Type XXIII collagen assay

Abstract

The present invention provides monoclonal antibodies targeting type XXIII collagen, and immunoassays and kits for detecting and quantifying epitopes using the antibodies. The present invention also provides a method for identifying and monitoring subjects with inflammatory bowel disease.

Description

Technical Field

[0001] The present invention relates to a monoclonal antibody targeting type XXIII collagen, and an immunoassay and a kit using the antibody. Background Art

[0002] Crohn's disease (CD) and ulcerative colitis (UC) are two major gastrointestinal diseases of inflammatory bowel disease (IBD) that share similar symptoms, such as increased inflammation and structural damage to the intestine. CD can affect the entire gastrointestinal tract, while UC is primarily confined to the colonic mucosa[1]. The etiology of IBD is not fully understood, but it is believed to have a genetic basis and an abnormal response of the immune system to environmental factors[2].

[0003] The epithelium of a healthy intestine is covered by a single layer of enterocytes that form a protective barrier against bacteria and other foreign substances including antigens and toxins and are selectively permeable only to dietary nutrients [3,4]. The epithelium is important for maintaining intestinal health. However, in CD and UC, intestinal permeability is impaired, resulting in a large number of bacteria invading the intestinal tissue from the intestinal lumen and increased influx of immune cells from the bloodstream into the tissue [5,6], leading to chronic gastrointestinal inflammation in IBD patients [7]. The tight connections between intestinal epithelial cells are mainly regulated by junctional complexes, which are composed of tight junctions, adherens junctions, and desmosomes [6]. Many studies have shown that some junctional proteins are significantly downregulated in inflamed intestinal tissues in IBD [8-11], which may be the main reason for the loss of intestinal permeability in IBD.

[0004] Type XXIII collagen is a member of the type II transmembrane proteins and was first discovered by Jacqueline Banyard et al. in rat metastatic tumor cells in 2003

[12] . In human non-small cell lung cancer cell lines and clear cell renal cell carcinoma cell lines, type XXIII collagen was found to be associated with cell adhesion and metastasis [13,14]. Knockout of type XXIII collagen in these cell lines resulted in altered expression of cell adhesion molecules and impaired cell adhesion [13,14], suggesting that type XXIII collagen may be a regulator of cell adhesion. Type XXIII collagen is not only expressed in cancer cells. In staining of embryonic mouse intestinal sections, type XXIII collagen was found to be expressed on the epithelial surface of the intestine, suggesting that it may play an important role in cell interactions

[15] .

[0005] Type XXIII collagen consists of a short cytoplasmic domain, a transmembrane domain, and a long extracellular domain

[12] . The extracellular domain has several collagen domains and is truncated by short non-collagenous domains

[12] . Newly synthesized type XXIII collagen can be transported to the cell surface as a transmembrane protein or cleaved intracellularly by furin, releasing the extracellular domain into the extracellular matrix (ECM)

[16] . However, the function of the cleavage is still unclear. Studies have shown that type XXIII is upregulated in a range of cancers[17-19] and can be used as a potential biomarker for prostate cancer

[18] , non-small cell lung cancer

[17] , and clear cell renal cell carcinoma

[14] .

[0006] However, the use of type XXIII collagen as a biomarker in other diseases has not yet been established.

[0007] US 7,993,863 B (Zetter et al.) describes a collagen-like gene (CLG) product that is expressed in human prostate and breast cancer cell lines, but not in normal adult, placental, lung, liver, skeletal muscle, kidney or pancreatic tissue. A C-terminal non-collagenous region having the sequence LDQPCPVGPDGLPVPGCWHK (expressed as SEQ ID No. 14) is also described. The authors note that this sequence has a high degree of identity with transmembrane collagens XMII and XXV and therefore conclude that it is likely a poor epitope.

[0008] There is evidence that intestinal permeability is increased in patients with active IBD compared with inactive disease

[20] . Junctional / adhesion proteins, such as E-cadherin and β-catenin, are significantly downregulated in actively inflamed tissues of patients with IBD [8]. Therefore, methods to assess intestinal permeability could be used to assess disease burden [5]. However, only a few non-invasive biomarkers are available. Fecal calprotectin, a protein expressed primarily by neutrophils, is the most promising biomarker in IBD. It correlates with endoscopic disease activity

[21] , can predict relapse

[22] , and monitor response to treatment

[23] . However, fecal calprotectin only measures intestinal inflammation, not tissue damage directly, as calprotectin is a small calcium-binding protein found primarily in neutrophils. Claudin-3 is a tight junction protein that can be measured in urine and may reflect loss of tight junctions

[24] . Information on this biomarker is limited and further studies in humans are needed. Therefore, there remains a great need for non-invasive biomarkers for the assessment of intestinal permeability in IBD. Summary of the invention

[0009] Since type XXIII collagen is expressed in the intestinal epithelial layer, it may be cleaved from the cell surface during epithelial damage in IBD. In addition, since type XXIII collagen plays an important role in cell adhesion, its levels may be modulated and contribute to changes in cell adhesion in IBD, and therefore could be used as a biomarker.

