Biomarkers of fibrosis

Abstract

The present invention relates to a sandwich immunoassay and kit for detecting cross-linked CTX-III in biological samples and its use for evaluating the efficacy of drugs targeting lysyl oxidase (LOX). Cross-linked CTX-III can be used as a biomarker in diseases associated with fibrosis, including liver fibrosis, chronic bowel disease, eosinophilic esophagitis, and cancer.

Description

Technical Field

[0001] Technical Field of the Invention The present invention relates to a sandwich immunoassay for detecting cross-linked CTX-III in a biological sample and its use for evaluating the efficacy of a drug targeting lysyl oxidase (LOX). The present invention also relates to a kit for performing the sandwich immunoassay.

Background Art

[0002] Description of Related Art Fibrotic diseases (including those listed in Table A), for example, cirrhosis, which is associated with 800,000 deaths worldwide per year, is a major cause of morbidity and mortality.

[0003] Table A. Different Fibrotic Diseases

Table 1

[0004] "Fibrotic disease" is any disease in which fibrosis occurs, whether as a primary or secondary symptom. Fibrosis is the end result of a chronic inflammatory response induced by various stimuli including persistent infection, autoimmune reaction, allergic reaction, chemical injury, radiation, and tissue damage. Fibrosis is characterized by the accumulation and reorganization of the extracellular matrix (ECM). Despite having distinct etiologies and clinical distinctions, most chronic fibrotic disorders share persistent stimulators that sustain the production of growth factors, proteolytic enzymes, angiogenic factors, and fibrogenic cytokines, which together stimulate the deposition of connective tissue elements, particularly collagen and proteoglycans, gradually removing and destroying the normal tissue structure. Despite having a significant impact on human health, currently, there are no approved treatments that directly target the mechanism of fibrosis.

[0005] Role of Type III Collagen and the Liver 1 , intestine 2 , kidney 3and the lungs 4 Its involvement in pulmonary fibrosis showed a change in turnover. Since it is one of the main fibrous collagens produced by fibroblasts, type III collagen is thought to be involved in fibrosis-promoting crosslinking events. Here, when crosslinks are formed excessively, the stiffness of the matrix 6 increases, resulting in a matrix that proteases cannot access 5 . As a result, the activation, differentiation 7 , and migration 8 of (myo)fibroblasts, leading to ECM accumulation via cell:ECM interactions that activate the fibrosis-promoting signaling cascade 9 .

[0006] Crosslinking of fibrous collagen: One of the final steps in the maturation of collagen fibrils is the formation of intramolecular and intermolecular crosslinks catalyzed by enzymes such as lysyl oxidase (LOX), lysyl oxidase-like enzymes (LOXL), and transglutaminase (TG) 10 . The formation of these crosslinks provides both mechanical and functional properties, including tensile strength 11 and elasticity, to their respective tissues and affects several cellular functions 12~14 . Various types of enzymatic and non-enzymatic crosslinks are present within fibrous collagens (types I, II, III, V, IX, and XI collagen), 10 but the enzymatic crosslink formation catalyzed by LOX(L) and TG is of particular interest. This is because it is involved in both physiological and pathological crosslinking events 15、16 . LOX(L) catalyzes the oxidative deamination of specific lysine or hydroxylysine within fibrous collagen telopeptides that leads to spontaneous crosslink formation, while TG catalyzes the formation of isopeptide bonds between glutamine and lysine. The final biochemical properties of LOX(L)-derived crosslinks vary depending on their tissue expression, but the crosslinking sites within major fibrous collagens appear to be conserved, indicating tissue specificity rather than collagen specificity 10 .

[0007] The importance of physiological crosslinking is evident as their absence has been observed to indicate a decrease in tissue strength and elasticity in animal models and certain genetic diseases. In addition to the detrimental effects of decreased or weakened crosslinking, excessive formation and biochemical changes can also be harmful to tissue function, as observed in both tissue fibrosis and cancer. In the context of ongoing tissue injury, repeated activation of the wound healing cascade leads to upregulation and accumulation of profibrotic factors, including fibrillar collagens such as type I and type III collagen 17 , as well as LOX(L) and TG. Excessive accumulation of collagen and their crosslinking enzymes mediates the formation of a stiff fibrotic extracellular matrix (ECM).

[0008] Assessment of fibrosis resolution: To determine the efficacy of antifibrotic therapies, sensitive, reliable, and minimally invasive assessment methods are needed. As current therapeutic options, nintedanib and pirfenidone only slow the progression as observed in patients with idiopathic pulmonary fibrosis 18、19 or, like the cases in the study of systemic sclerosis 20 , cannot have a significant effect on skin fibrosis, the goal of completely inhibiting and reversing fibrosis has not yet been achieved. Similarly, the use of anti-inflammatory drugs may not affect fibrosis, which is often seen in intestinal fibrosis in patients with inflammatory bowel disease (IBD) 21、22 . However, as knowledge and evaluation of the involvement of fibrillar collagen and their crosslinks in organ fibrosis increase, new potential targets have been identified. These include small molecule inhibitors of LOXL2 / 3 23 and TG2 24 , as well as inhibitors that inhibit the profibrotic intracellular signaling cascade by targeting Rho kinase 25 . Currently, the gold standard in liver fibrosis 26 and kidney fibrosis 27 is tissue biopsy, and in intestinal fibrosis 28 and pulmonary fibrosis 29 ​​​​The novel serological biomarker can be applied together with any of the other complicated or invasive methods used. Providing an important part of fibrosis that targets fragments of cross-linked fibrous collagen can potentially assist in the accurate assessment of fibrosis resolution. This is CTX-I which measures the degradation fragments of the cross-links of type I and type II collagen respectively 30 and CTX-II 31 The application of biomarkers has been partially shown in the clinical settings of bone resorption and cartilage destruction. Therefore, it is necessary to develop a novel biomarker of cross-linked type III collagen (CTX-III) as a biomarker for fibrosis resolution.

[0009] Eosinophilic esophagitis Eosinophilic esophagitis (EoE) is a food allergen-induced chronic inflammation of the esophagus characterized by a marked influx of eosinophils and Th2 cell-promoted inflammation. Activation of the Th2 inflammatory pathway leads to the recruitment of eosinophils, which in turn secrete inflammation-promoting and fibrosis-promoting mediators such as transforming growth factor β. Over time, the persistent inflammation and secretion of fibrosis-promoting mediators initiate the differentiation of fibroblasts into myofibroblasts, resulting in fibrosis. [46~48] Myofibroblasts are the main cells in tissue fibrosis and significantly secrete collagen as well as cross-linking enzymes such as lysyl oxidase (LOX) and LOX-like enzymes (LOXL). Histological evaluation by Masson's trichrome staining of esophageal biopsies shows a marked deposition of collagen in the subepithelial compartment of the lamina propria. [46、49] .

[0010] Since progressive fibrostenosis can lead to esophageal stricture and stenosis formation, common clinical symptoms include dysphagia and food impaction in which fibrosis plays an essential role. The diagnosis of EoE is mainly based on endoscopic evaluation and esophageal biopsy in patients experiencing dysphagia.

[50] Due to the patchiness of EoE, a single biopsy is insufficient with a sensitivity of 55%.

[51] Therefore, when a total of six biopsies are obtained, the sensitivity increases up to 99%.

[52] In endoscopy, clinical symptoms can be visually confirmed. However, since up to 10% of EoE patients do not show endoscopic findings, biopsies are necessary for all patients. Esophageal biopsies require upper endoscopy and sedation, so alternative methods such as blood-based biomarkers are being developed. Several serum biomarkers have been evaluated. [53、54] However, the use of blood-based biomarkers is not currently commonly used clinically for EoE. Therefore, there is a medical need for minimally invasive tools such as blood-based biomarkers. Similarly, for EoE, data obtained from inflammatory bowel diseases [55、56] can be applied to blood-based biomarkers that target collagen metabolites reflecting either fibrosis or fibrinolysis. These biomarkers can help identify patients with asymptomatic fibrosis that cannot be observed by endoscopy, enabling early intervention. Furthermore, the breakdown of metabolites can reflect the resolution of subepithelial inflammation or fibrosis and thus the treatment effect. Therefore, the introduction of validated blood-based biomarkers of collagen remodeling can provide an essential tool for evaluating deep tissue esophageal remodeling, potentially limiting the need for tissue biopsies.

[0011] Additional tools include high-resolution manometry, which provides information on the physical properties of the esophagus where pressures associated with fibrotic strictures increase.

[57] Furthermore, the results of brush cytology using a Cytosponge correlated with endoscopic findings and showed good sensitivity and specificity for both proximal and distal esophageal eosinophilia.

[58] .

[0012] Inflammatory bowel disease The pathological heterogeneity of inflammatory bowel disease (IBD) and in particular the pathology of subtypes of Crohn's disease (CD) require the creation of several disease classification systems. Clinical parameters, including patient evaluation by clinicians and pathological evaluation, have led to the determination of clinically inactive or active disease [63, 64]. Furthermore, the use of the Montreal classification based on endoscopy provides a visual and more objective classification of the disease that enables stratification of patients based on endoscopic symptoms such as non-stricturing and non-penetrating (B1), stricturing (B2), or penetrating (B3)

[65] . Strictures and fistulas are two severe complications of CD characterized by either fibrous strictures, excessive accumulation of tissue, particularly collagen, or severe tissue and collagen breakdown resulting in a penetrating wound. Collagen in the extracellular matrix occupies a significant proportion in intestinal tissue and has important structural and signaling instructions in both healthy and inflamed intestinal tissue

[66] . As a result of chronic inflammation in IBD, activated myofibroblasts in the interstitial matrix, in combination with cross-linking enzymes such as LOX (L) and TG2, deposit significant amounts of fibrous collagen such as type I, III, and V collagen. This process ultimately enhances matrix rigidity through extensive collagen cross-linking that spreads the fibrosis process independent of inflammation

[67] . Furthermore, myofibroblasts and recruited inflammatory cells produce large amounts of ECM-degrading proteases such as matrix metalloproteinases (MMPs) that promote collagen remodeling. Stricture disease caused by fibrous strictures is the result of extensive deposition and cross-linking of collagen, while penetrating disease is characterized by the predominance of collagen proteolysis.

[0013] Clinical parameters and endoscopy represent standardized methods in the field of IBD. Questionnaires for determining clinical parameters lack the objective and appropriate identification of tissue symptoms. Furthermore, currently, the gold-standard endoscopy causes discomfort to patients, has limited access to the small intestine, and lacks a validated histopathological system

[68] , often affecting CD patients due to the limited access to the small intestine region. More recent techniques such as MRE have become non-invasive and high-precision tools for assessing disease symptoms, but the handling costs are increasing

[69] .

[0014] Therefore, there is an urgent need to develop non-invasive biomarkers that can evaluate the underlying molecular processes that drive the symptoms of various diseases, particularly markers that reflect intestinal fibrosis and the resolution of intestinal fibrosis.

[0015] Cancer Despite years of intensive research, cancer remains the second leading cause of death worldwide. An important factor in the survival of cancer patients is the ability to predict early diagnosis and response to treatment.

[0016] Similar to organ fibrosis, cancer is characterized by the pathological degree of ECM remodeling

[72]

[73] . Here, cancer and stromal cells secrete large amounts of MMPs that degrade the surrounding ECM components including collagen. In addition to ECM degradation, cancer-associated fibroblasts (CAFs) significantly deposit collagen in the tumor stroma, which is subject to many enzyme cross-linkings by LOX (L) and TG2, enhancing tumor progression

[74]

[75] . This process regulates cell signaling, proliferation, differentiation, gene expression, migration, invasion, and metastasis

[76]

[77] . Thus, CAF-led alignment and enzymatic cross-linking of fibrous collagen open the way for tumor cells to invade. Furthermore, the highly cross-linked ECM is thought to impede T cell migration, thereby shielding tumor cells from the host immune system

[78] . This shielding effect may explain why some cancer patients have an insufficient response to immunotherapy

[78]

[79] .

[0017] Evidence has been found highlighting the important role of fibrillar collagen deposition and subsequent enzymatic crosslinking in tumor progression and treatment response. Therefore, the CTX-III biomarker was investigated in a wide range of cancer types. Quantification of CTX-III and subsequent patient stratification may help evaluate the molecular processes within the tumor stroma and identify patients who may potentially benefit from immunotherapy.

[0018] WO20178 / 34172 describes measuring the cross-linked N-terminal propeptide of type III collagen (PIIINP) in a suitable sample as a biomarker for fibrosis. This method utilizes monoclonal antibodies disclosed in WO2014 / 170312. This biomarker is produced alone during the formation process.

[0019] The applicant developed a highly sensitive immunoassay that targets the neoepitope of the C-terminal telopeptide of cross-linked type III collagen, which is generated by C protease and then releases fragments by additional unknown proteases, and can accurately evaluate the degradation of fibrillar ECM.

