Biomarker of Fibrosis

Abstract

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

Claims

1: A monoclonal antibody that specifically recognises and binds to a peptide having the C-terminus amino acid sequence KAGGFAPYYG (SEQ ID NO: 1).

2: The monoclonal antibody of claim 1, wherein the monoclonal antibody is a monoclonal antibody raised against a synthetic peptide having the C-terminus amino acid sequence KAGGFAPYYG (SEQ ID NO: 1).

3: The monoclonal antibody of claim 1, wherein the antibody does not specifically recognise or bind to a peptide having the C-terminus amino acid sequence KAGGFAPYYGX (SEQ ID NO: 2), wherein X represents any amino acid, the C-terminus amino acid sequence KAGGFAPYYGD (SEQ ID NO: 4) or the C-terminus amino acid sequence KAGGFAPYY (SEQ ID NO: 5).

4-5. (canceled)

6: A sandwich immunoassay for detecting in a biological sample cross-linked CT-III, said cross-linked CT-III comprising at least two strands of CT-III joined together by inter-strand cross-linking, said method comprising: contacting said biological sample comprising said cross-linked CT-III with a first monoclonal antibody bound to a surface, wherein each strand of CT-III comprised in the cross-linked CT-III comprises a C-terminal neo-epitope of CT-III generated by C-protease cleavage of intact type III collagen; adding a second monoclonal antibody; and determining the amount of binding of said second monoclonal antibody; wherein both said first monoclonal antibody and said second monoclonal antibody are specifically reactive with said C-terminal neo-epitope of CT-III, said neo-epitope being comprised in a C-terminal amino acid sequence KAGGFAPYYG-COOH (SEQ ID NO: 1).

7. (canceled)

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

9: The sandwich immunoassay of claim 6, wherein the biological sample is a biofluid selected from the group consisting of serum, plasma, urine, amniotic fluid, tissue supernatant, and cell supernatant.

10-11. (canceled)

11: The sandwich immunoassay of claim 6, wherein the second monoclonal antibody is labeled, is linked to a fluorophore or is an enzyme-linked antibody.

15. (canceled)

16: The sandwich immunoassay of claim 6, wherein a further labeled antibody which recognises the second monoclonal antibody is used to determine the amount of binding of said second monoclonal antibody.

17: The sandwich immunoassay of claim 8, further comprising correlating the quantity of cross-linked CTIII determined by said method with standard disease samples of known disease severity to evaluate the severity of a disease.

18: The sandwich immunoassay of claim 8, further comprising quantifying the amount of PRO-C3 present in the biofluid sample, determining the ratio of cross-linked type III collagen (CTX-III) to N-terminal propeptide of type III collagen (PRO-C3).

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

20-23. (canceled)

24: The sandwich immunoassay of claim 20, wherein the disease is non-alcoholic fatty liver disease, an HCV related liver disease, Crohn's disease, ulcerative colitis, breast cancer, bladder cancer, colorectal cancer, head and neck cancer, kidney cancer, lung cancer, pancreatic cancer, stomach cancer, ovarian cancer, liver cancer, prostate cancer, or melanoma.

25. (canceled)

26: A method for evaluating the efficacy of an antagonist drug targeting lysyl oxidases (LOXs), wherein said method comprises using the sandwich immunoassay of claim 6 to quantify the amount of cross-linked CT-III in at least two biological samples, said biological samples having been obtained from a subject at a first time point and at at least one subsequent time point during a period of administration of the antagonist drug to said subject, and wherein a reduction in the quantity of cross-linked CT-III from said first time point to said at least one subsequent time point during the period of administration of the antagonist drug is indicative of an efficacious antagonist drug targeting LOXs.

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

28: A kit for use in a sandwich assay, the kit comprising: a solid support to which is bound the first monoclonal antibody as defined in claim 1; and the second monoclonal antibody as defined in claim 1, said second monoclonal antibody comprising a label.

29: A method of identifying a patient with a fibrotic disease, the method comprising using the sandwich immunoassay of claim 6 to quantify the amount of cross-linked CT-III in a biofluid sample obtained from the patient, and correlating said amount of cross-linked CT-III with i) values associated with known patients with the fibrotic disease and/or normal healthy controls and/or ii) a predetermined cut-off value.

30: The method of claim 29, wherein the fibrotic disease is fibrosis and the method identifies the fibrosis response phenotype of the patient, said method further comprising quantifying the amount of N-terminal propeptide of type III collagen (PRO-C3) present in the biofluid sample, determining the ratio of cross-linked type III collagen (CTX-III) to N-terminal propeptide of type III collagen (PRO-C3), and correlating said ratio of cross-linked type III collagen (CTX-III) to N-terminal propeptide of type III collagen (PRO-C3) with a predetermined cut-off value.

31: The method of claim 29, wherein the cut-off value is at least 3.8 ng/mL cross-linked CT-III and/or a ratio of cross-linked type III collagen (CTX-III) to N-terminal propeptide of type III collagen (PRO-C3) of at least 0.5.

32: The method of claim 29, wherein the fibrotic disease is eosinophilic esophagitis, a chronic intestinal disease, a cancer, or fibrosis.

35. (canceled)

36: The method of claim 29, wherein said method identifies a patient who would benefit from a treatment with a medicament which targets collagen cross-linking.

37. (canceled)

Description

FIGURES

[0105] The invention will be demonstrated in the examples below which refer to the following figures:

[0106] FIG. 1: Sequence alignment of the type III collagen 1-chain between rat (top), mouse (middle), and human (bottom) sequence. The red box frames the target sequence of neo-epitope in CT-III. The alignment was made using the corresponding species sequences from uniprot.org and the CLUSTALW 2.1 multiple alignment tool.

[0107] FIG. 2: Final inhibition of the monoclonal NBH-242 antibody in an indirect competitive ELISA. The antibody specificity was tested against the selection peptide, elongated peptide, truncated peptide, immunogenic peptide, and the buffer.

[0108] FIG. 3: Depicts a 4-parameter logistical model of antibody specificity towards the human selection peptide, the elongated peptide, the truncated peptide, and the rat selection peptide.

[0109] FIG. 4: Depicts the CTX-III levels of the healthy human plasma EDTA donors, and the bariatric surgery patients at baseline and 6 months follow-up. Levels below LLMR was defined as the LLMR level (1.8 ng/mL). Asterisks indicate the following: *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001.