[0010] The present inventors have developed a monoclonal antibody that specifically recognizes the extracellular domain of type XXIII collagen, in particular the C-terminus of the α chain, and an immunoassay, in particular an enzyme-linked immunosorbent assay (ELISA) for detecting the extracellular domain of type XXIII collagen in a biological fluid sample. The present inventors have determined that type XXIII collagen can be used as a biomarker for changes in cell adhesion and provide new diagnostic information for patients with inflammatory bowel diseases such as Crohn's disease (CD) and ulcerative colitis (UC).

[0011] Therefore, in a first aspect, the present invention relates to a monoclonal antibody which specifically recognizes and binds to the C-terminus of type XXIII collagen α1 chain (also referred to herein as target peptide), which has the amino acid sequence GLPVPGCWHK (SEQ ID No. 1) (also referred to herein as target sequence).

[0012] Preferably, the monoclonal antibody is a monoclonal antibody raised against a synthetic peptide having a C-terminal amino acid sequence of GLPVPPGCWHK (SEQ ID No. 1). The synthetic peptide used to raise the antibody may be a synthetic peptide linked to a carrier protein at its N-terminus. Exemplary carrier proteins include proteins such as, but not limited to, keyhole limpet hemocyanin (KLH). The synthetic peptide may be linked to the carrier protein by any suitable bond, which may include one or more additional amino acid residues at the N-terminus of the peptide. The monoclonal antibody may be raised by suitable techniques known to those skilled in the art, such as, but not limited to, immunizing a mouse or other mammal, isolating splenocytes from the immunized mammal and fusing them with hybridoma cells, and then culturing the resulting hybridoma cells to allow monoclonal growth.

[0013] In a preferred embodiment, the monoclonal antibody does not specifically recognize or bind to a peptide having a C-terminal amino acid sequence of GLPVPPGCWHKX (SEQ ID No. 2), wherein X represents any amino acid. Therefore, the monoclonal antibody preferably does not specifically recognize or bind to an extended variant of the target peptide in which the target amino acid sequence is extended by one or more amino acids at the C-terminus.

[0014] In a preferred embodiment, the monoclonal antibody does not specifically recognize or bind to a peptide having the C-terminal amino acid sequence GLPVPGCWH (SEQ ID No. 3). Therefore, the monoclonal antibody preferably does not specifically recognize or bind to a shortened variant of the target peptide in which the target amino acid sequence is truncated by one or more amino acids at the C-terminus.

[0015] In a preferred embodiment, the monoclonal antibody does not specifically recognize or bind to a peptide having the C-terminal amino acid sequence GLPVQGCWNK (SEQ ID No. 4). Therefore, the monoclonal antibody preferably does not specifically recognize or bind to a peptide from type XIII collagen.

[0016] In a preferred embodiment, the monoclonal antibody does not specifically recognize or bind to a peptide having the C-terminal amino acid sequence GLPMPGCWQK (SEQ ID No. 5). Therefore, the monoclonal antibody preferably does not specifically recognize or bind to a peptide from type XXV collagen.

[0017] The monoclonal antibody or fragment thereof may preferably comprise one or more complementarity determining regions (CDRs) selected from:

[0018] CDR-H1: SYAMS (SEQ ID No. 6)

[0019] CDR-H2:SISTAGRTYYPDTVR(SEQ ID No.7)

[0020] CDR-H3:PDYDYDGYIN(SEQ ID No.8)

[0021] CDR-L1:RSSKSLLHSNGVTYLY(SEQ ID No.9)

[0022] CDR-L2: QMSNLAS (SEQ ID No.10) and

[0023] CDR-L3: AQNLELLPLT (SEQ ID No. 11).

[0024] Preferably, the antibody or fragment thereof comprises at least 2, 3, 4, 5 or 6 of the CDR sequences listed above.

[0025] Preferably, the monoclonal antibody or fragment thereof has a light chain variable region comprising the following CDR sequences:

[0026] CDR-L1:RSSKSLLHSNGVTYLY(SEQ ID No.9)

[0027] CDR-L2: QMSNLAS (SEQ ID No.10) and

[0028] CDR-L3: AQNLELLPLT (SEQ ID No. 11).