[0020] A direct sandwich enzyme-linked immunosorbent assay (ELISA) was developed using a highly specific monoclonal antibody that targets the C-terminal telopeptide neoepitope of cross-linked type III collagen. This assay can be used in a clinical setting for the quantitative evaluation of fibrinolysis. Summary of the Invention

[0021] The present invention is directed to a sandwich immunoassay for detecting cross-linked C-terminal telopeptide III collagen (CT-III) in a biological sample, which comprises at least two CT-III chains linked together by interchain cross-linking. The method involves contacting a biological sample containing cross-linked CT-III, wherein each chain of CT-III contained in the cross-linked CT-III has a C-terminal neoepitope of CT-III generated by N-protease cleavage of intact type III procollagen, with a first monoclonal antibody bound to a surface, and adding a second monoclonal antibody. Both monoclonal antibodies specifically react with the C-terminal neoepitope of CT-III, and the neoepitope is contained in the C-terminal amino acid sequence KAGGFAPYYG-COOH (SEQ ID NO: 1). The method further comprises determining the amount of binding of the second monoclonal antibody.

[0022] As used herein, the term "CT-III" refers to the C-terminal telopeptide of type III collagen.

[0023] The present invention is also directed to a method for evaluating the efficacy of an antagonist drug targeting lysyl oxidase (LOX). The method comprises quantifying the amount of cross-linked CT-III in at least two biological samples obtained from a subject at an initial time point and at least one subsequent time point during the administration period of the antagonist drug to the subject, using the sandwich immunoassay described herein. A decrease in the amount of cross-linked CT-III from the initial time point to at least one subsequent time point during the administration period of the antagonist drug indicates an effective antagonist drug targeting LOX.

[0024] The present invention further relates to a kit for use in the sandwich immunoassay described herein. The kit comprises a solid support bound with the above-mentioned first monoclonal antibody and a labeled second monoclonal antibody as described herein.

[0025] The present invention also relates to a method for identifying the fibrosis response phenotype of a patient having fibrosis, comprising quantifying the amount of cross-linked CT-III in a biological fluid sample obtained from the patient using the sandwich immunoassay described herein, and correlating the amount of said cross-linked CT-III with i) a value associated with a known fibrotic response phenotype and / or ii) a predetermined cut-off value. The method may further comprise quantifying the amount of N-terminal type III collagen propeptide (PRO-C3) present in the biological fluid sample, determining the ratio of cross-linked type III collagen (CTX-III) to PRO-C3, and correlating said ratio of cross-linked type III collagen (CTX-III) to PRO-C3 with a predetermined cut-off value.

Advantages of the Invention

[0026] Description of the Invention Accordingly, in a first aspect, the present invention relates to a monoclonal antibody that specifically recognizes and binds to a C-terminal telopeptide neoepitope of cross-linked type III collagen (also referred to herein as the target peptide) having a C-terminus with the amino acid sequence KAGGFAPYYG (SEQ ID NO: 1) (also referred to herein as the target sequence).

[0027] Preferably, the monoclonal antibody is a monoclonal antibody made against a synthetic peptide having a C-terminal amino acid sequence KAGGFAPYYG (SEQ ID NO: 1). The synthetic peptide used to make the antibody can be a synthetic peptide linked to a carrier protein at its N-terminus. Exemplary carrier proteins include, but are not limited to, proteins such as keyhole limpet hemocyanin (KLH). The synthetic peptide can be linked to a carrier protein that may contain one or more additional amino acid residues at the N-terminus of the peptide via any suitable linkage. The monoclonal antibody can be made, for example, by immunizing a mouse or other mammal, isolating spleen cells from the immunized mammal, fusing them with hybridoma cells, and then culturing the resulting hybridoma cells to ensure monoclonal growth, through appropriate techniques known to those skilled in the art.

[0028] In a preferred embodiment, the monoclonal antibody does not specifically recognize or bind to a peptide having a C-terminal amino acid sequence KAGGFAPYYGX (SEQ ID NO: 2), where X represents any amino acid. Therefore, the monoclonal antibody preferably does not specifically recognize or bind to an extended variant of the target peptide whose target amino acid sequence is extended at the C-terminus by one or more amino acids. Preferably, the monoclonal antibody is KAGGFAPYYGDZ-COOH (SEQ ID NO: 3), where Z is absent or is one or more amino acids of the type III collagen sequence and does not substantially recognize or bind to an extended version of the said C-terminal amino acid sequence. Preferably, the monoclonal antibody preferably does not specifically recognize or bind to a peptide having a C-terminal amino acid sequence KAGGFAPYYGD (SEQ ID NO: 4).

[0029] In a preferred embodiment, the monoclonal antibody does not specifically recognize or bind to a peptide having a C-terminal amino acid sequence of KAGGFAPYY (SEQ ID NO: 5). Therefore, the monoclonal antibody preferably does not specifically recognize or bind to a truncated variant of the target peptide in which the target amino acid sequence is truncated by one or more amino acids at the C-terminus.

[0030] Preferably, the monoclonal antibody or a fragment thereof is preferably one of the following: CDR-L1: RSSKSLLHSNGNTYLY (SEQ ID NO: 6) CDR-L2: RMSNLAS (SEQ ID NO: 7) CDR-L3: MQHLEFPLT (SEQ ID NO: 8) CDR-H1: DHGMH (SEQ ID NO: 9) CDR-H2: VISTYYGDATYNQKFKG (SEQ ID NO: 10) CDR-H3: SMGGNYVGTGFAY (SEQ ID NO: 11) It may contain one or more complementarity-determining regions (CDRs) selected from the following.

[0031] Preferably, the antibody or a fragment thereof contains at least 2, 3, 4, 5 or 6 of the above CDR sequences.

[0032] Preferably, the monoclonal antibody or a fragment thereof has a light chain variable region containing a CDR sequence. CDR-L1: RSSKSLLHSNGNTYLY (SEQ ID NO: 6) CDR-L2: RMSNLAS (SEQ ID NO: 7) and CDR-L3: MQHLEFPLT (SEQ ID NO: 8)

[0033] Preferably, the monoclonal antibody or a fragment thereof has a light chain containing a framework sequence between CDRs, and the framework sequence is substantially identical or substantially similar to the framework sequence between CDRs in the following light chain sequence (the CDRs therein are shown in bold, underlined, and the framework sequence is shown in italics). RSSKSLLHSNGNTYLY WFLQRPGQSPQLLIY RMSNLAS GVPDRFSGSGSGTAFTLRISRVEAEDVGVYYC MQHLEFPLT (SEQ ID NO: 12)

[0034] Preferably, the monoclonal antibody or fragment thereof has a heavy chain variable region containing a CDR sequence. CDR-H1: DHGMH (SEQ ID NO: 9) CDR-H2: VISTYYGDATYNQKFKG (SEQ ID NO: 10) and CDR-H3: SMGGNYVGTGFAY (SEQ ID NO: 11)

[0035] Preferably, the monoclonal antibody or fragment thereof has a heavy chain containing a framework sequence between CDRs, and the framework sequence is substantially identical or substantially similar to the framework sequence between CDRs in the following light chain sequence (the CDRs therein are shown in bold and underlined, and the framework sequence is shown in italic). DHGMH WVKQSQAKSLEWIG VISTYYGDATYNQKFKG KATMTVDKSSSTAYMELARLTSEDSAIYYCAR SMGGNYVGTGFAY (SEQ ID NO: 13)

[0036] As used herein, the framework amino acid sequences between the CDRs of an antibody are substantially identical or substantially similar to the framework amino acid sequences between the CDRs of another antibody if they have at least 70%, 80%, 90% or at least 95% similarity or identity. Similar or identical amino acids may or may not be adjacent.

[0037] The framework array may include one or more amino acid substitutions, insertions and / or deletions. The amino acid substitutions may be conservative, which means that the substituted amino acid has similar chemical properties to the original amino acid. Those skilled in the art understand which amino acids share similar chemical properties. For example, the following groups of amino acids: 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 share similar chemical properties such as size, charge and polarity.

[0038] Programs such as the CLUSTAL program can be used to compare amino acid sequences. This program compares amino acid sequences and finds the optimal alignment by appropriately inserting spaces into either sequence. It is possible to calculate the amino acid identity or similarity (identity plus conservation of amino acid type) for the optimal alignment. Programs such as BLASTx align the longest stretches of similar sequences and assign values to the degree of fitness. Therefore, it is possible to obtain comparisons in which several similarity regions are found, each having a different score. Both types of analysis are contemplated in the present invention. Identity or similarity is preferably calculated over the entire length of the framework array.

[0039] In one preferred embodiment, the monoclonal antibody or fragment thereof has a light chain variable region sequence: DIVMTQAAPSVPVTPGESVSISC RSSKSLLHSNGNTYLY WFLQRPGQSPQLLIY RMSNLAS GVPDRFSGSGSGTAFTLRISRVEAEDVGVYYC MQHLEFPLT FGAGTKLELK (SEQ ID NO: 14) (CDRs are in bold and underlined; framework sequences are in italics) and / or a heavy chain variable region sequence: QVQLQQSGAELVRPGVSVKISCKGSGHTFT DHGMH WVKQSQAKSLEWIG VISTYYGDATYNQKFKGKATMTVDKSSSTAYMELARLTSEDSAIYYCAR SMGGNYVGTGFAY WGQGTLVTVSA (SEQ ID NO: 15) (CDRs are in bold and underlined; framework sequences are in italics) may include

[0040] The present invention is a sandwich immunoassay for detecting cross-linked C-terminal telopeptide of type III collagen (CT-III) in a biological sample, wherein the cross-linked CT-III comprises at least two CT-III chains covalently bound together by intermolecular cross-links, contacting the biological sample containing the cross-linked CT-III with a first monoclonal antibody bound to a surface, wherein each chain of CT-III contained in the cross-linked CT-III contains a C-terminal neoepitope of CT-III generated by C-proteinase cleavage of intact type III collagen; adding a second monoclonal antibody; and determining the amount of binding of the second monoclonal antibody; including both the first monoclonal antibody and the second monoclonal antibody specifically react with the C-terminal neoepitope of CT-III, and the neoepitope is contained in the C-terminal amino acid sequence KAGGFAPYYG-COOH (SEQ ID NO: 1), relating to a sandwich immunoassay.

[0041] Preferably, the monoclonal antibody is KAGGFAPYYGDZ-COOH (SEQ ID NO: 3), wherein Z is absent or does not substantially recognize or bind to an extended version of the C-terminal amino acid sequence of one or more amino acids of the collagen type III sequence.

[0042] Preferably, the monoclonal antibody does not substantially recognize or bind to a truncated version of the C-terminal amino acid sequence KAGGFAPYY-COOH (SEQ ID NO: 5).

[0043] The sandwich immunoassay described herein uses the same antibody as both the capture antibody and the detection antibody, so that double-stranded peptides (i.e., cross-linked) can be recognized by the assay.

[0044] Preferably, the sandwich immunoassay is used to quantify the amount of cross-linked CT-III in a biological fluid, which can be, but is not limited to, serum, plasma, urine, amniotic fluid, tissue supernatant or cell supernatant.

[0045] The sandwich immunoassay can be, but is not limited to, a radioimmunoassay, a fluorescence immunoassay, or an enzyme-linked immunosorbent assay.

[0046] In a preferred embodiment, the second monoclonal antibody can be labeled to determine the amount of binding of the second monoclonal antibody.

[0047] Preferably, the second monoclonal antibody can be an enzyme-conjugated antibody. The enzyme can be, but is not limited to, horseradish peroxidase (HRP).

[0048] Preferably, the second monoclonal antibody may be radiolabeled or conjugated to a fluorophore.

[0049] These are preferred labels used in the present invention, but it is contemplated that any suitable labeling system such as a DNA reporter or an electrochemiluminescent tag can be used.

[0050] Alternatively, the amount of binding of the second monoclonal antibody may be determined using an additional labeled antibody that recognizes the second monoclonal antibody. The additional labeled antibody can be labeled using the labels described above.

[0051] In a preferred embodiment of the present invention, the sandwich immunoassay may further include correlating the amount of cross-linked CT-III determined by the method with a standard disease sample of a known disease severity to evaluate the disease severity. The disease can be a fibrotic disease. Such fibrotic diseases can be, but are not limited to, liver diseases, particularly non-alcoholic fatty liver disease (NAFLD), or viral liver fibrosis such as HCV-related liver fibrosis. Alternatively, the disease can be a chronic intestinal disease. Such chronic intestinal diseases can be, but are not limited to, Crohn's disease or ulcerative colitis, preferably Crohn's disease. Alternatively, the disease can be cancer. Such cancers can be, but are not limited to, breast cancer, bladder cancer, colorectal cancer, head and neck cancer, kidney cancer, lung cancer, pancreatic cancer, stomach (gastric) cancer, ovarian cancer, liver cancer, prostate cancer, or melanoma. Preferably, the cancer is breast cancer.