[0110] FIG. 5: Represents the net deposition of type III collagen as the ratio between the biomarker levels of PRO-C3 (type III collagen formation) and CTX-III (cross-linked type III collagen degradation) at baseline and 6 months follow-up. Asterisks indicate the following: ****: p<0.0001.

[0111] FIG. 6. Depicts Patients with HCV related liver fibrosis was stratified according to their Ishak score (1-2, 3, and 4-5) and their CTX-III levels at screening plotted against CTX-III levels of healthy donors. Data plotted as median+IQR and significance depicted as: p<0.0001****

[0112] FIG. 7: Depicts levels of net fibrolysis of patients divided according to their Ishak score; 1-2, 3, and 4-5. Data plotted as median+IQR and significance depicted as: p<0.05*

[0113] FIG. 8: shows CTX-III (A) or net fibrolysis (B) levels at screening of patients within the placebo group stratified according to their spontaneous fibrotic phenotype; Regressive, Stable, or Progressive. Data plotted with median+IQR and significance depicted as: p<0.05*, p<0.01**, and p<0.001***

[0114] FIG. 9: Depicts the percentage change of patient Ishak score based on their CTX-III (A) or net fibrolysis (B) levels at screening by dividing patients into tertiles from lowest to highest levels (1st-, 2nd-, and 3rd-tertile). Data plotted as mean+SEM and significance depicted as: p<0.01**, and p<0.001***

[0115] FIG. 10: Shows the mean percentage change of patient Ishak score based on CTX-III (A) or net fibrolysis (B) levels at screening utilizing a cut-off level calculated by the Youden index. Data plotted as mean+SEM and significance depicted as: p<0.01**

[0116] FIG. 11: Depicts the odds ratio for being presenting with a regressive fibrotic phenotype based on Youden index determined levels of CTX-III or net fibrolysis at screening. Odds ratio plotted with 95% CI and significance depicted as: p<0.01**

[0117] FIG. 12: Serum CTX-III plotted for the healthy donors and EoE patients at baseline and after intervention. Significant differentiation calculated by one-way ANOVA and depicted as follows: p<0.0001****

[0118] FIG. 13: Plotted CTX-III levels of healthy donors, CD- and UC patients. The statistical difference was calculated by Kruskal-Wallis one-way ANOVA with significance depicted as: p<0.0001****

[0119] FIG. 14: Plasma levels of CTX-III (A), net cross-linked fibrolysis (log(CTX-III/PC3X)) (B), and net fibrolysis (log(CTX-III/PRO-C3)) of patients with either non-stricturing and non-penetrating (B1) or stricturing disease manifestations (B2) as determined by endoscopy. Statistical difference calculated by Mann-Whitney (A) and unpaired t-test (B+C) with the significance depicted as: p<0.05*, and p<0.01**.

[0120] FIG. 15: Box plot illustrating the statistical difference between CTX-III levels of Healthy individuals against 12 types of cancer plotted with the 10-90 percentile. The significance was calculated by ordinary one-way ANOVA and depicted as follows: p>0.05 NS; p<0.05*; p<0.01**; p<0.001***.

[0121] FIG. 16: The levels of CTX-III (A) and the net fibrolysis (B) was plotted for breast cancer patients in either stage II or stage III. Data is presented as a box plot with 10-90 percentile. The significant differentiation was calculated by parametric t-test and depicted as: p<0.05*; p<0.001***.

EXAMPLE 1

Methods and Materials

Reagents

[0122] 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 monoclonal antibody production and assay development and validation were 1) Immunogenic peptide: Keyhole Limpet Hemocyanin (KLH)-CGG-KAGGFAPYYG, 2) Coating peptide: Biotin-KAGGFAPYYG, 3) Selection peptide: KAGGFAPYYG (SEQ ID NO: 1) or CKAGGFAPYYGCKAGGFAPYYG (SEQ ID NO: 16) (dimer linked by a N-terminal disulphide bridge), 4) Elongated peptide: KAGGFAPYYGD (SEQ ID NO: 4) or CKAGGFAPYYGDCKAGGFAPYYGD (SEQ ID NO: 17) (dimer linked by a N-terminal disulphide bridge), 5) Truncated peptide: KAGGFAPYY (SEQ ID NO: 5) or CKAGGFAPYYCKAGGFAPYY (SEQ ID NO: 18) (dimer linked by a N-terminal disulphide bridge), and 6) Rat dimeric peptide: CKSGGFSPYYGCKSGGFSPYYG. The dimeric peptides were only used for the assay development and validation. All the synthetic peptides were purchased from Genscript, Piscataway, NJ, USA.

Monoclonal Antibody Production and Clone Characterization

[0123] The target neo-epitope (1212-KAGGFAPYYG-1221) located in the C-terminal telopeptide of type III collagen was analysed for its uniqueness and the sequence homology with rat and mouse using protein blast (FIG. 1).

[0124] Generation of monoclonal antibodies was carried out in four- to six week-old Balb/C mice. The mice were immunized subcutaneously with 200 L emulsified antigen and 50 g immunogenic peptide (KLH-CGG-KAGGFAPYYG) using Freund's incomplete adjuvant (Sigma-Aldrich). The mice were immunized with two-week intervals until stable serum titer levels were reached. The mouse with the highest serum titer was selected for fusion. The mouse rested one month and immunized intravenously with 50 g immunogenic peptide in 100 L 0.9% NaCl solution. After 3 days, splenocytes were isolated for cell fusion. In brief, splenocytes were fused with SP2/0 myeloma cells to produce hybridoma cells and then cloned in culture dishes using the semi-medium method. The clones were plated into 96-well microtiter plates, and limited dilution was used to secure monoclonal growth. The supernatants were screened for reactivity against the selection peptide (KAGGFAPYYG (SEQ ID NO: 1)) and the elongated peptide (KAGGFAPYYGD (SEQ ID NO: 4)) an indirect competitive ELISA using streptavidin precoated plates (Roche, Hvidovre, Denmark, cat. No 11940279), which were coated with 4 ng/mL of the coating peptide (biotin-KAGGFAPYYG). All reagents were diluted in 50 mM PBS, 1% BSA, 1% Tween-20, 150 mM NaCl, pH 7.4. The two best monoclones were selected for final inhibition, testing their reactivity towards the selection peptide (KAGGFAPYYG (SEQ ID NO: 1)) and not the elongated (KAGGFAPYYGD (SEQ ID NO: 4)), the truncated peptide (KAGGFAPYY (SEQ ID NO: 5)), or the immunogenic peptide (KLH-CGG-KAGGFAPYYG. Prior to purification of the most optimal monoclone, the antibody was isotype tested using the sandwich ELISA kit, the SBA Clonotyping System-HRP (Southern Biotech, Birmingham, AL, USA). The monoclone with the best reactivity was purified using protein-G column according to the manufacturer's instructions (GE healthcare Life Sciences, Little Chalfont, Buckinghamshire, UK).