[0029] Preferably, the monoclonal antibody or fragment thereof has: a light chain comprising a framework sequence between the CDRs, wherein the framework sequence is substantially identical or substantially similar to the framework sequence between the CDRs in the following light chain sequence (wherein the CDRs are shown in bold and underlined, and the framework sequence is shown in italics):

[0030]

[0031] Preferably, the monoclonal antibody or fragment thereof has a heavy chain variable region comprising the following CDR sequences:

[0032] CDR-H1: SYAMS (SEQ ID No. 6)

[0033] CDR-H2:SISTAGRTYYPDTVR(SEQ ID No.7)

[0034] CDR-H3: PDYDYDGYIN (SEQ ID No. 8).

[0035] Preferably, the monoclonal antibody or fragment thereof has: a heavy chain comprising a framework sequence between the CDRs, wherein the framework sequence is substantially identical or substantially similar to the framework sequence between the CDRs in the following light chain sequence (wherein the CDRs are shown in bold and underlined, and the framework sequence is shown in italics):

[0036]

[0037] As used herein, if the framework amino acid sequence between the CDRs of one antibody has at least 70%, 80%, 90% or at least 95% similarity or identity with the framework amino acid sequence between the CDRs of another antibody, they are substantially identical or substantially similar. Similar or identical amino acids can be continuous or discontinuous.

[0038] The framework sequence may contain one or more amino acid substitutions, insertions and / or deletions. Amino acid substitutions may be conservative, meaning that the substituted amino acid has chemical properties similar to the original amino acid. A skilled person would understand which amino acids have similar chemical properties. For example, the following groups of amino acids have similar chemical properties, such as size, charge, and polarity: Group 1 Ala, Ser, Thr, Pro, Gly; Group 2 Asp, Asn, Glu, Gln; Group 3 His, Arg, Lys; Group 4 Met, Leu, Ile, Val, Cys; Group 5 Phe, Thy, Trp.

[0039] Amino acid sequences can be compared using programs such as the CLUSTAL program. The program compares amino acid sequences and finds the best alignment by inserting spaces appropriately in either sequence. Amino acid identity or similarity (identity plus conservation of amino acid types) can be calculated to obtain the best alignment. Programs like BLASTx will align the longest stretch of similar sequences and assign a value to the fit. Thus, a comparison can be made where there are several similar regions, each with a different score. The present invention contemplates two types of analysis. Identity or similarity is preferably calculated over the entire length of the framework sequence.

[0040] In certain preferred embodiments, the monoclonal antibody or fragment thereof may comprise a light chain variable region sequence:

[0041]

[0042] And / or heavy chain variable region sequence:

[0043]

[0044] (CDRs are bold and underlined; framework sequences are italicized).

[0045] In a second aspect, the present invention relates to an immunoassay method for detecting type XXIII collagen in a human biological fluid sample, the method comprising contacting the human biological fluid sample with a monoclonal antibody according to the first aspect of the invention, and detecting binding between the monoclonal antibody and a peptide in the sample.

[0046] Preferably, the detection is quantitative. Thus, the method may comprise detecting and determining the amount of binding between the monoclonal antibody and the peptide in the sample.

[0047] Preferably, the immunoassay is a competitive immunoassay.

[0048] Preferably, the immunoassay is an enzyme-linked immunosorbent assay (ELISA).Preferably, the ELISA is a competitive ELISA.

[0049] The human biological fluid sample can be, for example, blood, serum, plasma or urine. Preferably, the sample is serum or plasma.

[0050] The human biological fluid sample may be a sample from a human patient having medical signs or symptoms indicative of inflammatory bowel disease. Preferably, the biological fluid sample is a sample from a human patient having medical signs or symptoms indicative of Crohn's disease (CD) or ulcerative colitis (UC). Preferably, the biological fluid sample is a sample from a human patient having medical signs or symptoms indicative of active inflammatory bowel disease, such as active Crohn's disease (CD) or active ulcerative colitis (UC).

[0051] The method may be an immunoassay for diagnosing and / or monitoring and / or assessing the likelihood of an inflammatory bowel disease in a patient, the method comprising contacting a biological fluid sample obtained from the patient with a monoclonal antibody, detecting and determining the amount of binding between the monoclonal antibody and a peptide in the sample, and correlating the amount of binding with a value associated with a normal healthy subject and / or a value associated with a known disease severity and / or a value obtained from the patient at a previous time point. Preferably, the inflammatory bowel disease is Crohn's disease or ulcerative colitis. Preferably, the inflammatory bowel disease is an active inflammatory bowel disease, such as active Crohn's disease (CD) or active ulcerative colitis (UC).

[0052] In a third aspect, the present invention relates to an assay kit comprising a monoclonal antibody according to the first aspect of the invention and at least one of:

[0053] - Streptavidin-coated well plates;

[0054] - an N-terminal biotinylated peptide having a C-terminal amino acid sequence of GLPVPGCWHK (SEQ ID No. 1); and

[0055] - A calibrator peptide having the C-terminal amino acid sequence GLPVPGCWHK (SEQ ID No. 1).