[0052] The sandwich immunoassay can also be used to monitor disease progression by comparing the amount of cross-linked CT-III determined by the method with the amount of cross-linked CT-III determined in a second sample obtained from the same patient at different time points. The second sample can be obtained hours, days, weeks, or years before and after the sample is tested. Multiple samples can be obtained at different time points to determine the amount of cross-linked CT-III and the results can be compared. The sandwich immunoassay can be used to monitor disease progression after treatment to identify whether the treatment was successful. Preferably, the disease is a fibrotic disease. Such fibrotic diseases can be, but are not limited to, liver diseases, particularly non-alcoholic fatty liver disease (NAFLD), or viral liver fibrosis such as HCV-related liver fibrosis. Alternatively, the disease can be eosinophilic esophagitis. Alternatively, the disease can be a chronic intestinal disease. Such chronic intestinal diseases can be, but are not limited to, Crohn's disease or ulcerative colitis, preferably Crohn's disease. Alternatively, the disease can be cancer. Such cancers can be, but are not limited to, breast cancer, bladder cancer, colorectal cancer, head and neck cancer, kidney cancer, lung cancer, pancreatic cancer, stomach (gastric) cancer, ovarian cancer, liver cancer, prostate cancer, or melanoma. Preferably, the cancer is breast cancer.

[0053] The sandwich assay described herein may further comprise determining the amount of type III collagen formation, preferably by determining the amount of PRO-C3 present in a sample.

[0054] The ratio of PRO-C3 to cross-linked CT-III (CTX-III) can be used to determine the net deposition of type III collagen, which is significantly elevated in several disease states. Pro-C3 is generated during collagen formation and is a measure of collagen formation, while CTX-III is generated during degradation and is a measure of degradation. Thus, the ratio of cross-linked CT-III (CTX-III) to PRO-C3 can be used to determine the net fibrinolysis, which is significantly elevated in several disease states. For example, patients with a lower Ishak score (an indicator of the severity of fibrosis) among HCV-related liver fibrosis patients had a higher net fibrinolysis solubility compared to patients with a higher Ishak score. Further, in patients with Crohn's disease and ulcerative colitis, patients with a less severe disease, i.e., non-stricturing and non-penetrating disease, as indicated by the Montreal classification of B1, had a higher net fibrinolysis solubility compared to patients with the Montreal classification of B2. Further, in breast cancer patients, patients with stage III (an indicator of the severity of cancer) had a higher net fibrinolysis solubility compared to stage II patients.

[0055] In a further aspect, the sandwich immunoassay described herein can be used in a method for evaluating the efficacy of a drug targeting lysyl oxidase (LOX), such as an antagonist drug targeting LOX.

[0056] Accordingly, the present invention also provides a method for evaluating the effectiveness of an antagonist drug targeting lysyl oxidase (LOX), which comprises quantifying the amount of cross-linked CT-III in at least two biological samples using the sandwich immunoassay described herein, wherein the biological samples are obtained from a subject at an initial time point during the administration period of the antagonist drug to the subject and at least one subsequent time point, and a decrease in the amount of cross-linked CT-III from the initial time point to at least one subsequent time point during the administration period of the antagonist drug indicates an effective antagonist drug targeting LOX.

[0057] Preferably, the method quantifies the effectiveness of the antagonist drug.

[0058] Preferably, the method evaluates the effectiveness of an antagonist drug targeting LOXL2.

[0059] In another aspect, the present invention provides a kit for use in the sandwich immunoassay described herein, the kit comprising a solid support bound to the first monoclonal antibody as described above; and the labeled second monoclonal antibody as described above.

[0060] In another aspect, the present invention also provides a method for identifying a fibrosis response phenotype in a patient having fibrosis, which comprises quantifying the amount of cross-linked CT-III in a biological fluid sample obtained from the patient using the sandwich immunoassay described herein, and correlating the amount of cross-linked CT-III with i) a value associated with a known fibrotic response phenotype and / or ii) a predetermined cut-off value. The method may further comprise quantifying the amount of PRO-C3 present in the biological fluid sample, determining the ratio of cross-linked type III collagen (CTX-III) to PRO-C3, and correlating the ratio of cross-linked type III collagen (CTX-III) to PRO-C3 with a predetermined cut-off value.

[0061] The determined amount of cross-linked CT-III can be compared with a predetermined cut-off value. The predetermined cut-off value is preferably at least 3.5 ng / mL, more preferably at least 3.8 ng / mL, even more preferably at least 4.0 ng / mL, even more preferably at least 4.2 ng / mL, and most preferably at least 4.5 ng / mL. In this regard, by using a combination of various statistical analyses, it has been found that the measured binding amount between the monoclonal antibody (above) and the C-terminal CTX-III biomarker of at least 3.5 ng / mL or more can be used to determine patients with a "natural regression" phenotype. By having a statistical cut-off value of at least 3.5 ng / mL, more preferably at least 3.8 ng / mL, even more preferably at least 4.0 ng / mL, even more preferably at least 4.2 ng / mL, and most preferably at least 4.5 ng / mL, it is possible to identify patients with a natural regression phenotype with a high level of reliability using the method of the present invention.

[0062] The ratio of the measured cross-linked type III collagen (CTX-III) and type III collagen (PRO-C3) in the sample can be compared with a predetermined cut-off value. The predetermined cut-off value is preferably at least 0.5, more preferably at least 0.6, even more preferably at least 0.75, even more preferably at least 0.8, and most preferably at least 0.9. In this regard, by using a combination of various statistical analyses, it has been found that the ratio of cross-linked type III collagen (CTX-III) and type III collagen (PRO-C3) of at least 0.5 or more can be used to determine patients with a natural regression phenotype. By having a statistical cut-off ratio value of preferably at least 0.5, more preferably at least 0.6, even more preferably at least 0.75, even more preferably at least 0.8, and most preferably at least 0.9, it is possible to identify patients with a natural regression phenotype with a high level of reliability using the method of the present invention.

[0063] In another aspect, the invention also relates to a method for identifying a patient having a fibrotic disease, comprising quantifying the amount of cross-linked CT-III in a biological fluid sample obtained from the patient using the sandwich immunoassay described herein, and correlating the amount of said cross-linked CT-III with i) a value associated with known fibrotic disease patients and / or normal healthy controls and / or ii) a predetermined cut-off value. The method may further comprise quantifying the amount of PRO-C3 present in the biological fluid sample, determining the ratio of cross-linked type III collagen (CTX-III) to PRO-C3, and correlating said ratio of cross-linked type III collagen (CTX-III) to PRO-C3 with i) a value associated with known fibrotic disease patients and / or normal healthy controls and / or ii) a predetermined cut-off value. Higher levels of cross-linked CT-III or a significantly different ratio as compared to normal healthy controls indicate a fibrotic disease.

[0064] The fibrotic disease may be selected from, but is not limited to, liver diseases, particularly non-alcoholic fatty liver disease (NAFLD), or viral liver fibrosis such as HCV-related liver fibrosis.

[0065] In another aspect, the invention also relates to a method for identifying a patient having eosinophilic esophagitis, comprising quantifying the amount of cross-linked CT-III in a biological fluid sample obtained from the patient using the sandwich immunoassay described herein, and correlating the amount of said cross-linked CT-III with i) a value associated with known eosinophilic esophagitis patients and / or normal healthy controls and / or ii) a predetermined cut-off value. The method may further comprise quantifying the amount of PRO-C3 present in the biological fluid sample, determining the ratio of cross-linked type III collagen (CTX-III) to PRO-C3, and correlating said ratio of cross-linked type III collagen (CTX-III) to PRO-C3 with i) a value associated with known eosinophilic esophagitis patients and / or normal healthy controls and / or ii) a predetermined cut-off value. Higher levels of cross-linked CT-III or a significantly different ratio as compared to normal healthy controls indicate eosinophilic esophagitis.

[0066] In another aspect, the present invention also relates to a method for identifying a patient with a chronic intestinal disease, comprising quantifying the amount of cross-linked CT-III in a biological fluid sample obtained from the patient using the sandwich immunoassay described herein, and correlating the amount of said cross-linked CT-III with i) a value associated with known patients with chronic intestinal diseases and / or normal healthy controls and / or ii) a predetermined cut-off value. The method may further comprise quantifying the amount of PRO-C3 present in the biological fluid sample, determining the ratio of cross-linked type III collagen (CTX-III) to PRO-C3, and correlating said ratio of cross-linked type III collagen (CTX-III) to PRO-C3 with i) a value associated with known patients with chronic intestinal diseases and / or normal healthy controls and / or ii) a predetermined cut-off value. Higher levels of cross-linked CT-III or a significantly different ratio compared to normal healthy controls indicate the presence of a chronic intestinal disease.

[0067] The chronic intestinal disease may be selected from, but is not limited to, inflammatory bowel diseases such as Crohn's disease or ulcerative colitis. Preferably, the chronic intestinal disease is Crohn's disease or ulcerative colitis, more preferably Crohn's disease.

[0068] In another aspect, the invention also relates to a method for identifying a cancer patient, which comprises quantifying the amount of cross-linked CT-III in a biological fluid sample obtained from a patient using the sandwich immunoassay described herein, and correlating the amount of the cross-linked CT-III with i) a value associated with known cancer patients and / or normal healthy controls and / or ii) a predetermined cut-off value. The method may further comprise quantifying the amount of PRO-C3 present in the biological fluid sample, determining the ratio of cross-linked type III collagen (CTX-III) to PRO-C3, and correlating the ratio of cross-linked type III collagen (CTX-III) to PRO-C3 with i) a value associated with known cancer patients and / or normal healthy controls and / or ii) a predetermined cut-off value. A high level of cross-linked CT-III or a significantly different ratio compared to normal healthy controls indicates the presence of cancer.

[0069] Cancer can be selected from, but is not limited to, breast cancer, bladder cancer, colorectal cancer, head and neck cancer, kidney cancer, lung cancer, pancreatic cancer, stomach (gastric) cancer, ovarian cancer, liver cancer, prostate cancer, or melanoma. Preferably, the cancer is breast cancer.

[0070] In another aspect, the invention provides a method for identifying a patient who would benefit from treatment, which comprises quantifying the amount of cross-linked CT-III in a biological fluid sample obtained from a patient using the sandwich immunoassay described herein, and correlating the amount of the cross-linked CT-III with i) a value associated with known disease patients and / or normal healthy controls and / or ii) a predetermined cut-off value. The method may further comprise quantifying the amount of PRO-C3 present in the biological fluid sample, determining the ratio of cross-linked type III collagen (CTX-III) to PRO-C3, and correlating the ratio of cross-linked type III collagen (CTX-III) to PRO-C3 with i) a value associated with known disease patients and / or normal healthy controls and / or ii) a predetermined cut-off value. A high level of cross-linked CT-III or a significantly different ratio compared to normal healthy controls indicates the need for treatment.

[0071] The method may further comprise administering treatment to a patient.

[0072] The treatment is preferably administration of a drug targeting collagen crosslinking, such as an antagonist drug targeting lysyl oxidase (LOX).

[0073] The disease can be a fibrotic disease. Such fibrotic diseases can be, but are not limited to, liver diseases, particularly non-alcoholic fatty liver disease (NAFLD), or viral liver fibrosis such as HCV-related liver fibrosis. Alternatively, the disease can be eosinophilic esophagitis. Alternatively, the disease can be a chronic intestinal disease. Such chronic intestinal diseases can be, but are not limited to, Crohn's disease, or irritable bowel syndrome such as ulcerative colitis. Alternatively, the disease can be cancer. Such cancers can be, but are not limited to, breast cancer, bladder cancer, colorectal cancer, head and neck cancer, kidney cancer, lung cancer, pancreatic cancer, stomach (gastric) cancer, ovarian cancer, liver cancer, prostate cancer, or melanoma. Preferably, the cancer is breast cancer.

[0074] Applying a statistical cut-off value to the method of the present invention results in a single diagnostic assay, which is particularly advantageous. That is, it eliminates the need for a direct comparison with healthy individuals and / or patients with known disease severity to reach a diagnostic conclusion. This can also eliminate the need for more invasive procedures such as endoscopy or biopsy to demonstrate an initial prognosis, and can function as a rapid and definitive tool to facilitate the initiation of an appropriate treatment plan, and is thus particularly advantageous when using an assay to evaluate patients who already have medical signs or symptoms generally indicative of fibrosis (as determined, for example, by physical examination and / or consultation with a medical professional). Preferably, the predetermined cut-off value corresponds to a cut-off value measured in human blood, serum or plasma.

[0075] Definitions As used herein, the term "neoepitope" refers to the terminal of a polypeptide, i.e., the N-terminal or C-terminal N-terminal or C-terminal peptide sequence of a polypeptide, and should not be construed in its general sense.

[0076] As used herein, the term monoclonal antibody NBH-242 refers to a neoepitope-specific antibody made against a C-terminal neoepitope located at the C-terminal telopeptide of type III collagen (CT-III), and said neoepitope contains the C-terminal sequence KAGGFAPYYG-COOH (SEQ ID NO: 1).

[0077] As used herein, the term "PRO-C3" refers to the N-terminal propeptide of type III collagen.

[0078] As used herein, the term "PCX3" refers to the cross-linked N-terminal propeptide of type III collagen.