[0125] The antibodies generated were sequenced and the CDRs determined.

[0126] The sequence of the chains are as follows (CDRs underlined and in bold; Constant region in italics):

TABLE-US-00009 HeavyChainSequence(MouseIgGisotype) (SEQIDNO:19) QVQLQQSGAELVRPGVSVKISCKGSGHTFTDHGMHWVKQSQAKSLEWIG VISTYYGDATYNQKFKGKATMTVDKSSSTAYMELARLTSEDSAIYYCAR SMGGNYVGTGFAYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTL GCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSST WPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSV FIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQT QTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISK PKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKT ELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHH TTKSFSRTPGK CDR-H1: (SEQIDNO:9) DHGMH CDR-H2: (SEQIDNO:10) VISTYYGDATYNQKFKG CDR-H3: (SEQIDNO:11) SMGGNYVGTGFAY LightChainSequence(MouseKappaIsotype) (SEQIDNO:20) DIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGNTYLYWFLQRPGQSP QLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLE FPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPK DINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNS YTCEATHKTSTSPIVKSFNRNE CDR-L1: (SEQIDNO:6) RSSKSLLHSNGNTYLY CDR-L2: (SEQIDNO:7) RMSNLAS CDR-L3: (SEQIDNO:8) MQHLEFPLT.

Assay Development

Monoclonal Antibody Labelling

[0127] The monoclonal NBH-242 antibody used as the capture antibody was labelled with biotin by adding 110 L Na.sub.2CO.sub.3/NaHCO.sub.3 buffer, pH 9.6 to 1 mL (1 mg/mL) of the antibody, followed by 13.3 L of biotinamidohexanoic acid N-hydroxysuccinimide ester (Sigma Aldrich, St. Louis, MO, USA, cat. No B2643). The solution was incubated at 20 C. for 1 hour with end-over-end rotation. Subsequently, 110 L of 0.2 M ethanolamine, pH 8.0 was added to the solution and incubated as before. The solution was dialysed overnight in a Zeba 7k MWCO desalting column (Thermo Scientific, Waltham, MA, USA, cat No. 89889) submerged in 1PBS at 4 C. Additionally, a portion of the monoclonal antibody was labelled with horseradish peroxidase (HRP) and used as the detection antibody. HRP labelling was performed using the peroxidase labelling kit from Roche, purchased from Sigma Aldrich, St. Louis, MO, USA, cat 11829696001, and done according to the manufactures protocol.

Direct Sandwich ELISA Protocol

[0128] 96-well plates precoated with streptavidin (Roche Diagnostic's, Hvidovre, Denmark, cat. No. 11940279) were coated with 100 L of the biotinylated antibody targeting the CTX-III fragment diluted 1+100 in assay buffer (50 mM PBS, 1% BSA, 1% Tween-20, 150 mM NaCl, pH 7.4) for 30 minutes at room temperature rotating at 300 rounds-per-minute (rpm). Unbound biotinylated capture antibody was discarded, and the wells were washed with washing buffer (25 mM TRIZMA, 50 mM NaCl, 0.036% Bronidox L5, 0.1% Tween 20) using a standardized ELISA plate washing machine (BioTek Instruments, Microplate washer, ELx405 Select CW, Winooski, USA). All samples and the detection antibody were diluted in incubation buffer (50 mM PBS, 1% BSA, 1% Tween-20, 150 mM NaCl, 5% Liquid II, pH 7.4), with the detection antibody being diluted 1+100. 20 L of sample material and controls were incubated with 100 L HRP labelled detection antibody targeting the CTX-III fragment, at 4 C. for 20 hours and agitated at 300 rpm. Unbound primary antibody and sample was discarded, and the wells were washed with washing buffer. Subsequently, 100 L chemiluminescence substrate was added to the wells, plates were incubated for 3 minutes at 20 C. in the dark rotating 300 rpm. Finally, the luminescence emitted was quantified with an ELISA reader (VersaMAX; Molecular Devices, Wokingham Berkshire, UK) set to measure the luminescence at 450 nm and 650 nm. With the results obtained from a 2-fold serial dilution of the dimeric selection peptide (CKAGGFAPYYG x CKAGGFAPYYG (SEQ ID NO: 16)), a standard curve was plotted using a 4-parametric mathematical fit model. Unknown sample measurements were interpolated with the standard curve to obtain the concentration (ng/mL) of the CTX-III fragment.

Technical Validation

[0129] The lower limit of detection (LLOD) was determined from 21 zero samples (i.e. incubation buffer) and calculated as the mean+3 standard deviation, whereas the upper limit of detection (ULOD) was determined from 10 measurements of the dimeric selection peptide calculated as the mean+3 standard deviation. The inter- and intra-assay variation was determined by 10 independent runs of five quality control (QC) samples with minimum three of these being healthy human plasma EDTA samples (Valley Biomedical, Winchester, VA, USA), with each run consisting of double determinations of the samples. The acceptance criteria for the inter- and intra-assay variation was 15% and 10%, respectively. Assay linearity was determined by calculating the percentage recovery (100%20%) of a 1:6-fold dilution of healthy human plasma EDTA samples with a significant concentration, using the undiluted sample as reference. Specificity of the assay was determined by calculating the percentage recovery of the dimeric elongated peptide, the dimeric truncated peptide, and the dimeric rat peptide measurements towards the 100% sample of the dimeric selection peptide. The accuracy of sample measurements was determined by spiking two samples of healthy human plasma EDTA with a significant concentration, and subsequently calculating the percentage recovery between the theoretical and the actual measurements. Interference from biotin, lipids, and haemoglobin was tested by spiking healthy human plasma EDTA samples with a known concentration of the interferent. Subsequently, the percentage recovery between the control sample and the low or high interferent samples were calculated.