[0056] The kit can be used to diagnose or predict the risk of inflammatory bowel disease, preferably in combination with the method according to the second aspect of the invention.

[0057] Preferably, the inflammatory bowel disease is Crohn's disease or ulcerative colitis. Preferably, the inflammatory bowel disease is an active inflammatory bowel disease, such as active Crohn's disease or active ulcerative colitis.

[0058] definition

[0059] As used herein, the terms "peptide" and "polypeptide" are used synonymously.

[0060] As used herein, the term "monoclonal antibody" refers to an intact antibody and fragments thereof that retain the binding specificity of the intact antibody, such as a Fab fragment, a Fv fragment, or other such fragments known to those skilled in the art. Antibodies that retain the same binding specificity may contain the same complementary determining regions (CDRs). The CDRs of an antibody may be determined using methods known in the art, such as the method described by Rabat et al.

[28] .

[0061] Antibodies can be produced from B cell clones as described in the Examples. The isotype of the 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 isotypes.

[0062] The amino acid sequence of the antibody produced can be determined using standard techniques. For example, RNA can be isolated from cells and used to produce cDNA by reverse transcription. Then cDNA is subjected to PCR using primers for amplifying the heavy and light chains of the antibody. For example, primers specific to the leader sequence of all VH (variable heavy chain) sequences can be used together with primers previously determined to bind to sequences in the constant region of the isotype. The light chain can be amplified using primers that bind to the 3' end of the κ chain or λ chain and primers that anneal to the Vκ or Vλ leader sequence. Full-length heavy and light chains can be generated and sequenced.

[0063] As used herein, the term "C-terminus" refers to the end of a polypeptide, i.e., the end at the C-terminus of a polypeptide, and should not be interpreted as meaning in its general direction. Similarly, the term "N-terminus" refers to the end of a polypeptide, i.e., the end at the N-terminus of a polypeptide, and should not be interpreted as meaning in its general direction.

[0064] As used herein, the term "competitive immunoassay" refers to an immunoassay in which a target peptide (if any) present in a sample competes with a known amount of the target peptide (the target peptide is, for example, bound to a fixed substrate or labeled) for binding to an antibody, a technique known to those skilled in the art.

[0065] As used herein, the term "ELISA" (enzyme-linked immunosorbent assay) refers to an immunoassay in which an antibody linked to an enzyme (e.g., horseradish peroxidase or alkaline phosphatase) is used to detect a target peptide present in a sample (if any). The activity of the enzyme is then assessed by incubation with a substrate that produces a measurable product. The presence and / or amount of the target peptide in the sample can thereby be detected and / or quantified. ELISA is a technique known to those skilled in the art.

[0066] As used herein, the term "binding amount" refers to the quantification of the binding between the monoclonal antibody and the target peptide, which is determined by comparing the measured value of the target peptide in the biological fluid sample with a calibration curve, wherein the calibration curve is generated using a standard sample of the target peptide with a known concentration. In the specific assay disclosed herein for measuring a target peptide having a C-terminal amino acid sequence GLPVPPGCWHK (SEQ ID No. 1) in a biological fluid, the calibration curve is generated using a standard sample of a known concentration of a calibration peptide, which has a C-terminal amino acid sequence GLPVPPGCWHK (SEQ ID No. 1, and can specifically consist of the amino acid sequence GLPVPPGCWHK (SEQ ID No. 1)). The value measured in the biological fluid sample is compared with the calibration curve to determine the actual amount of the target peptide in the sample.

[0067] As used herein, the term "PRO-C23" refers to the extracellular domain of type XXIII collagen having a C-terminal amino acid sequence of GLPVPGCWHK (SEQ ID No. 1). BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Figure 1 : PRO-C23 antibody 10F6 specificity. A) Sequence alignment of the C-termini of collagen types XIII, XXIII and XXV. The antibody recognizes residues 531 to 540 of collagen type XXIII. B) Specificity of PRO-C23 antibody to different peptides. Reactivity to selected peptides (GLPVPGCWHK (SEQ ID No. 1)), extended peptides (GLPVPGCWHKA (SEQ ID No. 16)), truncated peptides (GLPVPGCWH), peptides from collagen type XIII (GLPVQGCWNK (SEQ ID No. 4)) and peptides from collagen type XXV (GLPMPGCWQK (SEQ ID No. 5)) were tested in the PRO-C23 assay. C) Western blot results of recombinant collagen type XXIII using 10F6 as primary antibody.