[0079] As used herein, the term "PRO-C3 assay" refers to a competitive ELISA for detecting and quantifying neoepitopes in the N-terminal propeptide as previously described 32 in a competitive ELISA for detecting and quantifying neoepitopes in the N-terminal propeptide.

[0080] As used herein, the term "PCX3 assay" refers to a competitive ELISA for detecting and quantifying cross-linked N-terminal propeptides as previously described in WO2017 / 134172.

[0081] As used herein, the term "CT-III" refers to the C-terminal telopeptide of type III collagen.

[0082] As used herein, the term "CTX-III" refers to the cross-linked C-terminal telopeptide of type III collagen comprising at least two chains of CT-III covalently linked by intermolecular cross-links.

[0083] As used herein, the term "CTX-III" assay refers to the sandwich assay described herein for detecting and quantifying cross-linked type III collagen, i.e., the neoepitope of the C-terminal telopeptide of cross-linked CT-III.

[0084] As used herein, the terms "peptide" and "polypeptide" are used synonymously.

[0085] As used herein, the term "monoclonal antibody" refers to both whole antibodies and fragments of antibodies that retain the binding specificity of the whole antibody, such as Fab fragments, Fv fragments, or other such fragments known to those of skill in the art. Antibodies that retain the same binding specificity may contain the same complementarity determining regions (CDRs). The CDRs of an antibody can be determined using methods known in the art, such as those described by Kabat et al. 45 and the like.

[0086] Antibodies can be made from B cell clones as described in the Examples. The isotype of an antibody can be determined by ELISA specific for human IgM, IgG, or IgA isotypes, or human IgG1, IgG2, IgG3, or IgG4 subclasses. Other suitable methods can be used to identify the isotype.

[0087] The amino acid sequence of an antibody produced can be determined using standard techniques. For example, RNA can be isolated from cells and used to make 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, for all VH (variable heavy chain) sequences, a primer specific for the leader sequence can be used in conjunction with a primer that binds to a sequence located in the constant region of a previously determined isotype. The light chain can be amplified using a primer that anneals to the V kappa or V lambda leader sequence in conjunction with a primer that binds to the 3' end of the kappa or lambda chain. Full-length heavy and light chains can be made and sequenced.

[0088] As used herein, the term "C-terminus" means the end of a polypeptide, i.e., the C-terminus of the polypeptide, and should not be construed as meaning in its general direction. Similarly, the term "N-terminus" refers to the end of a polypeptide, i.e., the N-terminus of the polypeptide, and should not be construed as meaning in its general direction.

[0089] As used herein, the term "competitive immunoassay" refers to an immunoassay in which a target peptide (if any) present in a sample competes for the binding of an antibody to a known amount of peptide (e.g., bound to a solid substrate or labeled), which is a technique known to those skilled in the art.

[0090] As used herein, the term "ELISA" (enzyme-linked immunosorbent assay) refers to an immunoassay that uses an antibody linked to an enzyme such as horseradish peroxidase or alkaline phosphatase to detect a target peptide (if any) present in a sample. The activity of the enzyme is then evaluated by incubation with a substrate that produces a measurable product. This allows the presence and / or amount of the target peptide in the sample to be detected and / or quantified. ELISA is a technique known to those skilled in the art.

[0091] As used herein, the term "sandwich immunoassay" refers to the use of at least two antibodies for the detection of an antigen in a sample, which is a technique known to those skilled in the art.

[0092] As used herein, the term "binding amount" refers to the quantification of the binding between a monoclonal antibody and a target peptide, and the quantification is determined by comparing the measured value of the target peptide in a biological fluid sample with a calibration curve, which is created using standard samples of known concentrations of the target peptide. In the specific assay disclosed herein for measuring a target peptide having the C-terminal amino acid sequence KAGGFAPYYG (SEQ ID NO: 1) in a biological fluid, the calibration curve is created using standard samples of a calibration peptide of known concentration having the C-terminal amino acid sequence KAGGFAPYYG (SEQ ID NO: 1) (in particular, which may consist of the amino acid sequence KAGGFAPYYG (SEQ ID NO: 1)). The measured value in the biological fluid sample is compared with the calibration curve to determine the actual amount of the target peptide in the sample.

[0093] As used herein, "cut-off value" means a binding amount or level of fibrinolysis that is statistically determined to indicate a high likelihood of a fibrotic disease such as liver fibrosis in a patient in that a measured value of a biomarker in a patient sample above the statistical cut-off value corresponds to a probability of at least 70%, preferably at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% of the presence or likelihood of a fibrotic disease such as liver fibrosis. "Cut-off value" can also mean a binding amount or fibrinolysis level that is statistically determined to indicate a high likelihood of a patient having a natural regression phenotype.

[0094] As used herein, "fibrosis response phenotype" refers to the phenotype of a patient indicating how the severity of fibrosis changes without treatment. Patients with a "natural regression" phenotype are those in whom the Ishak score decreased after 52 weeks of treatment with placebo. Patients with no change in the Ishak score after 52 weeks of treatment with placebo have a "stability" phenotype, while patients with an increased Ishak score after 52 weeks of treatment with placebo have a "progressive" phenotype. Patients with a "natural regression" phenotype may require a different treatment plan, i.e., a lower dosage or a shorter treatment cycle, compared to patients with a stability or progressive phenotype.

[0095] As used herein, the term "value associated with a normal healthy subject and / or value associated with the severity of a known disease" refers to a value determined by the above method for a subject considered to be healthy, i.e., free of disease (e.g., a fibrotic disease such as a liver disease, particularly non-alcoholic fatty liver disease (NAFLD), or viral liver fibrosis such as HCV-related liver fibrosis; a chronic intestinal disease such as Crohn's disease or ulcerative colitis; or free of cancer such as breast cancer, bladder cancer, colorectal cancer, head and neck cancer, kidney cancer, lung cancer, pancreatic cancer, stomach (gastric) cancer, ovarian cancer, liver cancer, prostate cancer, or melanoma), and the standardized amount of cross-linked type III collagen (CTX-III) or the standardized ratio of cross-linked type III collagen (CTX-III) to the N-terminal propeptide of type III collagen (PRO-C3), and / or a value determined by the above method for a subject known to have a disease of known severity (e.g., a fibrotic disease such as a liver disease, particularly non-alcoholic fatty liver disease (NAFLD), or viral liver fibrosis such as HCV-related liver fibrosis; a chronic intestinal disease such as Crohn's disease or ulcerative colitis; or cancer such as breast cancer, bladder cancer, colorectal cancer, head and neck cancer, kidney cancer, lung cancer, pancreatic cancer, stomach (gastric) cancer, ovarian cancer, liver cancer, prostate cancer, or melanoma).

[0096] As used herein, "fibrotic disease" refers to a liver disease, particularly, non-alcoholic fatty liver disease (NAFLD), or viral liver fibrosis such as HCV-related liver fibrosis, etc.

[0097] As used herein, "chronic intestinal disease" can be selected from hypersensitivity intestinal diseases such as, but not limited to, Crohn's disease or ulcerative colitis. Preferably, the chronic intestinal disease is Crohn's disease or ulcerative colitis, more preferably Crohn's disease.

[0098] As used herein, "cancer" may be selected from, but is not limited to, breast cancer, bladder cancer, colorectal cancer, head and neck cancer, kidney cancer, lung cancer, pancreatic cancer, stomach (gastric) cancer, ovarian cancer, liver cancer, prostate cancer, or melanoma. Preferably, the cancer is breast cancer.

[0099] The present invention is demonstrated in the following examples with reference to the following drawings.

Brief Description of the Drawings

[0100]

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Mode for Carrying Out the Invention

[0101] Example 1 Methods and Materials: Reagents All reagents used in the experiments were high-quality chemicals from companies such as Merck (Whitehouse Station, NJ, USA) and Sigma Aldrich (St. Louis, MO, USA). The synthetic peptides used for the production of monoclonal antibodies and the development and validation of assays were: 1) immunogenic peptide: keyhole limpet hemocyanin (KLH)-CGG-KAGGFAPYYG, 2) coating peptide: biotin-KAGGFAPYYG, 3) selection peptide: KAGGFAPYYG (SEQ ID NO: 1) or CKAGGFAPYYG×CKAGGFAPYYG (SEQ ID NO: 16) (dimer linked by an N-terminal disulfide bridge), 4) extension peptide: KAGGFAPYYGD (SEQ ID NO: 4) or CKAGGFAPYYGD×CKAGGFAPYYGD (SEQ ID NO: 17) (dimer linked by an N-terminal disulfide bridge), 5) cleavage peptide: KAGGFAPYY (SEQ ID NO: 5) or CKAGGFAPYY×CKAGGFAPYY (SEQ ID NO: 18) (dimer linked by an N-terminal disulfide bridge), and 6) rat dimer peptide: CKSGGFSPYYG×CKSGGFSPYYG. The dimer peptides were used only for the development and validation of the assays. All synthetic peptides were purchased from Genscript, Piscataway, NJ, USA.

[0102] Production of Monoclonal Antibodies and Characterization of Clones The target neoepitope (1212’-KAGGFAPYYG-‘1221) located in the C-terminal telopeptide of type III collagen was analyzed for its specificity using protein blast and sequence homology with rats and mice (Figure 1).

[0103] The monoclonal antibody was prepared in 4 - 6-week-old Balb / C mice. Using Freund's incomplete adjuvant (Sigma-Aldrich), 200 μL of emulsified antigen and 50 μg of immunogenic peptide (KLH-CGG-KAGGFAPYYG) were immunized subcutaneously into the mice. The mice were immunized at two-week intervals until a stable serum titer level was reached. Mice with the highest serum titer were selected for fusion. The mice were rested for one month and then immunized intravenously with 50 μg of immunogenic peptide in 100 μL of 0.9% NaCl solution. Three days later, spleen cells were isolated for cell fusion. Briefly, the spleen cells were fused with SP2 / 0 myeloma cells to generate hybridoma cells, which were then cloned in culture dishes using the semi-medium method. The clones were seeded into 96-well microtiter plates, and limiting dilution was used to ensure monoclonal growth. The supernatants were screened for reactivity against the selected peptide (KAGGFAPYYG (SEQ ID NO: 1)) and the extended peptide (KAGGFAPYYGD (SEQ ID NO: 4)) by indirect competitive ELISA using streptavidin precoated plates (Roche, Hvidovre, Denmark, catalog number 11940279) coated with 4 ng / mL of coating peptide (biotin-KAGGFAPYYG). All reagents were diluted in 50 mM PBS, 1% BSA, 1% Tween-20, 150 mM NaCl, pH 7.4. The selection for the final inhibition of the two best monoclonal clones was made by testing the reactivity against the selected peptide (KAGGFAPYYG (SEQ ID NO: 1)) rather than the extended peptide (KAGGFAPYYGD (SEQ ID NO: 4)), the cleaved peptide (KAGGFAPYY (SEQ ID NO: 5)), or the immunogenic peptide (KLH-CGG-KAGGFAPYYG). Before purifying the optimal monoclonal clone, the antibody was subjected to an isotype test using a sandwich ELISA kit, SBA Clonotyping™ System-HRP (Southern Biotech, Birmingham, AL, USA).Monoclonal antibodies with the best reactivity were purified using a Protein G column (GE healthcare Life Sciences, Little Chalfont, Buckinghamshire, UK) according to the manufacturer's instructions.

[0104] The sequence of the prepared antibody was determined, and the CDRs were determined.

[0105] The sequences of the chains are as follows (CDRs are underlined and bold; constant regions are italicized): Heavy chain sequence (mouse IgG isotype) QVQLQQSGAELVRPGVSVKISCKGSGHTFT DHGMH WVKQSQAKSLEWIG VISTYYGDATYNQKFKG KATMTVDKSSSTAYMELARLTSEDSAIYYCAR SMGGNYVGTGFAY WGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK (SEQ ID NO: 19) CDR-H1: DHGMH (SEQ ID NO: 9) CDR-H2: VISTYYGDATYNQKFKG (SEQ ID NO: 10) CDR-H3: SMGGNYVGTGFAY (SEQ ID NO: 11) Light chain sequence (mouse kappa isotype) DIVMTQAAPSVPVTPGESVSISC RSSKSLLHSNGNTYLY WFLQRPGQSPQLLIY RMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYC MQHLEFPLT FGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNE (SEQ ID NO: 20) CDR-L1: RSSKSLLHSNGNTYLY (SEQ ID NO: 6) CDR-L2: RMSNLAS (SEQ ID NO: 7) CDR-L3: MQHLEFPLT (SEQ ID NO: 8)

[0106] Development of the assay Monoclonal antibody labeling 110 μL of Na2CO3 / NaHCO3 buffer, pH 9.6 was added to 1 mL (1 mg / mL) of the antibody, and then 13.3 μL of biotinamidocaproic acid N-hydroxysuccinimide ester (Sigma Aldrich, St. Louis, MO, USA, catalog number B2643) was added to label the monoclonal NBH-242 antibody used as the capture antibody with biotin. The solution was incubated at 20°C for 1 hour with rotation. Then, 110 μL of 0.2 M ethanolamine, pH 8.0 was added to this solution and incubated as before. This solution was dialyzed overnight in a Zeba 7k MWCO desalting column (Thermo Scientific, Waltham, MA, USA, catalog number 89889) and immersed in 1xPBS at 4°C. Furthermore, a part of the monoclonal antibody was labeled with horseradish peroxidase (HRP) and used as the detection antibody. The HRP labeling was purchased from Sigma Aldrich, St. Louis, MO, USA, catalog number 11829696001 and carried out according to the manufacturer's protocol using a peroxidase labeling kit manufactured by Roche.