Analyte and Reagent Stability

[0130] The stability of the CTX-III fragment was determined by calculating the percentage recovery of three healthy human plasma EDTA samples from the non-stressed sample. The samples underwent up to four cycles of freeze and thaw or were subjected to 2, 4, 24, or 48 hours of incubation at either 4 C. or 20 C.

In vitro Cleavage Assay

Clinical Cohort Measurement

Biomarker Assay

[0131] Assessment of type III collagen formation was performed using the PRO-C3 (32) competitive ELISA targeting a neo-epitope in the N-terminal propeptide. In brief, streptavidin coated 96-well plates were incubated for 30 min at 20 C., 300 rpm with 100 L of biotinylated coater peptide diluted 1+100 in a PBS buffered coating solution containing protein stabilizers and preservatives. Following incubation, the coating solution was discarded, and the wells washed five times in washing buffer (25 mM TRIZMA, 50 mM NaCl, 0.036% Bronidox L5, 0.1% Tween 20) using a standardized ELISA plate washing machine (BioTek Instruments, Microplate washer, ELx405 Select CW, Winooski, USA). 20 L of sample material was added to the appropriate wells with subsequent addition of 100 L of HRP-labelled antibody diluted 1+100 in the incubation buffer. The plates were incubated for 20 hours at 4 C., 300 rpm after which they were washed as previously described. Tetramethylbenzidine (TMB, Kem-En-Tec cat. No. 438OH, Taastrup, Denmark) was used as the colorimetric reagent with 100 L/well with 15 min incubation agitating 300 rpm in the dark. The reaction was stopped with the addition of 100 L of 1% H.sub.2SO.sub.4 and the optical density was read at 450 nm with 650 nm as reference using an ELISA reader (VersaMAX; Molecular Devices, Wokingham, UK). The concentration of PRO-C3 within the analysed samples was determined by interpolation with the 4-parametric logarithmic standard curve generated through a 2-fold serial dilution of the selection peptide.

Results

Monoclonal Antibody Production and Characterization

[0132] Alignment of the rat-, mouse-, and human sequence of type III collagen revealed two amino acid differences (designated by : in FIG. 1). Isotype characterization determined the antibody NBH-242 used for the assay development to be an IgG2a -light chain. In the final inhibition the monoclonal antibody did not show any reactivity towards the elongated-, truncated-, or the immunogenic-peptide, observed by the lack of signal inhibition. Testing the reactivity towards the selection peptide demonstrated an increasing signal inhibition with the peptide concentration, thus illustrating its binding to the antibody in the indirect competitive ELISA (FIG. 2).

Technical Validation

[0133] In the development of the 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 the ULOD, which provided a range from 0.92-15.94 ng/mL. The technical performance of the assay determined by calculating the inter- and intra-assay variations was acceptable with variations of 14.8% and 5.4%, respectively (Table 4). Assay linearity was tested in healthy human plasma EDTA samples, which resulted in a mean recovery of 108.9%, thus within the acceptable range of 100%20%, (Table 5). Diluting the samples any further resulted in concentration below the measurement range. By plotting the calculated percentage recovery, the specificity of the antibodies in the direct sandwich ELISA towards the dimeric peptides was tested. Here, antibodies showed no reactivity towards either the dimeric elongated-, dimeric truncated-, or the dimeric rat-peptide, while showing reactivity towards an increasing concentration of the dimeric selection peptide illustrated by the increasing luminescence (y-axis) (FIG. 3). Spiking healthy human plasma EDTA samples with another healthy human plasma EDTA sample, resulted in mean recoveries of 102.2% (Table 6). None of the tested interferents demonstrated an effect on the sample measurements in the healthy human plasma EDTA samples with a mean recovery within the acceptable range (100%20%) (Table 7).

TABLE-US-00010 TABLE 4 Inter- and Intra-assay variation for the CTX-III assay by using five QC samples (three of these was healthy human plasma EDTA samples with the last two being the dimeric selection peptide). The variation (%) is calculated as the mean of 10 individual double determinations of each sample. CTX-III Mean value Intra-assay Inter-assay sample (ng/ml) variation (%) variation (%) #1 3.16 6.7 12.5 #2 9.13 8.3 12.9 #3 3.10 4.0 30.5 #4 4.03 2.2 2.6 #5 1.60 5.8 15.3 Average 5.4 14.8

TABLE-US-00011 TABLE 5 The sample dilution recovery of four healthy Human Plasma EDTA samples (HP). HP (n = 4) Undiluted 100% 5:6 101.5 4:6 100.8 1:2 124.3 Average 108.9

TABLE-US-00012 TABLE 6 Healthy human plasma EDTA samples with a significant concentration was spiked with each other and the spiking recovery (%) calculated between the measured- and theoretical-sample measurement. Samples spiked Spiking recovery (%) HP1 + HP2 117.3 HP2 + HP3 115.9 HP4 + HP2 90.2 HP5 + HP2 101.9 HP5 + HP6 85.6 Average 102.2

TABLE-US-00013 TABLE 7 The interference from haemoglobin, biotin, and lipids in three healthy human plasma EDTA samples. The recovery (%) is calculated from the low and high concentration of the interferent to the pure plasma EDTA sample. CTX-III Haemoglobin Biotin Lipid sample Conc. Recovery Conc. Recovery Conc. Recovery HP1 109.2 97.2 111.1 10.46 102.4 100.7 HP2 103.6 103.4 91.5 111.9 98.3 93.1 HP3 112.5 103.5 99.3 118.1 98.1 108.9

Analyte and Reagent Stability

[0134] The stability of the analyte in healthy human plasma EDTA samples was stable for up to four cycles of freeze/thaw (Table 8). The mean recovery for incubation at 4 C. of the healthy human plasma EDTA samples was 92.5% with stability up to 48 hours in all samples. At 20 C. the mean recovery was 114.7%, with three out of four samples demonstrating analyte stability up to 24 hours. (Table 9).

