MMP-9 and MMP-13 compared to uncleaved controls are plotted. The results were analyzed by two-way ANOVA (A) or one-way ANOVA (B), using Multiple comparison tests were performed to calculate statistical differences. Significance is indicated as **p<.01.

[0022] Figure 4: CTX-V is upregulated in patients with fibroinflammatory conditions. Serum CTX-V levels in healthy subjects (HS), ankylosing spondylitis (AS), psoriasis (PSO), and atopic dermatitis (AD) were plotted and presented as Tukey plots. Statistical differences between serum levels in HS and those in AS, PSO, and AD patients were performed by Kruskal-Wallis test using Dunn correction for multiple comparisons. Significance is indicated as *p<.05, **p<.001. DETAILED DESCRIPTION

[0023] As used herein, the term "neo-epitope" refers to an epitope formed at a protease cleavage site of type V collagen, specifically in the C-helical region. Preferably, the epitope is formed by cleavage with an MMP (eg, MMP-9).

[0024] As used herein, the term "competitive ELISA" refers to a competitive enzyme-linked immunosorbent assay. In a "competitive ELISA", the target peptide present in the sample (if any) competes with a known amount of the peptide target (e.g., bound to a fixed substrate or labeled) for binding to an antibody, and is a technique known to those skilled in the art.

[0025] As used herein, the term "sandwich immunoassay" refers to the use of at least two antibodies to detect an antigen in a sample, and is a technique known to those skilled in the art.

[0026] As used herein, the term "PRO-C5" refers to the C-terminal propeptide of type V collagen released during protein maturation, which produces the N-terminal epitope TAALGDIMGH (SEQ ID NO: 17).

[0027] As used herein, the term "C5M" refers to a fragment of type V collagen formed after cleavage by MMP-2 or MMP-9 in the α3 chain comprising epitopes 1317 "HMGREGREGE" 1329 (SEQ ID NO: 18).

[0028] As used herein, the term "CTX-V assay" refers to the sandwich immunoassay described herein for detecting and quantifying cross-linked type V collagen (also referred to herein as the "target peptide"). Preferably, the "target peptide" is an epitope contained in the C-terminal amino acid sequence PKGEKGHPGL-COOH (SEQ ID NO: 1), which is formed after cleavage of type V collagen by MMP (e.g., MMP-9) within the C-helical region of type V collagen (bases 559-1570 of COL5A1 human sequence P20908, https: / / www.uniprot.org / uniprotkb / P20908 / entry).

[0029] Disclosed herein is a monoclonal antibody suitable for use in the method of the present invention, which specifically reacts with a neo-epitope in the C-helical region of type V collagen, the neo-epitope being contained in the C-terminal amino acid sequence PKGEKGHPGL-COOH (SEQ ID NO: 1). Therefore, in a first aspect, the present invention provides a monoclonal antibody that specifically reacts with a neo-epitope in the C-helical region of type V collagen, the neo-epitope being contained in the C-terminal amino acid sequence PKGEKGHPGL-COOH (SEQ ID NO: 1).

[0030] Preferably, the monoclonal antibody does not substantially recognize or bind to the extended form of the C-terminal amino acid sequence, i.e., PKGEKGHPGLIZ-COOH (SEQ ID NO: 2), wherein Z is absent or is one or more amino acids in the C-helical region sequence of type V collagen following the new epitope in the intact type V collagen.

[0031] Preferably, the ratio of the affinity of the monoclonal antibody for the amino acid sequence PKGEKGHPGL-COOH (SEQ ID NO: 1) to the affinity of the monoclonal antibody for the extended amino acid sequence PKGEKGHPGLIZ-COOH (SEQ ID NO: 2) is at least 10 to 1, preferably at least 100 to 1, more preferably at least 1,000 to 1, more preferably at least 10,000 to 1, more preferably at least 100,000 to 1, and most preferably at least 1,000,000 to 1.

[0032] Preferably, the monoclonal antibody does not recognize or bind to a shortened form of the C-terminal amino acid sequence having the amino acid sequence PKGEKGHPG-COOH (SEQ ID NO: 3).

[0033] Preferably, the ratio of the affinity of the monoclonal antibody for the amino acid sequence PKGEKGHPGL-COOH (SEQ ID NO: 1) to the affinity of the monoclonal antibody for the shortened amino acid sequence PKGEKGHPG-COOH (SEQ ID NO: 3) is at least 10 to 1, preferably at least 100 to 1, more preferably at least 1,000 to 1, more preferably at least 10,000 to 1, more preferably at least 100,000 to 1, and most preferably at least 1,000,000 to 1.

[0034] As used herein, the term "monoclonal antibody" refers to both intact antibodies and fragments thereof that retain the binding specificity of intact antibodies, 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, intact antibodies generally have a "Y-shaped" structure of two pairs of identical polypeptide chains, each pair consisting of a "light" chain and a "heavy" chain. The N-terminal region of each light chain and heavy chain contains a variable region, while the C-terminal portion of each heavy chain and light chain constitutes a constant region. The variable region includes three complementary determining regions (CDRs), which are primarily responsible for antigen recognition. The constant region allows antibodies to recruit cells and molecules of the immune system. Antibody fragments that retain binding specificity contain at least CDRs and a sufficient portion of the remaining variable region to retain the binding specificity. Antibodies that retain the same binding specificity may contain the same complementary determining regions (CDRs). The CDRs of antibodies can be determined using methods known in the art (such as the methods described by Kabat et al.).