[0107] Direct sandwich ELISA protocol A 96-well plate pre-coated with streptavidin (Roche Diagnostic’s, Hvidovre, Denmark, catalog number 11940279) was coated with 100 μL of biotinylated antibody targeting the CTX-III fragment diluted 1+100 with assay buffer (50 mM PBS, 1% BSA, 1% Tween-20, 150 mM NaCl, pH 7.4) for 30 minutes at room temperature while rotating at 300 revolutions per minute (rpm). Unbound biotinylated capture antibody was discarded, and the wells were washed with wash buffer (25 mM TRIZMA, 50 mM NaCl, 0.036% Bronidox L5, 0.1% Tween 20) using a standardized ELISA plate washer (BioTek® instrument, microplate washer, ELx405 Select CW, Winooski, USA). All samples and detection antibodies were diluted with incubation buffer (50 mM PBS, 1% BSA, 1% Tween-20, 150 mM NaCl, 5% Liquid II, pH 7.4), and the detection antibody was diluted 1+100. 20 μL of sample material and controls were incubated with 100 μL of HRP-labeled detection antibody targeting the CTX-III fragment for 20 hours at 4°C while stirring at 300 rpm. Unbound primary antibody and sample were discarded, and the wells were washed with wash buffer. Then, 100 μL of chemiluminescent substrate was added to the wells, and the plate was incubated at 20°C for 3 minutes while rotating at 300 rpm in the dark. Finally, luminescence was quantified using an ELISA reader (VersaMAX; Molecular Devices, Wokingham Berkshire, UK) set to measure luminescence at 450 nm and 650 nm. A standard curve was plotted using a 4-parameter mathematical fitting model with results obtained from serial dilutions of the dimer-selective peptide (CKAGGFAPYYG×CKAGGFAPYYG (SEQ ID NO: 16)). Measured values of unknown samples were interpolated with the standard curve to obtain the concentration (ng / mL) of the CTX-III fragment.

[0108] Technical verification The lower limit of detection (LLOD) was determined from 21 zero samples (i.e., incubation buffer), calculated as the mean + 3×standard deviation, and the upper limit of detection (ULOD) was determined from 10 measurements of the dimer-selective peptide, calculated as the mean + 3×standard deviation. Intra-assay and inter-assay variability was determined by 10 independent runs of 5 quality control (QC) samples (at least 3 of which were healthy human plasma EDTA samples (Valley Biomedical, Winchester, VA, USA), with each run consisting of duplicate determinations of the samples). The acceptance criteria for inter-assay and intra-assay variability were 15% and 10%, respectively. The linearity of the assay was determined by calculating the recovery rate (100% ± 20%) of a 1:6 dilution of a healthy human plasma EDTA sample at a significant concentration, using the undiluted sample as a reference. The specificity of the assay was determined by calculating the recovery rates of the dimer-extension peptide, dimer-cleavage peptide, and dimer-rat peptide against a 100% sample of the dimer-selective peptide. The accuracy of sample measurements was determined by spiking two samples of healthy human plasma EDTA at significant concentrations, and then calculating the recovery rate between the theoretical and actual measured values. Interference from biotin, lipids, and hemoglobin was tested by spiking healthy human plasma EDTA samples with known concentrations of the interfering substances. The recovery rate between the control sample and the low or high interference sample was then calculated.

[0109] Stability of Analytes and Reagents The stability of the CTX-III fragment was determined by calculating the recovery rates of 3 healthy human plasma EDTA samples from non-stressed samples. These samples were subjected to up to 4 cycles of freeze-thaw or incubated at either 4°C or 20°C for 2, 4, 24, or 48 hours.

[0110] In Vitro Cleavage Assay Clinical Cohort Measurements Biomarker Assay The evaluation of type III collagen formation was performed using PRO-C3, which targets neoepitopes in the N-terminal propeptide32 It was performed using competitive ELISA. Briefly, a 96-well plate coated with streptavidin was incubated with 100 μL of biotinylated coater peptide diluted 1+100 with PBS coating solution containing a protein stabilizer and a preservative at 20 °C and 300 rpm for 30 minutes. After incubation, the coating solution was discarded, and the wells were washed 5 times with wash buffer (25 mM TRIZMA, 50 mM NaCl, 0.036% Bronidox L5, 0.1% Tween 20) using a standardized ELISA plate washer (BioTek® instrument, microplate washer, ELx405 Select CW, Winooski, USA). 20 μL of the sample substance was added to the appropriate wells, followed by 100 μL of HRP-labeled antibody diluted 1+100 with incubation buffer. The plates were incubated at 4 °C and 300 rpm for 20 hours and then washed as described above. Tetramethylbenzidine (TMB, Kem-En-Tec catalog number 438OH, Taastrup, Denmark) was used as a colorimetric reagent at 100 μL / well and incubated for 15 minutes in the dark with stirring at 300 rpm. 100 μL of 1% H2SO 4 was added to stop the reaction, and the optical density was read at 450 nm using an ELISA reader (VersaMAX; Molecular Devices, Wokingham, UK) with a reference of 650 nm. The concentration of PRO-C3 in the analyzed samples was determined by interpolation using a 4-parametric logarithmic standard curve created by serial 2-fold dilution of the selected peptide.

[0111] Results: Production and Characterization of Monoclonal Antibodies Alignment of type III collagen rat, mouse, and human sequences revealed two amino acid differences (indicated by ": " in Figure 1). By isotype characterization, the antibody NBH-242 used in assay development was determined to be IgG2a κ light chain. In the final inhibition, the monoclonal antibody showed no reactivity against the extended peptide, cleaved peptide, or immunogenic peptide, which was observed by the lack of signal inhibition. Testing for reactivity against the selected peptide demonstrated an increase in signal inhibition with peptide concentration, and thus binding to the antibody was shown in indirect competitive ELISA (Figure 2).

[0112] Technical Verification In the development of sandwich ELISA, the monoclonal NBH-242 antibody was used as both the capture and detection antibody. The measurement range of the human CTX-III ELISA was determined by calculating the LLOD and ULOD, providing a range of 0.92 - 15.94 ng / mL. The technical performance of the assay, determined by calculating the inter-assay and intra-assay variability, was within the acceptable range with variabilities of 14.8% and 5.4% respectively (Table 4). The linearity of the assay was tested with healthy human plasma EDTA samples, resulting in an average recovery rate of 108.9%, and thus the acceptable range was 100% ± 20% (Table 5). Further dilution of the sample resulted in a concentration lower than the measurement range. By plotting the calculated recovery rates, the specificity of the antibody in direct sandwich ELISA against the dimeric peptide was tested. Here, the antibody showed no reactivity against any of the dimeric extended peptide, dimeric cleaved peptide, or dimeric rat peptide, but showed reactivity against the increase in concentration of the dimeric selected peptide indicated by the increase in luminescence (y-axis) (Figure 3). Spiking healthy human plasma EDTA samples with another healthy human plasma EDTA sample resulted in an average recovery rate of 102.2% (Table 6). None of the tested interferents demonstrated an effect on the measured values of the samples in healthy human plasma EDTA samples, and the average recovery rate was within the acceptable range (100% ± 20%) (Table 7).

[0113] Table 4: Intra- and inter-assay variability for the CTX-III assay using five QC samples (three of these were healthy human plasma EDTA samples and the last two were dimer-selective peptides). Variability (%) was calculated as the mean of 10 individual duplicates for each sample. [Table 2]

[0114] Table 5: Sample dilution recovery rates for four healthy human plasma EDTA samples (HP) [Table 3]

[0115] Table 6: Spiked healthy human plasma EDTA samples at significant concentrations were spiked into each other and spike recovery rates (%) were calculated between the measured and theoretical values of the samples. [Table 4]

[0116] Table 7: Interference from hemoglobin, biotin, and lipids in three healthy human plasma EDTA samples. Recovery rates (%) were calculated from low and high concentrations of interfering substances relative to pure plasma EDTA samples. [Table 5]

[0117] Stability of analytes and reagents The stability of analytes in healthy human plasma EDTA samples was stable up to 4 cycles of freeze / thaw (Table 8). The mean recovery rate for incubation of healthy human plasma EDTA samples at 4 °C was 92.5% and was stable up to 48 hours in all samples. At 20 °C, the mean recovery rate was 114.7% and three of the four samples demonstrated analyte stability up to 24 hours (Table 9).

[0118] Table 8:

Table 6

[0119] Table 9:

Table 7

[0120] Investigation Based on the production of monoclonal antibodies targeting neoepitopes in the C-terminal telopeptide of type III collagen after cleavage by C protease, a novel CTX-III sandwich ELISA was developed by utilizing the NBH-242 monoclonal antibody to detect cross-linked fragments of type III collagen. Briefly, this assay demonstrated high specificity for the human neoepitope and had the ability to detect analytes in human plasma EDTA samples. Furthermore, this assay was shown to be technically stable using acceptable inter-assay and intra-assay variability, linearity, and accurate measurements.

[0121] Characterization and Technical Validation During the characterization of the antibody, the monoclonal antibody reactivity was tested by competitive ELISA against variations in the neoepitope sequence, which showed high specificity of the antibody, but the peptide used was monomeric and thus the use of the antibody in sandwich ELISA was not verified. Therefore, a set of dimeric peptides containing two identical sequences of the aforementioned different peptide variations cross-linked via a disulfide bond located at the N-terminus relative to the antibody binding site was designed. When testing the reactivity against the dimeric peptides, the antibody induced high specificity against the dimeric human selected peptides, thus re-verifying its high specificity against the human neoepitope sequence while also demonstrating its potential usefulness in the detection of cross-linked fragments.

[0122] Due to the high specificity and the inability of the antibody to detect rat sequence homologues, the development and validation of this assay focused on human sample materials. Natural reactivity was demonstrated in human plasma EDTA samples, but most of the measured sample values were at the lower end of the measurement range, as can be observed from the HD levels in Figure 3. Samples within the measurement range were selected for the technical validation of the assay to clarify its technical stability. Intra-assay and inter-assay variability was within acceptable limits, similar to the dilution recovery of healthy human plasma EDTA samples and spike recovery indicating assay accuracy.

[0123] Analyte and reagent stability Important factors in assay development and clinical use are the accurate measurement of sample materials after various freeze-thaw cycles and the stability of the analyte allowing incubation at higher or lower temperatures. This is particularly important when measuring clinical sample materials where the exact handling of the samples is not fully known. Although careful sample handling should always be a priority, stability testing of the CTX-III analyte did not suggest any major instability.

[0124] In the following examples, the levels of CTX-III were measured using the above assay. The levels of PROC3 were measured using the method described in WO2014 / 170312, and the levels of PC3X were measured using the assay described in WO2017 / 34172.

[0125] Example 2 - Bariatric surgery Blood samples were taken from 58 patients with non-alcoholic fatty liver disease (NAFLD) who had undergone bariatric surgery at baseline and at 6 months of follow-up. A schematic of the patient demographics is provided in Table 10, which includes the patients' BMI, non-alcoholic fatty liver disease activity score (NAS), fatty liver grade, inflammation grade, ballooning, and their fibrosis stage. Patient demographics were obtained only at baseline, and demographics were provided for only 45 - 48 patients. Prior to the surgical procedure, patients were put on a diet to promote preoperative weight loss. The samples measured were plasma EDTA and stored at -80 °C until CTX-III measurement.

[0126] Table 10: Represents the demographics of bariatric surgery patients at baseline.

Table 8

[0127] Statistical analysis The comparison of CTX-III levels between healthy plasma EDTA samples and plasma EDTA samples from the cohort of bariatric surgery patients was performed by applying one-way ANOVA (Kruskal-Wallis) with false discovery rate correction. Results are shown as median CTX-III level + interquartile range (IQR). All statistical analyses were performed using GraphPad Prism v.8.2.0 (Graph Pad Software, La Jolla, CA, USA). Asterisks indicate the following: * : p < 0.05; ** : p < 0.01; *** : p < 0.001; **** : p < 0.0001; ns = non-significant difference.

[0128] Results CTX-III levels are related to NAFLD The levels of CTX-III were significantly higher in patients with NAFLD who had undergone bariatric surgery at baseline (p < 0.0001) and at 6 months follow-up (p < 0.001), compared to levels in healthy human plasma EDTA donors (HD) (Figure 4). Furthermore, the baseline (p < 0.01) levels were significantly higher than the levels at 6 months follow-up (Figure).