TABLE-US-00014 TABLE 8 Freeze/Thaw cycle no. Recovery (%), HP (n = 4) 1 100 2 83.6 3 104.5 4 104.9 Average 97.7

TABLE-US-00015 TABLE 9 Incubation time and temperature Recovery (%), HP (n = 4) 0 h/4 100 2 h/4 93.8 4 h/4 93.0 24 h/4 91.7 48 h/4 83.8 0 h/20 100 2 h/20 109.7 4 h/20 115.8 24 h/20 121.5 48 h/20 126.6

Discussion

[0135] Based on the production of a monoclonal antibody targeting a neo-epitope in the C-terminal telopeptide of type III collagen following C-proteinase cleavage, a novel CTX-III sandwich ELISA was developed by utilizing the NBH-242 monoclonal antibody to detect cross-linked fragments of type III collagen. In brief, the assay demonstrated high specificity towards the human neo-epitope with the ability to detect the analyte in human plasma EDTA samples. Additionally, the assay showed to be technically stable with an acceptable inter- and intra-assay variation, linearity, and accurate measurements.

Characterization and Technical Validation

[0136] During the antibody characterization, the monoclonal antibody reactivity was tested in a competitive ELISA towards variations of the neo-epitope sequence, and while this showed the high specificity of the antibody, the peptides used were monomeric and did therefore not validate the antibodies use in a sandwich ELISA. Thus, a set of dimeric peptides was designed containing two identical sequences of the different peptide variations mentioned earlier, cross-linked through a disulphide bond located N-terminal to the antibody binding site. Testing the reactivity towards the dimeric peptides, the antibody elicited a high specificity towards the dimeric human selection peptide, thus re-validating its high specificity towards the human neo-epitope sequence, while also demonstrating its potential usefulness in the detection of cross-linked fragments.

[0137] Due to the high specificity and the inability of the antibody to detect the rat sequence homolog, the assay development and validation focused on human sample material. Native reactivity was shown in human plasma EDTA samples, though most of the measured sample values were in the lower end of the measuring range, as can be observed from the HD levels in FIG. 3. Samples within the measuring range was selected for the technical validation of the assay, which revealed its technical stability. The inter- and intra-assay variations was acceptable, as was the dilution recovery of healthy human plasma EDTA samples, and the spiking recovery showing the accuracy of the assay.

Analyte and Reagent Stability

[0138] An important factor in the development and clinical use of an assay is the stability of the analyte, allowing the accurate measurement of sample material after various freeze and thaw cycles, and incubation at higher or lower temperatures. This is especially important when measuring clinical sample material where the exact sample handling may not be fully known. Testing the stability of the CTX-III analyte did not suggest any major instabilities, although careful handling of samples should always be a priority.

[0139] In the following examples the level of CTX-III was measured using the assay described above. The level of PROC3 was measured using the method described in WO 2014/170312 and the level of PC3X was measured using the assay described in WO2017/34172.

EXAMPLE 2BARIATRIC SURGERY

[0140] 58 patients with non-alcoholic fatty liver disease (NAFLD) having undergone bariatric surgery were sampled for blood at baseline and 6 months follow-up. A schematic overview of the patient demographics is provided in table 10, which include patient BMI, non-alcoholic fatty liver disease activity score (NAS), steatosis grade, inflammation grade, ballooning, and their fibrosis stage. Patient demographics were only obtained at baseline with demographics only provided for 45-48 of the patients. Prior to the surgical procedure, patients were put on a diet to encourage preoperative weight loss. The measured samples were plasma EDTA which had been stored at 80 C. until CTX-III measurement.

TABLE-US-00016 TABLE 10 Represent the bariatric surgery patient demographics at baseline. BMI, mean kg/m.sup.2 range 51 38-66 NAS score <2, n % 28 59.6% 9 19.1% 3-4, n % 10 21.3% >5, n % Steatosis grade <5%, n % 18 37.5% 5-33%, n % 16 33.3% 33-66%, n % 8 16.7% >66%, n % 6 12.5% Inflammation grade No foci, n % 8 17.8% <2 foci/200 field, n % 22 48.9% 2-4 foci/200 field, n % 12 26.7% >4 foci/200 field, n % 3 6.7% Ballooning None, n % 30 65.2% Few ballooned cells, n % 16 34.8% Many ballooned cells, n % 0 0% Fibrosis stage F0, n % 17 36.2% F1, n % 25 53.2% F2, n % 5 10.6% F3, n % 0 0% F4, n % 0 0%

Statistical Analysis

[0141] Comparisons of the CTX-III levels between the healthy plasma EDTA samples and the plasma EDTA samples from the cohort of bariatric surgery patients were done by applying one-way ANOVA (Kruskal-Wallis) correcting for the false discovery rate. Results are shown as the median CTX-III levels+Inter Quartile Range (IQR). All statistical analyses were performed in 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.

Results

CTX-III Levels Relate to NAFLD

[0142] The levels of CTX-III were significantly higher in patients of NAFLD having undergone bariatric surgery, at baseline (p<0.0001), and 6 months follow-up (p<0.001) when compared with the levels of the Healthy human plasma EDTA Donors (HDs) (FIG. 4). Furthermore, the baseline (p<0.01) levels was significantly higher than the levels at the 6 months follow-up (Figure).

Deposition of Type III Collagen

[0143] The ratio of PRO-C3 and CTX-III depicting the net deposition of type III collagen was significantly elevated in the bariatric surgery patients at the 6 months follow-up in comparison with the patients at baseline (p<0.0001) (FIG. 5).

Discussion

[0144] The development of the CTX-III assay demonstrated an ability to differentiate between HDs and NAFLD patients, including the differences between baseline levels and the 6 months follow-up levels.