[0035] As described in the examples, antibodies can be produced by B cell cloning. The isotype of the antibody can be determined by ELISA with specificity for human IgM, IgG or IgA isotypes or human IgG1, IgG2, IgG3 or IgG4 subclasses. Isotypes can be identified by other suitable methods. In the present invention, monoclonal antibodies can include any constant region known in the art. Human constant light chains are divided into kappa and lambda light chains. Heavy chain constant chains are divided into μ, δ, γ, α or ε, and the isotype of the antibody is defined as IgM, IgD, IgG, IgA and IgE, respectively. IgG isotypes have several subclasses, including but not limited to IgG1, IgG2, IgG3 and IgG4. Monoclonal antibodies can preferably be IgG isotypes, including any one of IgG1, IgG2, IgG3 or IgG4.

[0036] The amino acid sequence of the antibody produced can be determined using standard techniques. For example, RNA can be isolated from cells, and the RNA is used to generate cDNA by reverse transcription. Then, cDNA is subjected to PCR using primers for amplifying antibody heavy and light chains. For example, primers specific to the leader sequences of all VH (variable heavy chain) sequences can be used together with primers that are bound to sequences in the constant region of the previously determined isotype. Primers that are bound to the 3' ends of κ or λ chains and primers that anneal to Vκ or Vλ leader sequences can be used to amplify light chains. Full-length heavy and light chains can be produced and sequenced.

[0037] Monoclonal antibodies that specifically bind to the C-terminal amino acid sequence PKGEKGHPGL-COOH (SEQ ID No.1) can be produced by any suitable technique known in the art. For example, monoclonal antibodies can be produced against a synthetic peptide having the amino acid sequence PKGEKGHPGL (SEQ ID No.1), for example by: immunizing a rodent (or other suitable mammal) with a synthetic peptide consisting of the sequence PKGEKGHPGL (SEQ ID No.1), which may optionally be linked to an immunogenic carrier protein (e.g., Aplysia hemocyanin); isolating and cloning individual antibody-producing cells; and assaying the resulting monoclonal antibodies to ensure that they have the desired specificity. An exemplary protocol for producing monoclonal antibodies that specifically bind to the N-terminal amino acid sequence PKGEKGHPGL (SEQ ID No.1) is described below.

[0038] Preferably, the monoclonal antibody or fragment thereof may preferably comprise one or more complementarity determining regions (CDRs) selected from the group consisting of:

[0039] CDR-L1:KSSQSLLYSDGKTYLN(SEQ ID No.4)

[0040] CDR-L2: LVSKLDS (SEQ ID No. 5)

[0041] CDR-L3:WQGTHLPYA(SEQ ID No.6)

[0042] CDR-H1: DYYMH (SEQ ID No. 7)

[0043] CDR-H2:WIDPENGDREYAPKFQG(SEQ ID No.8)

[0044] CDR-H3: RGHYEDH (SEQ ID No. 9)

[0045] Preferably, the antibody or fragment thereof comprises at least 2, 3, 4, 5 or 6 of the CDR sequences listed above.

[0046] Preferably, the monoclonal antibody or fragment thereof has a light chain variable region comprising a CDR sequence.

[0047] CDR-L1:KSSQSLLYSDGKTYLN(SEQ ID No.4)

[0048] CDR-L2: LVSKLDS (SEQ ID No. 5)

[0049] CDR-L3:WQGTHLPYA(SEQ ID No.6)

[0050] Preferably, the monoclonal antibody or fragment thereof has a light chain comprising a framework sequence between the CDRs, wherein the framework sequence is substantially identical or substantially similar to the framework sequence between the CDRs in the light chain sequence below (wherein the CDRs are shown in bold and underlined, and the framework sequence is shown in italics)

[0051]

[0052]

[0053] Preferably, the monoclonal antibody or fragment thereof has a heavy chain variable region comprising a CDR sequence.

[0054] CDR-H1: DYYMH (SEQ ID No. 7)

[0055] CDR-H2:WIDPENGDREYAPKFQG(SEQ ID No.8)

[0056] CDR-H3: RGHYEDH (SEQ ID No. 9)

[0057] Preferably, the monoclonal antibody or fragment thereof has a heavy chain comprising a framework sequence between the CDRs, wherein the framework sequence is substantially identical or substantially similar to the framework sequence between the CDRs in the heavy chain sequence below (wherein the CDRs are shown in bold and underlined, and the framework sequence is shown in italics)

[0058]

[0059] As used herein, if the framework amino acid sequence between the CDRs of an antibody has at least 70%, 80%, 90% or at least 95% similarity or identity with the framework amino acid sequence between the CDRs of another antibody, they are substantially identical or substantially similar. Similarity or identity can be measured over the entire length of each intervening framework sequence. Similar or identical amino acids can be continuous or non-continuous.

[0060] The framework sequence may contain one or more amino acid substitutions, insertions and / or deletions. Amino acid substitutions may be conservative, meaning that the substituted amino acid has similar chemical properties to the original amino acid. A skilled person will understand which amino acids have similar chemical properties. For example, the following groups of amino acids have similar chemical properties, such as size, charge, and polarity: Group 1: Ala, Ser, Thr, Pro, Gly; Group 2: Asp, Asn, Glu, Gln; Group 3: His, Arg, Lys; Group 4: Met, Leu, Ile, Val, Cys; Group 5: Phe, Thy, Trp.