[0069] Figure 2 :Correlation of PRO-C23 levels in three plasma and serum (N=16)

[0070] Figure 3 : PRO-C23 levels in the rat DSS model. A) PRO-C23 serum levels in control rats (n=8) and DSS rats with DAI≥5 (n=8). B) PRO-C23 serum levels increased upon induction of colitis and returned to baseline upon reversal of intestinal inflammation. Data are presented as median ± 95% confidence interval. *Asterisks (*) represent statistically significant differences. *P<0.05.

[0071] Figure 4 : PRO-C23 levels in human cohort 1. Data are presented as median ± 95% confidence interval. *Asterisk (*) indicates statistically significant difference. *P<0.05.

[0072] Figure 5 : PRO-C23 levels in human cohorts 2 (CD) and 3 (UC). Data are presented as median ± 95% confidence interval. *Asterisks (*) represent statistically significant differences. *(p<0.05)**(p<0.01,)***(p<0.001) DETAILED DESCRIPTION

[0073] Example

[0074] Various embodiments are described and disclosed in the following examples, which are presented to aid in understanding the present disclosure, and which should not be construed as limiting in any way the scope of the present invention as defined in the claims that follow. The following examples are presented to provide a complete disclosure and description of how to make and use the embodiments to those of ordinary skill in the art, and are not intended to limit the scope of the present disclosure, nor are they intended to represent that the following experiments are all or the only experiments performed. Efforts have been made to ensure the accuracy of the values ​​used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should be considered. Unless otherwise stated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is degrees Celsius, and pressure is atmospheric pressure or near atmospheric pressure.

[0075] method

[0076] Antibody Development of PRO-C23

[0077] The last 10 amino acids of the α1 chain of type XXIII collagen ( 531 'GLVPGCWHK' 540 , (SEQ ID No.1) Genscript, USA) was used as an immunogenic peptide to generate specific monoclonal antibodies. 4-6 week old Balb / C mice were subcutaneously immunized with 100 μg of immunogen (KLH-CGG-GLPVPGCWHK (SEQ ID No.1)) emulsified with Stimmune adjuvant (Thermo Fisher, USA). Continuous immunizations were performed at intervals of 2 weeks. Mice with the highest antiserum titer and best peptide reactivity were selected for fusion. Mouse spleen cells were fused with SP2 / 0 myeloma cells. The fused cells were cultured (raised) in 96-well plates and incubated in CO 2 Incubate in an incubator. Select cell lines that are specific for the selected peptide but have no cross-reactivity to the extended peptide (GLPVPGCWHKA (SEQ ID No.16)), the truncated peptide (GLPVPGCWH), and the deselected peptide (type XIII collagen GLPVQGCWNK (SEQ ID No.4), type XXV collagen GLPMPGCWQK (SEQ ID No.5)) (Genscript, USA) and perform subcloning. Finally, use an IgG column (GE health, USA) to purify the antibody.

[0078] The antibodies generated were sequenced and the CDRs were determined.

[0079] The sequence of the chain is as follows:

[0080] CDR is bold and underlined

[0081] Constant regions are in italics:

[0082] Heavy chain: amino acid sequence (454aa)

[0083]

[0084] Light chain: amino acid sequence (219aa)

[0085]

[0086] PRO-C23 Assay and Technology Evaluation

[0087] The ELISA plates used for assay development were coated with streptavidin from Roche (Catalog No.: 11940279). All ELISA plates were analyzed using an ELISA plate reader from Molecular Devices, SpectraMax M, (CA, USA). The selected monoclonal antibodies were labeled with horseradish peroxidase (HRP) using the Lightning link HRP labeling kit according to the manufacturer's instructions (Innovabioscience, Babraham, Cambridge, UK). The 96-well streptavidin plates were coated with biotin-GLPVPGCWHK (SEQ ID No. 1) (Genscript, USA) and incubated at 20°C for 30 minutes. 20 μL of standard peptides or samples were added to the appropriate wells, followed by 100 μL of HRP-conjugated monoclonal antibody 10F6, and incubated at 4°C for 20 hours. Finally, 100 μL of tetramethylbenzidine (TMB) (Kem-En-Tec, catalog number 4380H) was added and the plate was incubated at 20°C for 15 minutes in the dark. All of the above incubation steps included shaking at 300 rpm. After each incubation step, the plate was washed five times. The plate was then washed once more by adding 100 μL of stop solution (1% H 2 SO 4 ) to stop the TMB reaction and measure at 450 nm with 650 nm as reference.

[0088] The lower limit of detection (LLOD) is determined by 21 zero samples (i.e., buffer) and is calculated as mean + 3x standard deviation. The upper limit of detection (ULOD) is determined as the mean - 3xSD of 10 measurements of standard A. The intra- and inter-determination variation is the average variation of 10 QC samples independently repeated 10 times. The dilution recovery is determined in 4 serum samples and 4 plasma samples and is calculated as the percentage of the diluted sample recovered from 100% sample. The correlation between serum and plasma is determined in serum and matching plasma heparin, plasma citrate, plasma EDTA from 16 individuals (Innovative Research, USA).