CTX-III Levels Relate to NAFLD

[0145] NAFLD is one of the major chronic liver diseases affecting about one-quarter of the population (33), with one of the major causes being obesity which results in the accumulation of fat in the liver (34). This accumulation can then lead to initiation of the inflammatory cascade with the potential of formation of scar tissue, ultimately leading to a state of liver fibrosis (34). Here, the excessive accumulation of ECM components, including type III collagen (35) and the ECM related cross-linking enzyme LOXL2 (36) contribute to further the disease progression. As such, the potential and biological relevance of the novel CTX-III marker in a study of NAFLD patients having undergone bariatric surgery, was evaluated. By measuring the CTX-III levels in plasma EDTA samples obtained from the NAFLD patients at baseline and 6 months follow-up a significant difference between the patients at each timepoint was demonstrated. Furthermore, when comparing the CTX-III levels of the NAFLD patients with the HDs, patients diagnosed with NAFLD showed biomarker levels significantly higher at all timepoints. This increased level of the CTX-III marker suggests a significantly increased level of fibrolysis taking place in the bariatric surgery patients. In combination with CTX-III, the liver fibrosis relevant PRO-C3 biomarker of type III collagen formation was also measured. Combining the two biomarker measurements provided a measure of type III collagen net deposition. Here, the deposition was increased from baseline to the 6 months follow-up suggesting a switch from degradation of mature cross-linked type III collagen to the formation of new collagen within the tissue. These data indicate the potential of the CTX-III marker in distinguishing between individuals with a known active disease as well as monitoring their levels of CTX-III over time. The CTX-III marker was unable to differentiate between patients based on their individual disease scores, including steatosis grade, inflammation grade, ballooning, BMI, fibrosis stage, or NAS score (data not shown).

EXAMPLE 3 HCV RELATED LIVER FIBROSIS

Study Description

[0146] A total of 158 patients diagnosed with Hepatitis C Virus (HCV) related liver fibrosis were sampled for blood at the time of screening and after 52 weeks with the measured sample material being plasma EDTA. Table 11 provides an overview of the total patient population, while table 12 represents the 47 patients of the placebo group.

TABLE-US-00017 TABLE 11 Demographic overview of patients at screening diagnosed with HCV related liver fibrosis HCV RELATED FIBROSIS N 158 AGE, YR 51.6 MEN, N (%) 100 (63.3) BMI, KG/M.sup.2 28.6 BASELINE ISHAK SCORE 2.7 AST, IU/I 64 FIBROTEST, IU 0. CTX-III, NG/ML 4.5 NET FIBROLYSIS, RATIO 0.45

TABLE-US-00018 TABLE 12 Demographic overview of the patients within the placebo group showing data at time of screening REGRESSORS STABLE PROGRESSORS P VA N 5 31 11 AGE, YR 50.2 51.5 48.7 NS MEN, N (%) 5 (100) 21 (67.7) 7 (63.6) NS BMI, KG/M.sup.2 32 30.3 31 NS BASELINE 2.8 2.5 2.8 NS ISHAK SCORE DELTA 1 0 1.3 <0.0 ISHAK SCORE AST, IU/I 42.6 61 112 0.0 FIBROTEST, 0.6 0.6 0.7 NS IU CTX-III, 7.1 3.5 3 <0.0 NG/ML NET 0.63 0.43 0.31 <0.0 FIBROLYSIS, RATIO

Statistical Analysis

[0147] Comparisons of the CTX-III levels between the healthy plasma EDTA samples and the plasma EDTA samples from the cohort of HCV related liver fibrosis patients were done by applying non-parametric t-test or one-way ANOVA depending on the number of groups analysed (Mann-Whitney or Kruskal-Wallis). Data was corrected for False Discovery Rate. Results are shown as median CTX-III or net fibrolysis+Inter Quartile Range (IQR) unless otherwise stated. Biomarker tertial levels were defined as follow starting from lowest biomarker levels to highest: 1.sup.st-, 2.sup.nd-, and 3.sup.rd-tertile. All statistical analyses were performed in 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.

Results

[0148] Patients presenting with HCV related liver fibrosis was stratified according to their Ishak score at time of screening and their CTX-III levels compared to the levels of healthy donors. Independent of the degree of fibrosis, patients presenting with liver fibrosis had significantly (p<0.0001) elevated CTX-III levels at screening compared to the healthy donors (FIG. 6).

[0149] Calculating the degree of net fibrolysis (CTX-III/PRO-C3) at screening, a significant difference was discovered between patients presenting with various degree of fibrosis utilizing the Ishak classification (FIG. 7.). Patients with an Ishak score of 1-2 elicited a higher degree of net fibrolysis compared to patients with a score of 4-5 (p<0.05). The same was true for patients with a score of 3, in which the levels were significantly elevated compared to patients with a score of 4-5 (p<0.05)

[0150] A total of 47 patients were treated with a placebo for 52 weeks after which their Ishak score were determined. Patients were then stratified according to their change in the Ishak score from screening to 52 weeks and defined as having either a Regressive-, Stable-, or Progressive-fibrotic phenotype. Plotting the CTX-III levels at screening of each phenotype, biomarker levels were found significantly elevated in patients having a spontaneous regressive phenotype compared to a progressive phenotype (p<0.01). Additionally, those presenting with a stable fibrotic phenotype demonstrated significantly higher CTX-III levels at screening compared to the progressive phenotype (p<0.05) (FIG. 8A). By calculating net fibrolysis at screening allowed for a more significant differentiation between patient phenotypes as patients with a regressive phenotype displayed significantly higher levels of fibrolysis (p<0.001) compared to a progressive phenotype, while the same was true when comparing the stable and progressive phenotype (p<0.01) (FIG. 8B).

[0151] Dividing patients of the placebo group in tertiles based on either their CTX-III levels or levels of fibrolysis at screening, demonstrated a significant decrease of the Ishak score of patients with high levels of CTX-III at screening (3rd tertile) compared to patients with low initial CTX-III levels (1st tertile) (FIG. 9A). Observing the Ishak score changes based upon net fibrolysis, patients with levels in the 3rd tertile presented with a significant decrease compared to patients of the 1st tertile (p<0.001). Additionally, patients having levels of fibrolysis in the 2nd tertile at screening also presented with a significant decrease in their Ishak score compared to the 1st tertile (p<0.01) (FIG. 9B).

[0152] Based on the receiver operator characteristic (ROC) and its summary statistic, the Youden index, an optimal cut-off value for a regressive fibrotic phenotype was determined for both CTX-III and fibrolysis levels at screening (Table 13). Levels of CTX-III>3.8 ng/mL at screening resulted in a significant reduction in the Ishak score compared to patients with levels lower than the cut-off value (p<0.01) (FIG. 10A). The same was true for the net fibrolysis in which patients with a ratio of cross-linked type III collagen degradation (CTX-III) and type III collagen formation (PRO-C3) above 0.5 experienced a significant reduction in the Ishak score (p<0.01) compared to patients presenting with low levels of fibrolysis (FIG. 10B).