[0061] Amino acid sequences can be compared using programs such as the CLUSTAL program. This program compares amino acid sequences and finds the best alignment by appropriately inserting spaces in either sequence. Amino acid identity or similarity (identity plus the conservation of amino acid types) can be calculated to obtain the best alignment. Programs like BLASTx will align the longest parts of similar sequences and assign values ​​to the fit values. Therefore, a comparison can be obtained to find several similar regions, each with different scores. Two types of analysis are considered in the present invention. Preferably, identity or similarity is calculated over the entire length of the framework sequence.

[0062] In certain preferred embodiments, the monoclonal antibody or fragment thereof may comprise a light chain variable region sequence:

[0063]

[0064]

[0065] (CDRs are bold and underlined; framework sequences are italicized)

[0066] And / or heavy chain variable region sequence:

[0067]

[0068] (CDRs are bold and underlined; framework sequences are italicized)

[0069] In a second aspect, the invention relates to a sandwich immunoassay for detecting cross-linked type V collagen in a biological sample, the cross-linked type V collagen comprising at least two chains of a C-helical region of type V collagen linked together by interchain cross-links, the method comprising:

[0070] contacting a biological sample containing cross-linked type V collagen with a first monoclonal antibody bound to a surface, wherein each chain of type V collagen contained in the cross-linked type V collagen has a neo-epitope of a C-helical region generated by cleavage of intact type V collagen by a protease, and adding a second monoclonal antibody; and

[0071] determining the binding amount of the second monoclonal antibody;

[0072] Wherein, both the first monoclonal antibody and the second monoclonal antibody specifically react with a new epitope in the C-helical region of type V collagen, and the new epitope is contained in the C-terminal amino acid sequence PKGEKGHPGL-COOH (SEQ ID NO: 1).

[0073] Preferably, the sandwich immunoassay of the second aspect uses the monoclonal antibody of the first aspect of the invention. Therefore, the preferred features of the monoclonal antibody of the first aspect are also preferred features of the second aspect.

[0074] The sandwich immunoassay described herein uses antibodies that bind to the same epitope as both the capture antibody and the detection antibody (i.e., the first antibody and the second antibody), so double-stranded peptides (i.e., cross-linked) can be recognized by the assay. Since the two monoclonal antibodies bind to the same epitope, if the second monoclonal antibody binds, there must be at least two linked peptide chains.

[0075] Preferably, the sandwich immunoassay is used to quantify the amount of cross-linked type V collagen in a biological fluid, wherein the biological fluid may be, but is not limited to, serum, plasma, urine, amniotic fluid, tissue supernatant, or cell supernatant.

[0076] The human biological fluid sample can be a sample from a human patient having medical signs or symptoms indicative of a fibrotic disease. Examples of fibrotic diseases are shown in Table A below:

[0077]

[0078] Preferably, the fibrotic disease is selected from ankylosing spondylitis, psoriasis, atopic dermatitis or inflammatory bowel disease. Preferably, the biological fluid sample is a sample from a human patient with medical signs or symptoms indicative of Crohn's disease (CD) or ulcerative colitis (UC). Preferably, the biological fluid sample is a sample from a human patient with medical signs or symptoms indicative of active inflammatory bowel disease, such as active Crohn's disease (CD) or active ulcerative colitis (UC).

[0079] The sandwich immunoassay may be, but is not limited to, a radioimmunoassay, a fluorescent immunoassay, or an enzyme-linked immunosorbent assay.

[0080] As used herein, the term "ELISA" (enzyme-linked immunosorbent assay) refers to an immunoassay in which an antibody linked to an enzyme (such as horseradish peroxidase or alkaline phosphatase) is used to detect a target peptide (if any) present in a sample. The activity of the enzyme is then assessed by incubation with a substrate that produces a measurable product. Thus, the presence and / or amount of the target peptide in the sample can be detected and / or quantified. ELISA is a technique known to those skilled in the art.

[0081] The sandwich immunoassay can detect and determine the amount of binding between the monoclonal antibody and the peptide in the sample. The amount of binding can be a value associated with a normal healthy subject and / or a value associated with a known disease severity and / or a value obtained from the patient at a previous time point and / or related to a predetermined cutoff value.

[0082] As used herein, the term "amount of binding" refers to the quantification of the binding between the monoclonal antibody and the target peptide, which is determined by comparing the measured value of the target peptide in the biological fluid sample with a calibration curve, wherein the calibration curve is generated using standard samples of known concentrations of the target peptide.

[0083] As used herein, the term "specific binding" refers to that the antibody binding is selective for the antigen, and the binding can be distinguished from unwanted or non-specific interactions. The ability of a monoclonal antibody to bind to a specific epitope or peptide sequence can be determined by enzyme-linked immunosorbent assay (ELISA) described herein or other techniques familiar to those skilled in the art, such as surface plasmon resonance (SPR) technology (e.g., analysis on a BIAcore instrument) and traditional binding assays. The degree of binding of a monoclonal antibody to an unrelated protein is less than about 10% of the binding of the monoclonal antibody to an epitope or peptide, as measured by ELISA. "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an epitope binding region of an antibody) and its binding partner (e.g., an epitope or antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to intrinsic binding affinity, reflecting a 1:1 interaction between members of a binding pair (e.g., an antigen binding moiety and an antigen). The affinity of a molecule for its partner can generally be represented by a dissociation constant (Kd), which is the ratio of the dissociation rate constant and the association rate constant (koir and kon, respectively). Therefore, equivalent affinities can include different rate constants as long as the ratio of the rate constants remains the same. The dissociation constant represents the antigen concentration at which half of the binding sites on the antibody are occupied. A lower Kd indicates a higher binding affinity between the antibody and the antigen, while a higher Kd reflects weaker binding. Several methods can be used to measure the Kd of an antibody, including surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), and fluorescence-based assays. In certain aspects, a monoclonal antibody that binds to an epitope or peptide has the following dissociation constant (KD): <1 pM, <100 nM, <10 nM, <1 nM, <0.1 nM, <0.01 nM, or <0.001 nM (e.g., 10 8 M or smaller, e.g. 10 8 M-10 13 M, for example 10 9 M-10 13 M).