[0129] Deposition of type III collagen The ratio of PRO-C3 and CTX-III, which indicates the net deposition of type III collagen, was significantly increased in bariatric surgery patients at 6 months follow-up compared to patients at baseline (p < 0.0001) (Figure 5).

[0130] Discussion The development of the CTX-III assay demonstrated the ability to distinguish between HD and NAFLD patients, including the difference between baseline and 6-month follow-up levels.

[0131] CTX-III levels are related to NAFLD NAFLD is one of the main chronic liver diseases affecting approximately one quarter of the population 33 and one of the main causes is obesity, which leads to the accumulation of fat in the liver 34 This accumulation then leads to the initiation of an inflammatory cascade, which can form scar tissue and ultimately lead to the state of liver fibrosis 34 Here, type III collagen 35 and the ECM-related crosslinking enzyme LOXL2 36Excessive accumulation of ECM components, including , is involved in further disease progression. Thus, the potential and biological relevance of the novel CTX-III marker in the study of NAFLD patients who underwent bariatric surgery were evaluated. By measuring CTX-III levels in plasma EDTA samples obtained from NAFLD patients at baseline and at the 6-month follow-up time point, significant differences among patients at each time point were demonstrated. Furthermore, when comparing the CTX-III levels of NAFLD patients with those of HD, patients diagnosed with NAFLD showed significantly higher biomarker levels at all time points. This increase in the level of the CTX-III marker suggests that the level of fibrinolysis that occurs in bariatric surgery patients increased significantly. In combination with CTX-III, the liver fibrosis-related PRO-C3 biomarker of type III collagen formation was also measured. The combination of the two biomarker measurements provided a measure of the net deposition of type III collagen. Herein, the deposition increased from baseline to the 6-month follow-up, suggesting a switch from the degradation of mature cross-linked type III collagen to the formation of new collagen in the tissue. From these data, the potential of the CTX-III marker is shown in differentiating individuals with known active diseases and monitoring the level of CTX-III over time. The CTX-III marker could not distinguish between patients based on their individual disease scores, including fatty liver grade, inflammation grade, ballooning, BMI, fibrosis stage, or NAS score (not shown).

[0132] Example 3 HCV-related liver fibrosis Description of the study A total of 158 patients diagnosed with hepatitis C virus (HCV)-related liver fibrosis were screened and blood samples were collected and measured at the 52-week time point after screening. The sample substance to be measured was plasma EDTA. Table 11 provides an overall overview of the patient population, and Table 12 represents 47 patients in the placebo group.

[0133] Table 11: Overview of the demographics of patients at the screening time point diagnosed with HCV-related liver fibrosis

Table 9

[0134] Table 12: Summary of patient demographics within the placebo group showing data at the time of screening

Table 10

[0135] Statistical analysis Depending on the number of groups analyzed (Mann-Whitney or Kruskal-Wallis), the comparison of CTX-III levels between healthy plasma EDTA samples and plasma EDTA samples from a cohort of HCV-related liver fibrosis patients was performed by applying a non-parametric t-test or one-way ANOVA. The data were corrected for the false discovery rate. Results are shown as median CTX-III or net fibrinolysis + interquartile range (IQR), unless otherwise stated. Biomarker tertile levels were defined as the 1st, 2nd, and 3rd tertiles, starting from the lowest biomarker level to the highest biomarker level as follows: All statistical analyses were performed using GraphPad Prism v.8.4.3 (Graph Pad Software, La Jolla, CA, USA) and MedCalc v.19.3 (MedCalc Software Ltd, 8400 Ostend, Belgium). Asterisks indicate the following: * : p < 0.05; ** : p < 0.01; *** : p < 0.001; **** : p < 0.0001; ns = non-significant difference.

[0136] Results Patients presenting with HCV-related liver fibrosis were stratified according to their Ishak scores at the time of screening, and their CTX-III levels were compared to those of healthy donors. Independently of the degree of fibrosis, patients presenting with liver fibrosis had significantly (p < 0.0001) elevated CTX-III levels at the time of screening compared to healthy donors (Figure 6).

[0137] When calculating the net fibrotic solubility (CTX-III / PRO-C3) at the time of screening, significant differences were found among patients presenting with varying degrees of fibrosis using the Ishak classification (Figure 7). Patients with an Ishak score of 1-2 induced a higher net fibrotic solubility compared to patients with a score of 4-5 (p<0.05). The same was true for patients with a score of 3, with a significantly (p<0.05) elevated level compared to patients with a score of 4-5.

[0138] A total of 47 patients were treated with placebo for 52 weeks, after which their Ishak scores were measured. The patients were then stratified according to the change in Ishak score from screening to 52 weeks and defined as having either a regressive, stable or progressive fibrosis phenotype. Plotting the CTX-III levels at the time of screening for each phenotype, it was found that the biomarker levels were significantly elevated (p<0.01) in patients with a natural regressive phenotype compared to the progressive phenotype. Furthermore, patients presenting with a stable fibrosis phenotype showed significantly higher CTX-III levels (p<0.05) at the time of screening compared to the progressive phenotype (Figure 8A). Calculation of the net fibrinolysis at screening enabled a more distinct discrimination between patient phenotypes as patients with a regressive phenotype showed significantly higher levels of fibrinolysis (p<0.001) compared to the progressive phenotype, and the same was true when comparing the stable and progressive phenotypes (p<0.01) (Figure 8B).

[0139] When patients in the placebo group were divided into tertiles based on either their CTX-III levels or levels of fibrinolysis at the screening time point, a significant decrease in Ishak scores was demonstrated in patients with high levels of CTX-III at the screening time point (third tertile) compared to patients with low initial CTX-III levels (first tertile) (Figure 9A). Observation of Ishak scores varied based on net fibrinolysis, and patients with levels in the third tertile showed a significant decrease compared to patients in the first tertile (p < 0.001). Furthermore, patients with levels of fibrinolysis in the second tertile at the screening time point also showed a significant decrease in their Ishak scores compared to the first tertile (p < 0.01) (Figure 9B).

[0140] Based on receiver operating characteristic (ROC) and summary of its statistics, the Youden index and optimal cut-off values for the degenerative fibrosis phenotype were determined for both CTX-III levels and fibrinolysis levels at the screening time point (Table 13). Levels of CTX-III > 3.8 ng / mL at the screening time point significantly decreased Ishak scores compared to patients with levels below the cut-off value (p < 0.01) (Figure 10A). The same was true for net fibrinolysis, and patients with a ratio of cross-linked type III collagen degradation (CTX-III) and type III collagen formation (PRO-C3) greater than 0.5 experienced a significant decrease in Ishak scores compared to patients presenting with low levels of fibrinolysis (p < 0.01) (Figure 10B).

[0141] Table 13: Identification of spontaneous regressors using specific cut-off values for either CTX-III levels or net fibrinolysis solubility at the screening time point and their associated values from the ROC curve

Table 11

[0142] Patients were stratified based on the cutoff level determined by calculating the Youden index, and then the odds ratio of fibrosis regression was calculated from logistic regression. This yielded an odds ratio of fibrosis regression that was 19.4 times higher (p = 0.0088) for patients with CTX-III levels ≥ 3.8 ng / mL at screening compared to patients presenting with lower levels than this value. When observing the fibrinolytic levels of the patients, with a fibrinolytic rate ≥ 0.5, the odds ratio increased to 23.3 (p = 0.0057) (Figure 11).

[0143] Discussion Type III collagen in HCV fibrosis: During the progression of fibrosis, activation of pathogenic cells results in the excessive formation of collagen within the ECM, particularly the major fibrillar collagens of types I, III, and V. In combination with the increase in collagen cross-linking enzymes such as LOXL and TG, the deposition of fibrillar collagen and subsequent increase in cross-linking can increase tissue stiffness, cause tissue destruction, and potentially lead to organ failure 37 In HCV-related liver fibrosis, unchecked viral infection results in the loss of tissue homeostasis with resulting chronic inflammation and the expression of several inflammatory and fibrogenic cytokines including tumor growth factor-β1 (TGF-β1). This cascade of fibrogenic cytokines ultimately activates quiescent hepatic stellate cells and differentiates them into myofibroblasts 38 Myofibroblasts constitute the main effector cells of fibrosis involved in the regulation of ECM production and matrix stiffening mediated by extensive collagen cross-linking and ECM contraction 39 The accumulation of type III collagen in HCV-related liver fibrosis has previously been shown using the biomarker PRO-C3, which quantifies the formation of type III collagen via a sensitive monoclonal antibody targeting the NH2 propeptide of type III collagen 40、41Using the known relationship between PRO-C3 and the fibrotic phenotype, the relationship between fibrinolysis and the novel biomarker CTX-III was investigated. Similar to the findings of CTX-III levels in NAFLD, patients with HCV-related fibrosis showed higher levels of cross-linked type III collagen degradation compared to HD. Furthermore, an increase in the level of fibrinolysis was observed in patients with the lowest degree of fibrosis (Ishak score 1-2), suggesting an increase in fibrinolysis of mature cross-linked type III collagen and a decrease in type III collagen formation. These data indicate upregulated proteolysis of cross-linked fibrotic ECM, and net fibrinolysis can distinguish between patients based on the degree of fibrosis.

[0144] Resolution of fibrosis: For a long time, fibrosis was considered irreversible, but the understanding of fibrosis has changed in recent years and is now recognized as a dynamic process of fibroplasia and fibrinolysis. Novel antifibrotic agents, including targets such as LOXL2 / 3, are being developed 42 so the ultimate goal of fibrosis resolution is within reach. However, to optimize clinical management, there is a need for sensitive and specific tools that can, therefore, evaluate patients. Serological biomarkers of ECM turnover can provide non-invasive tools for this purpose, as demonstrated by the PRO-C3 biomarker that previously identified patients with a natural progressive fibrotic phenotype. 43、44 After 52 weeks, patients were divided according to whether their fibrotic phenotype regressed, remained stable, or progressed, and significant differences were observed in both CTX-III levels and net fibrinolysis levels between the regressive and progressive phenotypes at the time of screening. These data suggest the potential for prognostic diagnosis of biomarkers that patients with initial high levels of cross-linked type III collagen degradation, i.e., fibrinolysis, experience spontaneous resolution of fibrotic ECM compared to patients with lower fibrinolysis solubility.

[0145] Identification of regressors, results of treatment By calculating the cutoff values of the biomarkers, it was demonstrated in this study that patients with CTX-III levels of 3.8 ng / mL or higher, or fibrinolytic levels of 0.5 or higher at the time of screening, have a 19.4-fold and 23.3-fold higher likelihood of presenting a degenerative phenotype. Using the biomarker cutoff levels at the time of screening, natural regressors can be identified. Patients presenting spontaneous resolution of the fibrotic ECM may require lower treatment doses compared to patients with low initial fibrinolytic activity as determined by the biomarker. As a result, this leads to better stratification of patients in clinical trials, cost reduction, increased patient well-being, and the potential for assistance in treatment development.

[0146] Example 4 Eosinophilic Esophagitis Method: Patient demographics and clinical evaluation Twenty-nine adult EoE patients treated with a removal diet were included in the analysis. Dysphagia and total EREF scores were evaluated at baseline and after endoscopic intervention.

[0147] Table 14 shows the basic patient demographics of EoE patients at baseline and after intervention, including healthy donors. Demographics include age, gender, presence of dysphagia, and total eosinophilic esophagitis reference score (EREFS).

Table 12

[0148] Statistical analysis The statistical variance between patient demographics and patient clinical parameters at baseline and after intervention was determined by Fisher's exact test for two groups or chi-square test for multiple groups.

[0149] The calculation of the statistical difference between serum CTX-III of EoE patients and healthy donors at both time points was performed by one-way ANOVA applying Kruskal-Wallis for non-parametric data. A p-value of less than 0.05 was determined to be statistically significant.

[0150] Result: Cohort description: There was a significant difference in the mean age between EoE patients and healthy donors (p = 0.0039), with healthy donors being 9 years older on average. A significant decrease (p < 0.0001) was observed in the comparison of the total EREF score of EoE patients at baseline and after the elimination diet (Table 14).

[0151] The potential of CTX-III biomarker for differentiating EoE patients from healthy donors Serum CTX-III levels were significantly increased both at baseline and after the elimination diet (post-intervention) compared to healthy donors (p < 0.0001). No significant difference was shown between the baseline level and the post-intervention level (Figure 12).