[0089] Western blotting using recombinant human type XXIII collagen

[0090] Recombinant human type XXIII collagen (R&D system, 4165-CL) was diluted in a sample buffer containing 80mM DTT and run on a 10% SDS-PAGE gel and then transferred to a nitrocellulose membrane. The nitrocellulose membrane was then blocked for nonspecific binding by incubating in TBS-T containing 5% skim milk powder for 1 hour at room temperature. Then incubate overnight with 1 μg / ml10F6 or commercial type XXIII collagen antibody (R&D system, MAB4165) diluted in TBS-T milk. The membrane was then washed 3 times in TBS-T and then incubated in a peroxidase-conjugated secondary antibody. Finally, the membrane was washed 3 times in TBS-T and the results were observed using an ECL system (GE Healthcare, catalog number RPN2109).

[0091] DSS rat model

[0092] Male SD (Sprague-Dawley) rats aged 12 weeks were divided into 2 groups: a 6% DSS group (12 rats) and a control group (9 rats). Acute colitis was induced in the DSS group by adding 6% DSS to the drinking water for 5 days. After the 5th day, DSS was removed from the drinking water. Half of the rats in the 6% DSS group and 3 control rats were sacrificed on the 6th day, and the colons were removed. The remaining rats were allowed to recover from DSS-induced colitis until they were sacrificed on the 16th day. Blood samples were collected at baseline, on days 6, 7, and 16. The disease activity index (DAI) was scored daily to assess the progression of colitis. It is based on the following parameters: weight loss, stool consistency, and blood in the stool or rectal bleeding. The weight loss scores of DSS rats were compared with the average weight of age-matched controls: score 0 = 0-4% weight loss; score 1 = 5-10.9% weight loss; score 2 = 11-15.9% weight loss; score 3 = 16-20% weight loss. Stool consistency: score 0 = normal and well-formed; score 1 = soft, sticky stool visible at the base of the tail; score 2 = very soft and unformed; score 4 = diarrhea and watery stools. Blood in stool or rectal bleeding: score 0 = normal-colored stool; score 2 = reddish-colored stool; score 4 = bloody stool or rectal bleeding. The score for each parameter results in a total daily DAI score ranging from 0 to 12. A total score of 5 or more is considered high disease activity.

[0093] IBD cohort

[0094] Three different cohorts were measured to evaluate the biological relevance of the PRO-C23 assay. Serum samples were collected after obtaining informed consent and approval from the local ethics committee. In cohort 1, serum from patients with CD and UC was obtained from the commercial supplier Reprocell (USA), while serum from healthy donors was obtained from Valley Biomedical (USA) (Table 1). In cohorts 2 and 3, serum from patients with CD and UC was obtained, respectively (Table 2). For any cohort, there were no significant statistical differences in patient demographics (sex and age) between healthy donors, CD, and UC patients.

[0095] Table 1 Patient demographics of cohort 1

[0096]

[0097] Comparisons of age and sex were performed using the Kruskal-Wallis test. P values ​​below 0.05 were considered significant. Abbreviations: CD: Crohn's disease, UC: ulcerative colitis.

[0098] Table 2 Patient demographics of cohorts 2 and 3

[0099]

[0100] Comparisons of age and sex were performed using the Mann-Whitney U test or Fisher's exact test. P values ​​below 0.05 were considered significant. Abbreviations: CD: Crohn's disease.

[0101] Statistics

[0102] Statistical analysis was performed using MedCalc version 14 and GraphPad Prism version 7. Biomarker levels were expressed as median ± 95% confidence intervals. The difference in PRO-C23 between DSS rats and controls was determined by an unpaired t-test. In the human cohort, age and gender were compared using the Kruskal-Wallis test. The difference in PRO-C23 between patients and healthy controls was determined by the Mann-Whitney t-test. The diagnostic efficacy of biomarkers was studied by the area under the receiver operating characteristic (ROC) curve (AUC) with 95% confidence interval (CI). The sensitivity and specificity of the appropriate critical values ​​were determined based on the ROC curve. The significance threshold was set at P < 0.05.