[0084] As used herein, the term "predetermined cutoff value" means a statistically determined binding amount indicating a high likelihood of a disease or a specific severity of a disease (or its prognosis) in a patient, i.e., a measured value of the target peptide in a patient sample that is equal to or above the statistical cutoff value, corresponding to at least a 70% probability of the presence of the disease or a specific severity of the disease, 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.

[0085] As used herein, the term "value associated with normal healthy subjects" refers to the standardized binding amount of samples from subjects who are considered healthy (i.e., disease-free) determined by the above method; the term "value associated with a known disease severity" refers to the standardized binding amount of samples from patients known to have a disease of a known severity determined by the above method.

[0086] In a specific assay disclosed herein for measuring a target peptide having a C-terminal amino acid sequence of PKGEKGHPGL-COOH (SEQ. ID No. 1) in a biological fluid, a calibration curve is generated using a standard sample of a calibration peptide having a C-terminal amino acid sequence of PKGEKGHPGL (SEQ. ID No. 1) of known concentration, and the calibration peptide can specifically consist of the amino acid sequence PKGEKGHPGL (SEQ. ID No. 1). The value measured in the biological fluid sample is compared with the calibration curve to determine the actual amount of the target peptide in the sample.

[0087] In a preferred embodiment, the second monoclonal antibody can be labeled to determine the amount of binding of the second monoclonal antibody.

[0088] Preferably, the second monoclonal antibody can be an enzyme-linked antibody. The enzyme can be, but is not limited to, horseradish peroxidase (HRP).

[0089] Preferably, the second monoclonal antibody may be radiolabeled or linked to a fluorophore.

[0090] While these are the preferred labels to be used with the present invention, it is envisaged that any suitable labelling system may be used, such as but not limited to DNA reporters or electrochemiluminescent tags.

[0091] Alternatively, another labeled antibody that recognizes the second monoclonal antibody may be used to determine the amount of binding of the second monoclonal antibody.The further labeled antibody may be labeled with a label as described above.

[0092] In a preferred embodiment of the present invention, the sandwich immunoassay may further include correlating the amount of cross-linked type V collagen determined by the method with a standard fibrotic disease sample of known disease severity to assess the severity of the fibrotic disease. Such fibrotic disease may be, but is not limited to, inflammatory bowel disease (IBD), ankylosing spondylitis, psoriasis or atopic dermatitis.

[0093] The method can be an immunoassay for diagnosing and / or monitoring and / or assessing the possibility of a fibrotic disease in a patient, the method comprising performing the immunoassay of the second aspect of the present invention on a biological fluid sample obtained from the patient using a monoclonal antibody, detecting and determining the amount of binding between the monoclonal antibody and the peptide in the sample, and correlating the amount of binding with a value associated with a normal healthy subject and / or a value associated with a known disease severity and / or a value obtained from the patient at a previous time point. Preferably, the fibrotic disease is inflammatory bowel disease, ankylosing spondylitis, atopic dermatitis or psoriasis. Preferably, the inflammatory bowel disease is Crohn's disease or ulcerative colitis. Preferably, the inflammatory bowel disease is active inflammatory bowel disease, such as active Crohn's disease (CD) or active ulcerative colitis (UC).

[0094] In another aspect, the present invention provides a method of treating a fibrotic disease in a patient in need thereof, the method comprising:

[0095] (a) performing an immunoassay according to the second aspect of the present invention on a patient's blood, serum or plasma sample to detect whether the patient suffers from a fibrotic disease and / or to assess the severity of the patient's fibrotic disease; and

[0096] (b) if the patient is determined to suffer from the fibrotic disease or a certain severity thereof in step (a), administering to the patient a drug for treating the fibrotic disease.

[0097] Preferably, the fibrotic disease is an inflammatory bowel disease, such as Crohn's disease or ulcerative colitis, ankylosing spondylitis, atopic dermatitis or psoriasis. Preferably, the inflammatory bowel disease is an active inflammatory bowel disease, such as active Crohn's disease or active ulcerative colitis.

[0098] Suitable treatments for inflammatory bowel disease include anti-inflammatory agents such as aminosalicylates; immunosuppressants, corticosteroids, and antibodies such as infliximab or adalimumab. Suitable treatments for ankylosing spondylitis include physical therapy, analgesics (including acetaminophen), codeine, and nonsteroidal anti-inflammatory drugs (such as ibuprofen, diclofenac, etoricoxib, and naproxen); monoclonal antibody therapy such as secukinumab and ixekizumab; corticosteroids, disease-modifying antirheumatic drugs (DMARDs), and surgery. Suitable treatments for psoriasis include topical corticosteroids, emollients, vitamin D analogs, calcineurin inhibitors, coal tar, and anthracenol. Suitable treatments for atopic dermatitis include topical and / or oral corticosteroids, emollients, topical calcineurin inhibitors such as pimecrolimus or tacrolimus, wet dressings, phototherapy, and antihistamines.