TABLE-US-00019 TABLE 13 Identification of spontaneous regressors using a specific cut-off value of either CTX-III levels or degree of net fibrolysis as screening and their associated values from the ROC curve. BIOMARKER, SENSITIVITY, SPECIFICITY, PLR, NLR, CUT-OFF VALUE % (95% CI) % (95% CI) (95% CI) (95% CI) CTX-III, 3.8 NG/ML 80 (28.4-99.5) 85.4 (70.8-94-4) 5.5 (2.3-12.9) 0.2 (0.04-1.4) NET FIBROLYSIS, 80 (28.4-99.5) 85.4 (70.8-94.4) 5.5 (2.3-12.9) 0.2 (0.04-1.4) 0.5 ROC, RECIEVER OPERATING CHARACTERSTIC; C; CONFIDENCE INTERVAL; PLR, POSITIVE LIKELIHOOD RATIO; NLR, NEGATIVE LIKELIHOOD RATIO

[0153] Patients were stratified based on cutoff levels determined by calculating the Youden index, with subsequent logistic regression calculating the odds ratio for being a regressor of fibrosis. This resulted in an odds ratio for being a spontaneous regressor of fibrosis of 19.4 times higher (p=0.0088) for patients with CTX-III levels ng/mL at screening compared with patients presenting with levels lower than this value. Observing patient levels of fibrolysis, a fibrolytic ratio (:).5 the odds ratio increased to 23.3 (p=0.0057) (FIG. 11).

Discussion

Type III Collagen in HCV Fibrosis

[0154] During the progression of fibrosis, pathogenic cell activation results in excessive formation of collagen within the ECM particularly the main fibrillar collagens of type I, III, and V. Combined with an increase of collagen cross-linking enzymes such as LOXLs and TGs, the increased fibrillar collagen deposition and subsequent cross-linking leads to increased tissue stiffening causing tissue disruption which may lead to organ failure (37). In HCV related liver fibrosis, unchecked viral infection results in loss of tissue homeostasis with resulting chronic inflammation causing expression of several inflammatory and fibrogenic cytokines including tumour growth factor-1 (TGF-(1). This cascade of fibrogenic cytokines ultimately activates quiescent hepatic stellate cells to transdifferentiate to myofibroblast (38). Myofibroblast constitutes the main effector cells of fibrosis responsible for ECM production and modulation of matrix stiffness mediated by extensive collagen cross-linking and ECM contraction (39). Accumulation of type III collagen in HCV related liver fibrosis have previously been shown with the biomarker PRO-C3 quantifying type III collagen formation through highly sensitive monoclonal antibodies targeting the NH.sub.2 pro-peptide of type III collagen (40,41). With the known relation of PRO-C3 and a fibrogenic phenotype, the association between fibrolysis and the novel biomarker CTX-III was investigated. Similar to the results investigating CTX-III levels in NAFLD, patients suffering from HCV related fibrosis presented with elevated levels of cross-linked type III collagen degradation in comparison to HDs. Furthermore, increased levels of fibrolysis in patients with the lowest degree of fibrosis (Ishak score 1-2) were observed, suggesting an increase of fibrolysis of mature cross-linked type III collagen as well as a decrease in type III collagen formation. These data indicate upregulated proteolysis of the cross-linked fibrotic ECM, with net fibrolysis is capable of differentiating between patients based on the degree of fibrosis.

Resolution of Fibrosis

[0155] Though fibrosis was long thought to be irreversible, understanding of fibrosis has changed in recent years, now acknowledging fibrosis as a dynamic process of fibrogenesis and fibrolysis. As new anti-fibrotic therapeutics are being developed, which includes targets such as LOXL2/3 (42), the end goal of fibrotic resolution is within reach. However, in order to optimise clinical management highly sensitive and specific tools capable of assessing patients accordingly is needed. Serological biomarkers of ECM turnover could provide a non-invasive tool for this, as has previously been demonstrated with the PRO-C3 biomarker identifying patients with a spontaneous progressive fibrotic phenotype (43,44). Dividing patients according to whether their fibrotic phenotype regressed, remained stable, or progressed after 52 weeks, significant differences were observed in both CTX-III and net fibrolysis levels between a regressive and progressive phenotype at the time of screening. These data suggest a prognostic potential of the biomarkers, in which patients with initial high levels of cross-linked type III collagen degradation i.e. fibroylsis experience a spontaneous resolution of their fibrotic ECM compared to patients with a lower degree of fibrolysis.

Identification of Regressors. Consequences for Therapy

[0156] Calculating a cut-off value for the biomarkers, demonstrated how patients in this exploratory study with CTX-III levels ng/mL or fibrolysis levels5 at screening had 19.4- and 23.3-times higher chance of presenting with a regressive phenotype. Biomarker cut-off levels can be used at screening to identify spontaneous regressors. Patients presenting with a spontaneous resolution of fibrotic ECM may need a lower treatment dose compared to patients with low initial fibrolysis as determined by the biomarkers. Consequently, this would lead to better patient stratification during trials, decreased expenses, increased patient welfare, and potentially aid in treatment development.

EXAMPLE 4 EOSINOPHILIC ESOPHAGITIS

Methods:

Patient Demographics and Clinical Evaluation

[0157] Twenty-nine adult EoE patients treated with an elimination diet were included for analysis. Dysphagia and EREF total score were assessed at baseline and after intervention by endoscopy.

TABLE-US-00020 TABLE 14 Display basic patient demographics of EoE patients at baseline and after intervention, including healthy donors. Demographics include age, gender, presence of dysphagia, and the Eosinophilic Esophagitis Reference Score (EREFS) total. EoE patients EoE patients after Healthy at baseline intervention donors P value N 29 29 20 Age, year range 37 22-62 37 22-62 46 18-61 0.0039 Gender, male % 17 58.6% 17 58.6% 10 50% NS Dysphagia daily, n % 14 48.3% 3 10.3% ND <0.0001 EREF total, 4.5 1-8 3.0 1-5 ND <0.0001 mean range

Statistical Analysis

[0158] Statistical variance between patient demographics and clinical parameters of patients at baseline and after intervention was determined by Fischer's Exact test for two groups or Chi square test for multiple groups.