[0103] result

[0104] Characterization of the PRO-C23 assay

[0105] Like type XXIII collagen, types XIII and XXV collagen are also transmembrane collagens, and their C-termini have highly similar sequences ( Figure 1 A) In order to fully investigate the specificity of the antibody, a series of peptides were synthesized and included in the inhibition test. The selected antibody 10F6 specifically recognizes the last 10 amino acids of the C-terminus of type XXIII collagen 531 'GLPVPGCWHK' 540 (SEQ ID No. 1), but not the extended peptide GLPVPPGCWHKA (SEQ ID No. 16), the truncated peptide GLPVPPGCWH (SEQ ID No. 3), the type XIII collagen C-terminal peptide GLPVQGCWNK (SEQ ID No. 4) or the type XXV collagen C-terminal peptide GLPMPGCWQK (SEQ ID No. 5) ( Figure 1 B). Western blot of recombinant type XXIII collagen extracellular domain (4165-CL, R&D system) showed that the selected antibody 10F6 recognized the type XXIII collagen extracellular domain of about 60 kD, while the reference commercial antibody (MAB4165, R&D system) was also shown ( Figure 1 C).

[0106] The PRO-C23 competitive ELISA provided a measurement range from 0.38 ng / ml (LLOD) to 18.73 ng / ml (ULOD). The inter- and intra-assay variability were 8.1% and 3.5%, respectively. The dilution and spiking recoveries in human serum are shown in Table 3. The correlation between human serum and the three plasma values ​​was high ( Figure 2 , P<0.0001), indicating that PRO-C23 levels were not related to the blood preparation method.

[0107] Table 3 Technical performance of PRO-C23 assay

[0108] Detection range (LLOD-ULOD) 0.38ng / ml-18.73ng / ml Intra-assay variability 3.5％ Inter-assay variability 8.1％ Recovery rate in serum Within 100±20% Recovery rate of spiked serum 75.8％ interference No interference from HAMA, biotin, endotoxin and hemoglobin

[0109] PRO-C23 biomarker in the DSS rat model

[0110] To investigate the biological relevance of PRO-C23 fragments, a DSS-induced colitis rat model was used. PRO-C23 biomarkers were measured in serum samples. DSS rats with high disease activity index (DAI ≥ 5) had significantly higher PRO-C23 serum levels on days 6 and 7 compared with controls (p>0.05, Figure 3 A). Serum levels of PRO-C23 increased upon induction of colitis and returned to baseline upon reversal of intestinal inflammation ( Figure 3 B).

[0111] PRO-C23 Biomarkers in a Human IBD Cohort

[0112] PRO-C23 was measured in serum from three independent human cohorts. In cohort 1, PRO-C23 was quantified in 10 CD and 10 UC patients and 10 age-matched healthy donors. The results showed that PRO-C23 levels were significantly higher in CD and UC patients compared with healthy donors (p<0.05, Figure 4 No information on disease activity was provided for these patients. In cohorts 2 and 3, 44 patients with CD and 29 patients with UC, as well as 29 age-matched healthy donors, were included. PRO-C23 levels were elevated in patients with active CD and active UC compared with healthy donors (CD: p < 0.05, UC: p < 0.001, Figure 5 ).

[0113] discuss

[0114] It has been hypothesized that during epithelial injury, type XXIII collagen can be cleaved from the cell surface. Furthermore, loss of type XXIII collagen may contribute to epithelial adhesion changes in IBD and therefore serve as a biomarker for IBD. Therefore, a PRO-C23 ELISA was developed that measures the extracellular domain of type XXIII collagen.

[0115] The antibody only recognizes the C-terminal sequence of type XXIII collagen and has no cross-reaction with the C-termini of type XIII and type XXV collagen, which show similar sequences. This data clearly confirms the specificity of the antibody. Thereafter, the antibody was applied to competitive ELISA and optimized for human serum and plasma measurements. The data show that the extracellular domain of type XXIII in the circulation can be detected by competitive ELISA, which is independent of the blood preparation method.

[0116] To further validate the biomarker in in vivo studies, the PRO-C23 biomarker was measured in a DSS-induced colitis rat model. DSS can cause intestinal epithelial cell damage. These animals exhibit symptoms similar to IBD, such as diarrhea, rectal bleeding, and weight loss [25,26]. One study also showed that DSS can induce changes in the expression of tight junction proteins

[27] . Therefore, the DSS rat model may be a suitable animal model for the validation of the PRO-C23 biomarker. Type XXIII collagen was found to be elevated in rats with active disease and was weakly correlated with disease activity. This finding suggests that the extracellular domain of type XXIII collagen is found in the circulation that is associated with disease activity in DSS rats.

[0117] To further validate the PRO-C23 biomarker, it was measured in two human cohorts. PRO-C23 was elevated in human CD patients (cohort 2) and UC patients (cohort 3) with active disease. These data suggest that the release of the extracellular domain of type XXIII collagen is enhanced in active intestinal damage, which is consistent with the results in animal models.