[0099] In another aspect, the present invention relates to a kit for use in a sandwich immunoassay as described herein, the kit comprising a solid support bound to a first monoclonal antibody as described above; and a labeled second monoclonal antibody as described above.

[0100] The kit can be used to diagnose or predict the risk of a fibrotic disease, preferably in combination with the method according to the second aspect of the invention. Preferably, the fibrotic disease is an inflammatory bowel disease, such as Crohn's disease or ulcerative colitis, ankylosing spondylitis, atopic dermatitis or psoriasis. Preferably, the inflammatory bowel disease is an active inflammatory bowel disease, such as active Crohn's disease or active ulcerative colitis.

[0101] In another aspect, the sandwich immunoassay described herein can be used in a method for evaluating the efficacy of a drug targeting a cross-linking enzyme such as LH, P3H3 lysyl oxidase (LOX), and LOXL enzymes. For example, the method can be used to evaluate a drug, such as an antagonist drug targeting LH, P3H3, LOX, or LOXL.

[0102] Therefore, the present invention also relates to a method for evaluating the efficacy of an antagonist drug targeting a cross-linking enzyme selected from LH, P3H3, LOX or LOXL, wherein the method comprises quantifying the amount of CTX-V in at least two biological samples using the sandwich immunoassay described herein, the biological samples being obtained from the subject at a first time point and at least one subsequent time point during administration of the antagonist drug to the subject, and wherein a decrease in the amount of CTX-V from the first time point to the at least one subsequent time point during administration of the antagonist drug indicates that the antagonist drug targeting the cross-linking enzyme is effective.

[0103] Preferably, the method quantifies the effectiveness of said antagonist drug.

[0104] Preferably, the method assesses the efficacy of an antagonist drug targeting LH or LOXL.

[0105] Example 1 Development of Sandwich Assay

[0106] Reagents

[0107] All reagents used in the experiment were high quality chemicals from Merck (Whitehouse Station, NJ, USA) and Sigma (St. Louis, MO, USA). Synthetic peptides used for monoclonal antibody production, assay development and validation were 1) immunogenic peptide: keyhole limpet hemocyanin (KLH)-CGG-PKGEKGHPGL (SEQ ID No. 14), 2) coating peptide: biotin-PKGEKGHPGL (SEQ ID No. 15), 3) selection peptide: PKGEKGHPGL (SEQ ID No. 1) or CPKGEKGHPGL (SEQ ID No. 16) × CPKGEKGHPGL (dimers connected by N-terminal disulfide bridges), 4) extended peptide: PKGEKGHPGLI (SEQ ID No. 2), 5) truncated peptide: PKGEKGHPG (SEQ ID No. 3). Dimer selection peptides were used for assay development and validation. All synthetic peptides were purchased from Genscript (Piscataway, NJ, USA).

[0108] Monoclonal Antibody Production and Clone Characterization

[0109] The monoclonal antibody generated in Balb / C mice

[30] targets a new epitope located in the C-helical region of type V collagen (1478'-PKGEKGHPGL-'1487 (SEQ ID No. 1)). Briefly, 200 μL of emulsified antigen and 100 μg of immunogenic peptide with Sigma adjuvant system were injected. We repeated the intravenous injection of immunogenic peptide (100 μg in 100 μL 0.9% NaCl) one month later and isolated splenocytes 72 hours later for cell fusion in SP2 / 0 myeloma cells

[31] . In an indirect competitive enzyme-linked immunosorbent assay (ELISA), supernatants were screened for reactivity to the selected peptide and extended and truncated peptides using streptavidin pre-coated plates (Roche, Hvidovre, Denmark, catalog number 11940279). Clones were selected by testing the selectivity and unique reactivity of the antibodies to the selected peptide or immunogenic peptide. No extended or truncated peptides were detected ( Figure 1 ). Using SBA Clonotyping TMSelected antibodies were isotyped using the ELISA system-HRP (Southern Biotech, Birmingham, AL, USA) and purified using a protein-G column from GE Healthcare Life Sciences (Little Chalfont, Buckinghamshire, UK).

[0110] Selected antibodies were sequenced and the CDRs determined.

[0111] The sequences of the chains are as follows (CDRs in bold; framework sequences in italics; constant regions underlined):

[0112] Light chain: amino acid sequence (238aa)

[0113]

[0114] CDR-L1:KSSQSLLYSDGKTYLN(SEQ ID No.4)

[0115] CDR-L2: LVSKLDS (SEQ ID No. 5)

[0116] CDR-L3:WQGTHLPYA(SEQ ID No.6)

[0117] Heavy chain: Amino acid sequence (464aa):

[0118]

[0119]

[0120] CDR-H1: DYYMH (SEQ ID No. 7)

[0121] CDR-H2:WIDPENGDREYAPKFQG(SEQ ID No.8)

[0122] CDR-H3: RGHYEDH (SEQ ID No. 9)

[0123] The selected antibodies were used in the sandwich immunoassay described below for both capture and detection antibodies.

[0124] By mixing 110 μ L Na2CO3 / NaHCO3 buffer, pH 9.6, 1 mL (1 mg / mL) antibody and 13.3 μ L biotin aminocaproic acid-N-hydroxysuccinimide ester, the solution was incubated for 1 hour at 20 ° C in a manner of up and down rotation to generate capture antibodies. Subsequently, 110 μ L of 0.2 M ethanolamine (pH 8.0) was added to the solution and incubated as before. The solution was dialyzed overnight in a Zeba 7kMWCO desalting column (Thermo Scientific, Waltham, MA, USA, catalog number 89889) immersed in 4 ° C 1xPBS. According to the manufacturer's instructions, a portion of the solution was labeled with horseradish peroxidase using a kit from Sigma (catalog number 11829696001) for use as a detection antibody.