[0152] Discussion: In a study of 29 EoE patients and healthy donors who had blood samples taken at baseline and 6 weeks after a elimination diet, the application of the CTX-III biomarker demonstrated an increase in serum CTX-III in EoE patients. At the position in the interstitial matrix, during fibrosis in EoE, the deposition of type III collagen mainly occurs in the subepithelial layer by activated myofibroblasts [46, 49]. In the final step of collagen maturation, a large amount of secreted cross-linking enzymes mediate the extensive formation of intramolecular and intermolecular cross-links. The ability of the highly cross-linked pathological collagen matrix of EoE patients to initiate myofibroblast differentiation of healthy donor fibroblasts clearly shows the importance of the ECM in the increase of EoE-related fibrosis

[59] . The clinical symptoms caused by esophageal fibrosis occur later than the actual onset of subepithelial fibrosis, and the risk doubles every 10 years of the disease period

[60] . Therefore, early evaluation of fibrotic extracellular matrix remodeling is important for the early initiation of treatment

[61] . In this specification, the quantification of the protease degradation metabolites of cross-linked type III collagen demonstrated the diagnostic potential of the CTX-III biomarker. The serum levels of CTX-III were significantly increased in patients diagnosed with EoE, and the biomarker provides a supportive diagnostic tool in EoE and may potentially also serve as a pharmacodynamic biomarker for EoE. Anti-fibrotic therapeutic agents or markers for monitoring the development of fibrotic strictures or the resolution of fibrosis are currently not available for EoE, but research on therapeutic agents targeting particularly important pro-inflammatory cytokines in the etiology of EoE is ongoing. Current therapeutic options include the administration of topical steroids and elimination diet

[62] . These two therapies have demonstrated a decrease in esophageal eosinophilia but have not yet demonstrated a decrease in fibrosis.

[0153] Conclusion: In current research, giving 6 weeks of elimination diet to EoE patients did not significantly affect serum CTX-III levels. Although no change in CTX-III biomarker levels was observed with short-term dietary intervention, the significantly elevated levels of proteolytically degraded and cross-linked type III collagen in EoE patients indicate the potential of this as a biomarker in type III collagen remodeling in EoE-related fibrosis.

[0154] Example 5 - Inflammatory Bowel Disease Methods: Patient demographics and pathological evaluation: Patients were evaluated and scored endoscopically at the time of blood sampling according to the simple endoscopic CD score (SES-CD). Patients with a SES-CD score of 0 - 1 were determined to be endoscopic inactive, while scores above 1 were determined to be endoscopic active. Additionally, when the SES-CD score was not available, the determination of inactive or active disease was based on the Harvey Bradshaw Index (HBI) score of the patients, which was determined by clinical parameters. HBI scores of 0 - 4 represent patients with clinically inactive disease, while scores above 4 were determined to be patients with clinically active disease.

[0155] Patients were further stratified by using the Montreal classification for the disease behavior of patients divided into either non-stricturing and non-penetrating disease (B1) or stricturing disease behavior (B2). Patients with a disease onset age of 16 years or younger and / or patients with a Montreal B4 classification for perianal disease behavior were excluded from the analysis.

[0156] Table 15: Summary of patient demographics and stratification according to endoscopic or clinical disease activity and endoscopic disease behavior

Table 13

[0157] Statistical analysis: The statistical variance between the patient demographics and the patient's pathological parameters in the B1 and B2 Montreal classifications was determined by Fisher's exact test.

[0158] The evaluation of plasma CTX-III or net fibrinolysis (log(CTX-III / PRO-C3)) between healthy donors and patients diagnosed with either CD or ulcerative colitis (UC) was performed by applying a t-test or one-way ANOVA depending on the number of comparison groups. Statistical differences were calculated by either the Kruskal-Wallis for non-parametric one-way ANOVA and either the Mann-Whitney or unpaired t-test for non-parametric and parametric data. A p-value of less than 0.05 was determined to be statistically significant.

[0159] Results: Patient demographics and pathological evaluation There were statistically more patients in the B1 group compared to the number of patients in B2 (p = 0.008). The age of patients with B1 was significantly younger than that of patients with B2 classification (p = 0.0118). Furthermore, patients with B1 classification had a significantly higher number of patients presenting symptoms around the anus (B4) compared to patients with B2 (p = 0.0026). No significant differences were shown between the remaining demographic parameters and the pathological parameters.

[0160] Plasma CTX-III increases in patients with chronic intestinal inflammation Patients with CD and UC showed significantly higher levels of plasma CTX-III compared to healthy donors (p < 0.0001). No statistical differences were seen between patients with CD and UC (Figure 13).

[0161] Differentiation of symptoms of luminal and stricturing diseases by quantification of plasma CTX-III Plasma CTX-III was significantly elevated in CD patients presenting inactive disease and non-stricturing and non-penetrating disease (B1) behavior. The levels were increased compared to patients with stricturing disease (B2) symptoms (p<0.01) (Figure 14A). Furthermore, by calculating net cross-linked fibrinolysis (log(CTX-III / PC3X)) or net fibrinolysis (log(CTX-III / PRO-C3)), patients with non-stricturing and non-penetrating disease (B1) demonstrated higher levels of fibrinolysis compared to patients with stricturing disease (B2) (p<0.05, and p<0.01) (Figure 14B+C).

[0162] Discussion: In this study, fibrinolysis in IBD patients was investigated by quantifying the levels of proteolytic metabolites of cross-linked type III collagen (CTX-III), and by either net cross-linked fibrinolysis by the CTX-III / PC3X ratio or net fibrinolysis by the CTX-III / PRO-C3 ratio. The main findings in this study were as follows: 1) CTX-III biomarker levels were significantly elevated in IBD patients compared to healthy donors (Figure 13), and 2) quantification of CTX-III or net cross-linked or non-cross-linked fibrinolysis (log(CTX-III / PC3X or PRO-C3)) levels could distinguish patients in clinical remission presenting luminal or structuring disease behavior (Figure 14).

[0163] The characteristic chronic inflammation of IBD that maintains activation of pathological wound healing is recognized as an important promoter of extensive ECM remodeling. The expression of fibrous collagen in CD patients was significantly increased compared to healthy individuals

[70] , and histological evaluation showed excessive deposition in different tissue layers of the intestine from the submucosa to the muscularis mucosa

[68] . Furthermore, both inflammatory cells and activated fibroblasts produce increased amounts of MMP, resulting in increased collagen degradation and, in severe cases, fistula formation.

[0164] Previous studies by Haaften et al.

[71] demonstrated the use of type III collagen biomarkers that reflect MMP-mediated degradation and formation when differentiating CD patients based on the endoscopic evaluation of disease behavior. Patients with stricturing disease were associated with increased levels of collagen formation markers that reflect excessive deposition of collagen in the tissue, while collagen degradation was increased in patients with penetrating disease

[71] .

[0165] In this specification, an increase in the level of fibrinolysis is demonstrated by quantifying the proteolytic metabolites of cross-linked type III collagen and evaluating the overall net fibrinolysis in CD patients with inactive disease presenting non-stricturing and non-penetrating disease behavior (B1). These patients are considered to have less severe disease symptoms compared to patients with stricturing disease (B2) and can be combined with inactive disease, suggesting a low degree of active inflammation. The enzymatic formation of intra- and intermolecular cross-links of fibrous collagen deposited in the interstitial matrix of CD patients represents the final step in collagen maturation. Therefore, the proteolysis of cross-linked type III collagen is associated with the fibrinolysis of mature collagen fibrils. Patients with stricturing disease presenting a large amount of collagen formation causing stricture formation and combined with extensive collagen cross-linking can suppress proteolysis. This is observed as a decrease in the fibrinolysis level demonstrated in this study. Thus, patients with non-stricturing and non-penetrating disease with increased type III collagen deposition have an increased degradation and clearance of pathological type III collagen deposition, despite potentially being cross-linked to a lesser extent. Therefore, these differences in the molecular processes of type III collagen remodeling between non-stricturing and non-penetrating disease and stricturing disease can be quantified by the use of the ratio of either the CTX-III biomarker and PC3X or PRO-C3 for net fibrinolysis.

[0166] Due to the limitations of endoscopic examination and histological evaluation of intestinal fibrosis, it can be proven beneficial in a clinical setting to include biomarkers such as CTX-III that reflect true fibrinolysis. By evaluating type III collagen remodeling at the molecular level with minimally invasive biomarkers, data can be provided to support endoscopy and histology. These biomarkers can identify subclinical disease behavior and provide subclinical information on treatment response. As treatment progresses in fibrotic strictures in CD, biomarkers such as CTX-III can be utilized for the evaluation of fibrosis resolution.

[0167] Conclusion: The data presented indicate an increase in the degree of proteolytic activity that releases cross-linked metabolites of type III collagen into the circulation of IBD patients. This was demonstrated for both CD and UC patients compared to healthy individuals. Furthermore, CD patients were stratified based on being in endoscopic and / or clinical remission (inactive), and then further stratified according to behavior of non-stricturing and non-penetrating disease (B1) or stricturing disease (B2) of the Montreal classification. Herein, patients with the B1 Montreal classification showed the highest fibrinolysis solubility compared to patients with the B2 classification.

[0168] Example 6 - Cancer Method: The assay procedures were performed as described above. These assays included CTX-III and PRO-C3.

[0169] The cohort included 20 patients each with cancers of the pancreas, colorectal, kidney, stomach, ovary, breast, bladder, lung, melanoma, head and neck, and prostate. It also included 3 patients with liver cancer and 33 healthy controls. All cancer samples were obtained from Proteogenex (Los Angeles, CA, USA), and healthy controls were obtained from BioIVT (Westbury, NY, USA).

[0170] Table 16 Cohort Demographics

Table 14

[0171] Result: Blood levels of protease cleavage fragments of cross-linked type III collagen in cancer Serum CTX-III in healthy individuals and patients diagnosed with cancer was found to be at significantly elevated levels in 7 out of 12 types of cancer when compared to healthy individuals. Biomarker levels were found to be elevated in bladder cancer (p<0.01), breast cancer (p<0.05), CRC (p<0.001), kidney cancer (p<0.05), lung cancer (p<0.05), pancreatic cancer (p<0.05), and gastric cancer (p<0.05).

[0172] Patients with H&N cancer, liver cancer, ovarian cancer, prostate cancer, and melanoma did not show significantly elevated levels of CTX-III compared to healthy individuals (p>0.05). However, the median levels of CTX-III in all 12 types of cancer were elevated compared to healthy individuals, and liver cancer showed the highest median level of 11.96 (Table 1).

[0173] Stage III breast cancer is associated with increased fibrinolysis When stratifying patients with breast cancer according to cancer stage, significantly elevated levels of CTX-III (p<0.001) were observed in patients with stage III breast cancer compared to patients with stage II. Furthermore, by calculating the net fibrinolysis using the ratio of CTX-III to PRO-C3, significantly higher levels (p<0.05) of net fibrinolysis were observed in patients with stage III compared to stage II (Figure 16).

[0174] Discussion The potential of the CTX-III biomarker in evaluating the degree of proteolysis of cross-linked type III collagen and the investigation of net fibrinolysis in various types of cancer were as follows: (1) The level of CTX-III was significantly elevated in 7 out of 12 types of cancer compared to healthy individuals, and (2) patients with stage III breast cancer exhibited higher serum CTX-III and net fibrinolysis compared to stage II patients.

[0175] Healthy individuals experience balanced ECM remodeling in which old collagen is degraded and replaced to maintain tissue homeostasis, but this process is severely distorted in the tumor stroma. In the tumor stroma, cells such as CAFs promote the formation of an increasingly rigid ECM through the deposition and cross-linking of mainly type I collagen, but also type II, III, V, and XI collagen. The major cause of the increased matrix rigidity is the amount of intra- and intermolecular cross-links within fibrillar collagen mediated by the enzymatic action of LOXL(L) and TG2

[80] .

[0176] Since Lys, which is implicated in LOX(L)-mediated cross-linking, is embedded within the CTX-III neoepitope

[81] , it is possible to specifically quantify the cross-link fragments released after the proteolysis of type III collagen. Thus, the increased release of MMPs, the deposition of type III collagen, and LOX(L)-mediated cross-linking that characterizes the tumor stroma are indicated by the increased levels of the CTX-III biomarker.

[0177] In line with this theory, in this study, an increase in the level of the CTX-III biomarker was observed, which can distinguish healthy individuals from cancer patients and identify individuals with underlying pathological degradation and cross-linking of type III collagen. However, among the 12 types of cancers investigated, H&N cancer, liver cancer, ovarian cancer, prostate cancer, and melanoma did not show significantly higher levels of fibrinolysis than healthy individuals. The levels in this study were not statistically different from the CTX-III levels of healthy individuals, but an increase was observed in the median CTX-III of cancer patients. This indicates an overall increase in the degree of proteolysis of cross-linked type III collagen in these patients. The lack of statistical distinction may be caused by the limited sample size, which is particularly true for the observation of liver cancer patients consisting of only three patients.

[0178] Furthermore, both the CTX-III biomarker level and the overall net fibrinolysis solubility (CTX-III / PRO-C3) were elevated in the later stages of breast cancer. These data indicate the use of the CTX-III biomarker in the diagnosis of cancer patients and its potential ability as a biomarker to stratify patients according to the severity of their diseases. Regarding fibrinolysis, a significant difference was observed between stage III and stage IV breast cancer patients.