[0118] Type XXIII collagen has been suggested as a potential biomarker for prostate cancer recurrence

[18] , non-small cell lung cancer

[17] , and clear cell renal cell carcinoma

[14] . It shows significantly higher expression in those cancer tissues, especially in metastatic tissues. Type XXIII collagen is believed to promote cell-cell adhesion and cell-matrix adhesion

[13] . Silencing of type XXIII collagen in lung cancer and clear cell renal cell lines showed altered adhesion protein expression and reduced cell adhesion and migration ability[13,14]. However, type XXIII collagen is also present in other tissues, and its function and use in other diseases are still unclear. To the best of the inventors' knowledge, this is the first study to show that type XXIII collagen levels are regulated in IBD. The results suggest that type XXIII collagen may also play an important role in intestinal cell adhesion and contribute to the pathology of IBD.

[0119] in conclusion

[0120] The data suggest that the epithelial cell PRO-C23 biomarker could be used as a non-invasive surrogate for disease activity in CD patients, thereby aiding in monitoring patients. Higher levels of PRO-C23 were measured in the serum of CD and UC patients with active disease compared to inactive disease.

[0121] All prior teachings acknowledged in this specification are hereby incorporated by reference. Acknowledgment of any prior published document herein is not to be taken as an acknowledgment or representation that its teachings are common general knowledge in Australia or elsewhere as of the date of publication of the document.

[0122] References

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Claims

1. A monoclonal antibody or an antigen-binding fragment thereof, which specifically binds to a peptide consisting of the C-terminal amino acid sequence GLPVPGCWHK (SEQ ID No. 1), wherein: The monoclonal antibody or antigen-binding fragment thereof comprises the following complementarity determining regions (CDRs): CDR-H1: SYAMS (SEQ ID No. 6) CDR-H2:SISTAGRTYYPDTVR(SEQ ID No.7) CDR-H3:PDYDYDGYIN(SEQ ID No.8) CDR-L1:RSSKSLLHSNGVTYLY(SEQ ID No.9) CDR-L2: QMSNLAS (SEQ ID No.10) and CDR-L3: AQNLELPLT (SEQ ID No. 11).

2. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody does not specifically bind to a peptide having a C-terminal amino acid sequence of GLPVPPGCWHKX (SEQ ID No. 2), wherein X represents any amino acid, and wherein the antibody does not specifically bind to a peptide having a C-terminal amino acid sequence of GLPVPPGCWH (SEQ ID No. 3).

3. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein The monoclonal antibody is a monoclonal antibody generated against a synthetic peptide consisting of the C-terminal amino acid sequence GLPVPGCWHK (SEQ ID No. 1).

4. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody does not specifically bind to a peptide having the C-terminal amino acid sequence GLPVQGCWNK (SEQ ID No. 4).

5. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody does not specifically bind to a peptide having the C-terminal amino acid sequence GLPMPGCWQK (SEQ ID No. 5).

6. Use of a monoclonal antibody or an antigen-binding fragment thereof in the preparation of a kit for detecting type XXIII collagen in a human biological fluid sample using an immunoassay, the immunoassay comprising contacting the human biological fluid sample with the monoclonal antibody or the antigen-binding fragment thereof according to any one of claims 1 to 5, and detecting the binding between the monoclonal antibody or the antigen-binding fragment thereof and a peptide in the sample.

7. The use according to claim 6, wherein The detection is quantitative.

8. The use according to claim 6 or 7, wherein The immunoassay is a competitive immunoassay.

9. The use according to claim 6, wherein The human biological fluid sample is from a human patient having medical signs or symptoms indicative of inflammatory bowel disease.

10. The use according to claim 6, wherein: The immunoassay is an immunoassay for diagnosing and / or monitoring and / or assessing the likelihood of inflammatory bowel disease in a patient, the immunoassay comprising contacting a biological fluid sample obtained from the patient with the monoclonal antibody, detecting and determining the amount of binding between the monoclonal antibody and peptides in the sample, and correlating the amount of binding to values ​​associated with normal healthy subjects and / or values ​​associated with known disease severity and / or values ​​obtained from the patient at a previous time point.

11. The use according to claim 9 or 10, wherein The inflammatory bowel disease is Crohn's disease or ulcerative colitis.

12. An assay kit comprising: a monoclonal antibody or an antigen-binding fragment thereof according to any one of claims 1 to 5 that specifically binds to a peptide consisting of the C-terminal amino acid sequence GLPVPGCWHK (SEQ ID No. 1) and at least one of the following: - Streptavidin-coated well plates; - an N-terminally biotinylated peptide consisting of the C-terminal amino acid sequence GLPVPGCWHK (SEQ ID No. 1); and - A calibrator peptide consisting of the C-terminal amino acid sequence GLPVPGCWHK (SEQ ID No. 1).

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