[0125] CTX-V direct sandwich ELISA protocol

[0126] Streptavidin pre-coated 96-well plates (Roche Diagnostic's, Hvidovre, Denmark, catalog number 11940279) were coated with 0.250 μg / mL of capture antibody diluted 1 / 200 in assay buffer (50 mM PBS, 1% BSA, 0.1% Tween-20, 150 mM NaCl, pH 7.4) and incubated at 20°C for 30 minutes with rotation at 300 rpm. 20 μL of standards, controls or samples were added, followed by 100 μL of incubation buffer (25 mM PBS, 1% BSA, 0.1% Tween-20, 75 mM NaCl, 5% Liquid II, pH. 7.4). The plates were then rotated at 20°C for 1 hour. After sample incubation, the plates were coated with 0.500 μg / mL of detection antibody diluted 1 / 100 in incubation buffer for 1 hour. The plates were washed with 25 mM TRIZMA, 50 mM NaCl, 0.036% Bronidox L5, 0.1% Tween 20 buffer between each incubation. Subsequently, 100 μL of chemiluminescent substrate was added to the wells, incubated for 3 minutes at 20 ° C in the dark, and measured at 470 nm. A standard curve was drawn using a 4-parameter mathematical fitting model based on the results obtained from 1.5-fold serial dilutions of the dimer-selected peptide. The unknown sample assay values ​​were inserted into the standard curve to obtain the concentration (ng / mL) of CTX-V.

[0127] Technical Validation

[0128] The lower limit of detection (LLOD) was determined based on 21 zero samples (i.e., incubation buffer). LLOD was calculated as mean + 3 × standard deviation (SD). The upper limit of quantification was determined based on the highest point of the standard curve, and the recovery rate <20% was determined based on ten independent runs. The inter-assay and intra-assay variations were determined by performing ten independent runs of five quality control samples with double determination, wherein at least three healthy human serum or plasma EDTA samples (Valley Biomedical, Winchester, VA, USA) were used. The acceptance criteria for inter-assay and intra-assay variations were 15% and 10%, respectively. The linearity, specificity, accuracy and interference of the assay were determined by calculating the recovery percentage, and the validation standard was 100% ± 20% of the reference sample. Using undiluted samples as references, linearity was evaluated by diluting four healthy serum and human plasma EDTA samples 1:2 times. The accuracy of the assay was determined by adding two healthy human serum or plasma EDTA samples and calculating the recovery rate between the actual measured value and the theoretical measured value. Interferences were assessed by adding known concentrations of biotin (low = 5 ng / mL, high 100 ng / mL), hemoglobin (low = 0.078 mM, high = 0.155 mM), or lipids (low = 2.42 mM, high = 5.49 mM) to healthy human serum or plasma EDTA samples. The recovery between the unspiked sample and the low or high interferent sample was calculated.

[0129] Analyte and reagent stability

[0130] Analyte stability was determined by calculating percent recovery of three healthy human serum and plasma EDTA samples from non-stressed samples. The samples were subjected to five freeze and thaw cycles.

[0131] CTX-V technical performance

[0132] No reactivity was observed to extended or truncated peptides, as confirmed by lack of signal inhibition. As the concentration of selected and immunogenic peptides increased, increased signal inhibition was observed, demonstrating the specificity of the monoclonal antibodies for the selected CTX-V neoepitopes ( Figure 1 ). Based on the validation criteria (Table 2), technical validation and analytical as well as reagent stability were acceptable.

[0133]

[0134]

[0135] Example 2 Analysis of samples from patients with inflammatory bowel disease

[0136] Clinical cohort

[0137] Study subjects were enrolled in a prospective observational study at Odense University Hospital as previously described in

[29] (ClinicalTrials.gov ID: NCT02612103). The analyses performed here included patients with UC (n = 47) and CD (n = 39), as well as healthy subjects (n = 32). Informed consent was collected from each patient before blood sampling. The Regional Ethics Committee of Southern Denmark (journal number: S-20150107) approved this study and was conducted in accordance with the Declaration of Helsinki. Patient demographics are listed in Table 1. Biomarker levels were assessed at baseline in UC, CD, and healthy subjects.

[0138] Statistical analysis

[0139] The classified patient characteristics are presented as frequencies (percentages), and continuous variables are mean ± SD. Fisher's exact test (for two groups) or chi-square test (for more than two groups) are used to calculate the statistical differences in the classified patient characteristics. The differences in continuous variables (including biomarker levels between healthy subjects and patients with UC or CD) were determined by Kruskal-Wallis application of Dunn's test correction for multiple comparisons. Biomarker data are expressed in ng / mL and plotted as Tukey graphs. Receiver operating characteristic (ROC) analysis was performed to evaluate the diagnostic potential of CTX-V biomarkers. All statistical analyses were performed in GraphPad Prism v.9.1.1 (Graph Pad Software, La Jolla, CA, USA) or MedCalcv.19.3 (MedCalc Software, Ostend, Belgium). Asterisks indicate the following: *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001; ns==no significant difference.