[0179] Standardized cross-linked collagen fragments for the evaluation of pathological collagen remodeling in cancer patients were recently shown in a study by Christina Jensen et al. Here, the researchers examined the blood-based biomarker PC3X in the study of hepatocellular carcinoma together with the PRO-C3 biomarker

[82] . PRO-C3 quantifies the cross-linked and non-cross-linked N-terminal propeptides of type III collagen and reflects type III collagen formation, while the PC3X biomarker specifically targets the cross-linked N-terminal propeptide. The PC3X levels in hepatocellular carcinoma patients compared to PRO-C3 showed an increase in the level of cross-linked type III collagen, supporting an increase in the amount of collagen cross-linking in the tumor stroma.

[0180] In recent years, enzymatic cross-linking of collagen has gained interest in the field of cancer therapy

[83] . As described above, enzymes such as LOX(L) and TG2 promote an increase in the amount of cross-linking, but the biochemical nature of the cross-linking is also critically important. In particular, the enzymatic action of lysyl hydroxylase 2, which is expressed intracellularly and extracellularly, has been shown to promote metastasis and reduce survival. LH2 mediates the hydroxylation of specific Lys within the collagen α-chain, and the degree of cross-linking promoted by Lys hydroxylation is higher. Due to their important mechanistic role in enhancing tumor progression by governing matrix stiffness, LOXL2 and LH2 have been identified as targets for future therapeutic options

[84] .

[0181] Therefore, using blood-based biomarkers that specifically target the metabolites of cross-linked collagen can provide a quantitative measurement that reflects CAF activity and the enzymatic activity of cross-linking enzymes. In a clinical setting, biomarkers can potentially be used for diagnostic and prognostic purposes, to stratify patients based on fibrin solubility, and to identify patients for whom therapeutic options targeting collagen cross-linking would be beneficial.

[0182] Conclusion: Proteolytic fragments of cross-linked type III collagen that reflect fibrinolysis were demonstrated to be released and quantifiable in 12 types of cancer, with median levels elevated in all types of cancer compared to healthy individuals. Although elevated in cancer patients, CTX-III levels were found to be significantly elevated in only 7 types of cancer. Furthermore, quantification of cross-linked type III collagen fibrinolysis enabled the discrimination of breast cancer patients at either stage II or stage III, with elevated levels being associated with late-stage breast cancer.

[0183] The CTX-III biomarker can be used for the quantification of cross-linked type III collagen fragments released into the circulation after proteolysis, and it can thus be concluded that it can be used in the clinical setting of cancer patients.

[0184] Summary The development and validation of a highly neoepitope-specific ELISA capable of measuring cross-linked fragments of type III collagen was demonstrated. This assay was able to distinguish between HD and obese patients with NAFLD, HD and patients with liver fibrosis, HD and patients with EoE, HD and patients with chronic intestinal diseases, and HD and cancer patients, and showed the relevance of the CTX-III marker as a disease marker in conditions associated with known accumulation of type III collagen and increased levels of cross-linking enzymes.

[0185] Furthermore, the calculation of the net fibrinolysis ratio (CTX-III / PRO-C3) using the CTX-III biomarker and the PRO-C3 biomarker demonstrated increased levels in HCV-related liver fibrosis by its ability to distinguish patients according to their natural fibrosis phenotypes. The calculation of net fibrinolysis was also able to distinguish patients according to the severity of those diseases in chronic intestinal diseases, particularly Crohn's disease, and cancers such as breast cancer. Therefore, the CTX-III biomarker and the associated net fibrinolysis ratio can not only identify patients with HCV-related liver fibrosis, chronic intestinal diseases or cancer, but can also be applied as prognostic biomarkers potentially predicting the response at the screening time point.

[0186] As used herein, unless expressly stated otherwise, the word "or" is used in the sense of an operator that returns a true value if either or both of the stated conditions are met, as opposed to the "exclusive or" operator that requires only one of the conditions to be met. The word "comprising" is used in the sense of "including" rather than in the sense of "consisting of". All prior teachings previously recognized are incorporated herein by reference. No admission of any prior art document published herein should be construed as an admission or representation that the teaching is part of the common general knowledge in Australia or anywhere else as of the date of this document.

[0187] The following references are cited herein: TIFF0007699608000018.tif242162TIFF0007699608000019.tif242162TIFF0007699608000020.tif242162TIFF0007699608000021.tif242162TIFF0007699608000022.tif242162TIFF0007699608000023.tif242162TIFF0007699608000024.tif242162TIFF0007699608000025.tif242162TIFF0007699608000026.tif242162TIFF0007699608000027.tif227162

Claims

1. A monoclonal antibody that specifically recognizes and binds to a peptide having a C-terminal amino acid sequence KAGGFAYYYG (SEQ ID NO: 1).

2. The monoclonal antibody according to claim 1, wherein the monoclonal antibody is a monoclonal antibody produced against a synthetic peptide having a C-terminal amino acid sequence KAGGFAYYYG (SEQ ID NO: 1).

3. The monoclonal antibody according to claim 1 or claim 2, wherein the antibody does not specifically recognize or bind to a peptide having a C-terminal amino acid sequence KAGGFAYYGX (SEQ ID NO: 2), wherein X represents any amino acid.

4. The monoclonal antibody according to any one of claims 1 to 3, wherein the antibody does not specifically recognize or bind to a peptide having a C-terminal amino acid sequence KAGGFAYYGD (SEQ ID NO: 4).

5. The monoclonal antibody according to any one of claims 1 to 4, wherein the antibody does not specifically recognize or bind to a peptide having a C-terminal amino acid sequence KAGGFAYY (SEQ ID NO: 5).

6. A sandwich immunoassay for detecting cross-linked CT-III in a biological sample, wherein the cross-linked CT-III comprises at least two chains of CT-III covalently linked by an interchain cross-link, the sandwich immunoassay comprising the step of contacting the biological sample containing the cross-linked CT-III with a first monoclonal antibody bound to a surface, wherein each chain of CT-III contained in the cross-linked CT-III comprises a C-terminal neoepitope of CT-III generated by C-protease cleavage of intact type III collagen; the step of adding a second monoclonal antibody; and the step of determining the amount of binding of the second monoclonal antibody comprising, a sandwich immunoassay, wherein both the first monoclonal antibody and the second monoclonal antibody specifically react with the C-terminal neoepitope of CT-III, and the neoepitope is contained in the C-terminal amino acid sequence KAGGFAYYG-COOH (SEQ ID NO: 1).

7. The sandwich immunoassay according to claim 6, wherein the monoclonal antibody is the monoclonal antibody according to any one of claims 1 to 5.

8. The sandwich immunoassay according to claim 6 or 7, which is used to quantify the amount of cross-linked CT-III in a biological sample.

9. The sandwich immunoassay according to any one of claims 6 to 8, wherein the biological sample is a biological fluid.

10. The sandwich immunoassay according to claim 9, wherein the biological fluid is serum, plasma, urine, amniotic fluid, tissue supernatant or cell supernatant.

11. The sandwich immunoassay according to any one of claims 6 to 10, wherein the sandwich immunoassay is a radioimmunoassay, a fluorescence immunoassay, or an enzyme-linked immunosorbent assay.

12. The sandwich immunoassay according to any one of claims 6 to 11, wherein the second monoclonal antibody is labeled.

13. The sandwich immunoassay according to claim 12, wherein the second monoclonal antibody is an enzyme-conjugated antibody.

14. The sandwich immunoassay according to claim 13, wherein the enzyme is horseradish peroxidase (HRP).

15. The sandwich immunoassay according to claim 14, wherein the second monoclonal antibody is radiolabeled or linked to a fluorophore.

16. The sandwich immunoassay according to any one of claims 6 to 10, wherein the binding amount of the second monoclonal antibody is determined using a further labeled antibody that recognizes the second monoclonal antibody.

17. The sandwich immunoassay according to any one of claims 8 to 16, further comprising correlating the amount of cross-linked CTIII determined by the sandwich immunoassay with a standard disease sample of the severity of a known disease to evaluate the severity of the disease.

18. Quantifying the amount of PRO-C3 present in the biological sample, Determining the ratio of cross-linked type III collagen (CTX-III) to the N-terminal propeptide of type III collagen (PRO-C3), The sandwich immunoassay according to any one of claims 8 to 16, further comprising.

19. The sandwich immunoassay according to claim 18, further comprising correlating the ratio of cross-linked type III collagen (CTX-III) to the N-terminal propeptide of type III collagen (PRO-C3) determined by the sandwich immunoassay with a standard disease sample of the severity of a known disease to evaluate the severity of the disease.

20. The sandwich immunoassay according to claim 17 or claim 19, wherein the disease is a fibrotic disease.

21. The sandwich immunoassay according to claim 20, wherein the fibrotic disease is a liver disease.

22. The sandwich immunoassay according to claim 21, wherein the liver disease is non-alcoholic fatty liver disease or HCV-related liver disease.

23. The sandwich immunoassay according to claim 17 or claim 19, wherein the disease is a chronic intestinal disease or cancer.

24. The sandwich immunoassay according to claim 23, wherein the chronic intestinal disease is Crohn's disease or ulcerative colitis.

25. The sandwich immunoassay according to claim 23, wherein the cancer is breast cancer, bladder cancer, colorectal cancer, head and neck cancer, kidney cancer, lung cancer, pancreatic cancer, stomach (gastric) cancer, ovarian cancer, liver cancer, prostate cancer, or melanoma.

26. A method for evaluating the efficacy of an antagonist drug targeting lysyl oxidase (LOX), comprising quantifying the amount of cross-linked CT-III in at least two biological samples using the sandwich immunoassay according to any one of claims 6 to 16, wherein the biological samples are obtained from the subject at an initial time point during the administration period of the antagonist drug to the subject and at least one subsequent time point, and a decrease in the amount of cross-linked CT-III from the initial time point to the at least one subsequent time point during the administration period of the antagonist drug indicates an effective antagonist drug targeting LOX.

27. The method according to claim 26, wherein the method evaluates the efficacy of an antagonist drug targeting LOXL2.

28. A kit for use in a sandwich immunoassay, comprising: a solid support bound to the first monoclonal antibody according to claim 1; and the second monoclonal antibody according to claim 1 wherein the second monoclonal antibody comprises a label.

29. A method for identifying the fibrosis response phenotype of a patient having fibrosis, comprising using the sandwich immunoassay according to any one of claims 8 to 19 to quantify the amount of cross-linked CT-III in a biological fluid sample obtained from the patient, and correlating the amount of the cross-linked CT-III with i) a value associated with a known fibrotic response phenotype and / or ii) a predetermined cut-off value.

30. Quantifying the amount of the N-terminal propeptide (PRO-C3) of type III collagen present in a biological fluid sample, Determining the ratio of cross-linked type III collagen (CTX-III) to the N-terminal propeptide (PRO-C3) of type III collagen, and Correlating the ratio of cross-linked type III collagen (CTX-III) to the N-terminal propeptide (PRO-C3) of type III collagen with a predetermined cut-off value, The method according to claim 29, further comprising.

31. The method according to claim 29 or claim 30, wherein the cut-off value is at least 3.8 ng / mL of cross-linked CT-III and / or the ratio of cross-linked type III collagen (CTX-III) to the N-terminal propeptide (PRO-C3) of type III collagen is at least 0.

5.

32. A method for identifying a patient having eosinophilic esophagitis, comprising using the sandwich immunoassay according to any one of claims 8 to 19 to quantify the amount of cross-linked CT-III in a biological fluid sample obtained from the patient, and correlating the amount of the cross-linked CT-III with i) a value associated with a known eosinophilic esophagitis patient and / or a normal healthy control and / or ii) a predetermined cut-off value.

33. A method for identifying a patient having a chronic intestinal disease, comprising using the sandwich immunoassay according to any one of claims 8 to 19 to quantify the amount of cross-linked CT-III in a biological fluid sample obtained from the patient, and correlating the amount of the cross-linked CT-III with i) a value associated with a known patient having a chronic intestinal disease and / or a normal healthy control and / or ii) a predetermined cut-off value.

34. A method for identifying a patient having cancer, comprising quantifying the amount of cross-linked CT-III in a biological fluid sample obtained from the patient using the sandwich immunoassay according to any one of claims 8 to 19, and correlating the amount of the cross-linked CT-III with i) a value associated with known cancer patients and / or normal healthy controls and / or ii) a predetermined cut-off value.

35. A method for identifying a patient having a fibrotic disease, comprising quantifying the amount of cross-linked CT-III in a biological fluid sample obtained from the patient using the sandwich immunoassay according to any one of claims 8 to 19, and correlating the amount of the cross-linked CT-III with i) a value associated with known fibrotic disease patients and / or normal healthy controls and / or ii) a predetermined cut-off value.

36. A method for identifying a patient who benefits from treatment, comprising quantifying the amount of cross-linked CT-III in a biological fluid sample obtained from the patient using the sandwich immunoassay according to any one of claims 8 to 19, and correlating the amount of the cross-linked CT-III with i) a value associated with known disease patients and / or normal healthy controls and / or ii) a predetermined cut-off value.

37. The method according to claim 36, wherein the treatment comprises administration of a drug targeting collagen cross-linking.

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