[0140] result

[0141] Cohort Demographics

[0142]

[0143] UC and CD patients have increased proteolytic degradation of cross-linked type V collagen

[0144] Plasma CTX-V levels were significantly increased in patients with UC or CD compared with healthy subjects (all: p < 0.0001, mean difference [3.87 ng / mL and 2.19 ng / mL]) ( Figure 2 ).

[0145] Quantification of proteolysis and cross-linking of type V collagen can identify patients with UC or CD.

[0146] ROC analysis was performed to evaluate the discriminatory ability of CTX-V, demonstrating that the area under the curve for healthy subjects and patients with UC or CD was 0.99 (sensitivity: 100%, specificity: 96.87%) and 0.99 (sensitivity: 100%, specificity: 96.87%), respectively. Table 3 summarizes the results of the ROC analysis.

[0147]

[0148]

[0149] Example 3 - Analysis of samples from patients with ankylosing spondylitis, psoriasis and atopic dermatitis

[0150] Clinical cohort

[0151] Commercial serum samples were obtained from patients diagnosed with ankylosing spondylitis (AS) (n=19), psoriasis (PSO) (n=9), atopic dermatitis (AD) (n=10) and healthy subjects (HS) (n=32). Samples from AS, PSO and AD patients were obtained from Protogenex (Inglewood, California, USA), while samples from HS were obtained from Valley Biomedical (Winchester, Virginia, USA). Patient demographic data are presented in Table 4.

[0152] Scar-in-a-Jar, an in vitro model of fibrosis

[0153] Human primary intestinal fibroblasts purchased from Cellbiologics (Chicago, Illinois, USA) (Catalog No. H-6025) were grown to confluence and seeded at 30,000 cells / well at passages 6-8 in 48-well plates on day -2 in high serum medium (10% fetal bovine serum (FBS) (Catalog No. F7524, Sigma-Aldrich, St. Louise, Missouri, USA), Dulbecco's Modified Eagle Medium (DMEM) + Glutamax (Catalog No. 31966, Gibco, Life Technologies, Carlsbad, California, USA)). On day -1, cells were serum starved in low serum medium (0.4% FBSDMEM) to avoid interference with biomarker measurements. After induction of fibrogenesis on day 0, cells were cultured in low serum medium containing ficoll-70 (112.5 mg / mL, catalog number F2878, Sigma-Aldrich, St. Louise, Missouri, USA) and -400 (75 mg / mL, catalog number F4375, Sigma-Aldrich, St. Louise, Missouri, USA) supplemented with 1.0% L-ascorbic acid, magnesium phosphate, n-hydrate (catalog number 013-19,641, Wako, Osaka, Japan) in the absence or presence of 20 ng / mL TGF-β1 (catalog number 100-B-010 / CF, R&D systems, Minneapolis, Minnesota, USA). Cell culture was performed by incubation at 37°C, 95% O2 and 5% CO2 for 12 days, with freshly prepared medium (without or with TGF-β1 stimulation) replaced on days 0, 4 and 8, and supernatants saved for subsequent CTX-V measurements on days 4, 8 and 12. Three replicates were performed without and with stimulation.

[0154] Cell-matrix cleavage

[0155] On day 12, after removal of the supernatant, the scar-in-a-jar (SIAJ) cell model wells were washed twice in PBS and stored at -20°C. For in vitro cleavage of wells containing matrix, the plates were thawed and gently washed with digestion buffer (50 mM Tris-HCl, 200 mM NaCl, 10 mM CaCl2, 100 uM ZnCl, pH 7.5). By diluting to 100ng / μL protease precursor and 1mM 4-aminophenylmercuric acetate (APMA, catalog number A9563, Sigma-Aldrich, St.Louis, Missouri, USA) in digestion buffer, incubating precursor-MMP-9 for twenty-four hours and incubating precursor-MMP-13 for two hours at 37°C, to activate the precursor form of MMP-9 (catalog number 911-MP-010, R&D system, Minneapolis, Minnesota, USA) and MMP-13 (catalog number 511-MM-010, R&D system, Minneapolis, Minnesota, USA). 500μL of 0.5μg activated MMP diluted in digestion buffer was added to the wells and the plates were incubated at 37°C for 72 hours. Subsequently, proteases were inhibited by adding 1μM EDTA to the wells. The cleavage supernatant was stored at -20°C until CTX-V measurement. Wells incubated with the corresponding digestion buffer alone were used as controls.

[0156] Statistical analysis

[0157] Data from unstimulated and stimulated cells were compared by two-way ANOVA, with correction for multiple comparisons. The biomarker data of the SIAJ cell model were analyzed by ELISA. Data are presented as mean ± SD. The exact biomarker measurements (ng / mL) of three replicates were used. Statistical analysis of CTX-V fold changes in cell-matrix lysis was performed by one-way ANOVA corrected for multiple comparisons comparing the presence and absence of active MMPs. Fold changes in CTX-V were calculated based on the respective medium controls for SIAJ and cell-matrix lysis experiments.

[0158] result

[0159] Cohort Demographics

[0160]

[0161]

[0162] In vitro generation of CTX-V fragment

[0163] Analysis of supernatants from human primary intestinal fibroblasts stimulated with or without TGF-β1 did not demonstrate any proteolytic release of CTX-V fragments. However, incubation with activated MMP-9 resulted in a 3.2-fold increase in CTX-V compared to uncleaved controls ( Figure 3 ).

[0164] Proteolysis of cross-linked type V collagen in fibro-inflammatory pathologies

[0165] Serum levels of CTX-V were measured in patients with AS, PSO, and AD and compared with HS levels. Patients with AS, PSO, or AD had significantly elevated levels of the biomarker (AS: p < 0.001, PSO: p < 0.001, AD: p < 0.05) ( Figure 4 ).

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Claims

1. A monoclonal antibody that specifically recognizes and binds to a neo-epitope in the C-helical region generated by protease cleavage of intact type V collagen, wherein: The new epitope is contained in the amino acid sequence PKGEKGHPGL-COOH (SEQ ID No. 1).

2. The monoclonal antibody according to claim 1, wherein The monoclonal antibody is a monoclonal antibody generated against a synthetic peptide having a C-terminal amino acid sequence of PKGEKGHPGL-COOH (SEQ ID No. 1).

3. The monoclonal antibody according to claim 1 or claim 2, wherein The antibody non-specifically recognizes or binds to a peptide having a C-terminal amino acid sequence of PKGEKGHPGLIZ (SEQ ID No. 2), wherein Z is absent or is one or more amino acids of the sequence of the C-helical region of type V collagen.

4. The monoclonal antibody according to any one of the preceding claims, wherein The antibody does not specifically recognize or bind to a peptide having a C-terminal amino acid sequence of PKGEKGHPG (SEQ ID No. 3).

5. A sandwich immunoassay for detecting cross-linked type V collagen in a biological sample, the cross-linked type V collagen comprising at least two chains of a C-helical region of type V collagen linked together by interchain cross-links, the method comprising: contacting the biological sample comprising the cross-linked type V collagen with a first monoclonal antibody bound to a surface, wherein each chain of type V collagen contained in the cross-linked type V collagen has a neo-epitope of a C-helical region generated by cleavage of intact type V collagen by a protease; adding a second monoclonal antibody; and determining the amount of binding of the second monoclonal antibody; Wherein, both the first monoclonal antibody and the second monoclonal antibody specifically react with a new epitope in the C-helical region produced by protease cleavage of intact type V collagen, and the new epitope is contained in the amino acid sequence PKGEKGHPGL-COOH (SEQ ID NO: 1).

6. The sandwich immunoassay according to claim 5, wherein The monoclonal antibody does not substantially recognize or bind to an extended form of the C-terminal amino acid sequence, which is PKGEKGHPGLIZ-COOH (SEQ ID NO: 2), wherein Z is absent or is one or more amino acids of the sequence of the C-helical region of type V collagen.

7. The sandwich immunoassay according to claim 5 or 6, wherein The sandwich immunoassay is used to quantify the amount of cross-linked type V collagen in a biological sample.

8. The sandwich immunoassay of claim 7, further comprising correlating the amount of cross-linked type V collagen determined by the method with standard fibrotic disease samples of known disease severity to assess the severity of the fibrotic disease.

9. The sandwich immunoassay according to claim 8, wherein The fibrotic disease is inflammatory bowel disease (IBD), ankylosing spondylitis, psoriasis or atopic dermatitis.

10. The sandwich immunoassay according to any one of claims 7 to 9, wherein The biological sample is a biological fluid.

11. The sandwich immunoassay according to claim 10, wherein The biological fluid is serum, plasma, urine, amniotic fluid, tissue supernatant or cell supernatant.

12. The sandwich immunoassay according to any one of claims 5 to 11, wherein The sandwich immunoassay is a radioimmunoassay, a fluorescent immunoassay or an enzyme-linked immunosorbent assay.

13. The sandwich immunoassay according to any one of claims 5 to 12, wherein The second monoclonal antibody is labeled.

14. The sandwich immunoassay according to claim 13, wherein The second monoclonal antibody is an enzyme-linked antibody.

15. The sandwich immunoassay according to claim 14, wherein The enzyme is horseradish peroxidase (HRP).

16. The sandwich immunoassay according to claim 13, wherein The second monoclonal antibody is radiolabeled or linked to a fluorophore.

17. The sandwich immunoassay according to any one of claims 5 to 12, wherein The amount of bound second monoclonal antibody is determined using a further labeled antibody that recognizes the second monoclonal antibody.

18. A kit for sandwich assay, comprising: a solid support, which is bound to a first monoclonal antibody as defined in any one of claims 1 to 4; as well as The second monoclonal antibody as defined in any one of claims 1 to 4, comprising a label.

19. A method for diagnosing a fibrotic disease, the method comprising: i) measuring the level of CTX-V in a biological fluid sample from a patient using a sandwich immunoassay according to any one of claims 5 to 17; ii) comparing the level of CTX-V in a biological fluid sample from a healthy patient with the level of CTX-V in a biological fluid sample from a patient believed to have a fibrotic disease, iii) determining whether the patient believed to have a fibrotic disease actually has a fibrotic disease based on the comparison in step ii).

20. The method according to claim 19, further comprising: iv) treating a patient believed to be suffering from a fibrotic disease with a drug to treat said fibrotic disease.

21. A method for evaluating the efficacy of an antagonist drug targeting a cross-linking enzyme selected from LH, P3H3, LOX or LOXL, wherein: The method comprises quantifying the amount of CTX-V in at least two biological samples using a sandwich immunoassay according to any one of claims 5 to 17, the biological samples being obtained from the subject at a first time point and at least one subsequent time point during administration of the antagonist drug to the subject, and wherein a decrease in the amount of CTX-V from the first time point to the at least one subsequent time point during administration of the antagonist drug indicates that the antagonist drug targeting LOX is effective.

22. The method according to claim 21, wherein: The method evaluates the efficacy of antagonist drugs targeting LH or LOXL.

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