[0159] Calculating the statistical differentiation between serum CTX-III of EoE patients at both timepoints and healthy donors were done by one-way ANOVA applying Kruskal-Wallis for non-parametric data. A p value below 0.05 was determined as being statistically significant.

Results

Cohort Description

[0160] There was a significant difference in the mean age of the EoE patients and healthy donors (p=0.0039), with the healthy donors being on average nine years older. Comparison of the EREF total score of the EoE patients at baseline and following the elimination diet, a significant reduction (p<0.0001) was observed (Table 14).

Diagnostic Potential of the CTX-III Biomarker, Distinguishing EoE Patients and Healthy Donors

[0161] Serum CTX-III levels were significantly elevated at both baseline and after the elimination diet (after intervention) compared to healthy donors (p<0.0001). No significant differentiation was demonstrated between baseline and the after-intervention levels (FIG. 12).

Discussion

[0162] By applying the CTX-III biomarker in a study of 29 EoE patients with blood-sampling at baseline and following a 6-week elimination diet as well as healthy donors, elevated serum CTX-III in EoE patients was demonstrated. Located in the interstitial matrix, deposition of type III collagen during fibrosis in EoE would primarily occur in the subepithelial layer by activated myofibroblast (46,49). In the final steps of collagen maturation, high amounts of secreted cross-linking enzymes mediate an extensive formation of intra- and inter-molecular cross-links. The ability of the heavily cross-linked and pathological collagen matrix of EoE patients to initiate myofibroblast differentiation of healthy donor fibroblast, underlines the importance of the ECM in propagating EoE related fibrosis (59). Clinical symptoms caused by esophageal fibrosis occur later than the actual onset of subepithelial fibrosis with the risk doubling for every 10-year of disease duration (60). Thus, early evaluation of fibrotic extracellular matrix remodelling is critical for early initiation of treatment (61). Here, quantification of protease degraded metabolites of cross-linked type III collagen demonstrated a diagnostic potential of the CTX-III biomarker. With significantly elevated serum levels of CTX-III in patients diagnosed with EoE, the biomarker could provide a supportive diagnostic tool in EoE, and potentially also to serve as pharmacodynamic marker for EoE. Though no anti-fibrotic treatment or markers to monitor fibrostenosis development or resolution of fibrosis is currently available for EoE, studies are ongoing for therapeutics targeting especially key pro-inflammatory cytokines of EoE pathogenesis. Current therapeutic options include administration of topical steroids and elimination diets (62). These two therapies have demonstrated reduction of esophageal eosinophilia but have yet to demonstrate reduction of fibrosis.

Conclusion

[0163] In the current study, EoE patients were placed on a 6-week elimination diet which did not result in any significant effect on the serum CTX-III levels. Although no changes in CTX-III biomarker levels by short term dietary intervention was observed, the significantly elevated levels of protease degraded and cross-linked type III collagen in EoE patients indicates the biomarker's potential in type III collagen remodelling of EoE related fibrosis.

EXAMPLE 5INFLAMMATORY BOWEL DISEASE

Methods

Patient Demographics and Pathological Evaluation

[0164] At the time of blood sampling patients were endoscopically assessed and scored according to the simple endoscopic score for CD (SES-CD). Patients with a SES-CD score of 0-1 was determined as endoscopically inactive, whereas a score>1 was determined as endoscopically active. Additionally, when a SES-CD score was unavailable, determination of an inactive or active disease was based patients Harvey-Bradshaw Index (HBI) score, which was determined on clinical parameters. A HBI score of 0-4 represented patients with a clinically inactive disease, while a score>4 determined patients with a clinically active disease.

[0165] Further patient stratification occurred by utilizing the Montreal classification for disease behaviour with patients separated into either the non-stricturing and non-penetrating (B1) or stricturing disease behaviour (B2). Patients in the Montreal Al group for patients with an age of disease onset of 16 years or below, and/or patients with a Montreal B4 classification for perianal disease behaviour was excluded from the analysis.

TABLE-US-00021 TABLE 15 Summary of patient demographics and stratification according to endoscopic-, or clinical disease activity as well as endoscopic disease behaviour. B1 (non- Healthy Ulcerative Crohn's stricturing non- B2 P value (B1 donors Colitis Disease penetrating) (stricturing) vs. B2) N 20 102 61 37 19 0.008 Age, mean 46 45.1 36.1 32.4 42.9 0.0118 range 18-61 18-80 19-76 19-76 18-72 Sex, male 10 57 33 21 10 NS (n %) 50% 55.9 54.1% 56.8% 52.6% BMI, mean ND 26.3 25.4 26.1 24.7 NS range 17.4-43.6 15.6-66.6 15.6-66.6 18.0-33.6 Smoking, ND 8 18 9 5 NS yes (n %) 7.8% 29.5% 24.3% 26.3% SES-CD ND ND 4 2 1 NS 0-1 (n %) 5.4% 5.3% HBI 0-4 (n ND ND 29 19 10 NS %) 51.4% 52.6 Montreal A1 ND ND 10 5 4 NS (n %) 16.4% 13.5% 21.1% Montreal B4 ND ND 5 3 0 0.0026 (n %) 8.2% 8.1% 0% Not determined (ND); Not significant, p > 0.05 (NS)

Statistical Analysis

[0166] Statistical variance between patient demographics and pathological parameters of patients in the B1 and B2 Montreal classification was determined by Fischer's Exact test.

[0167] Evaluation of plasma CTX-III or net fibrolysis (log(CTX-III/PRO-C3)) between healthy donors and patients diagnosed with either CD or ulcerative colitis (UC) was done by applying t-test or one-way ANOVA dependent on the number of comparing groups. Statistical difference was calculated by Kruskal-Wallis for non-parametric one-way ANOVA and either Mann-Whitney or unpaired t-test for non-parametric and parametric data. A p value below 0.05 was determined as being statistically significant.

Results

Patient Demographics and Pathological Evaluation

[0168] There were statistical higher number of patients in the B1 group compared to the number of patients in B2 (p=0.008). The age of patients with B1 were significantly younger than patients with a B2 classification (p=0.0118). Furthermore, patients with a B1 classification had a significant higher number of patients who also presented with perianal manifestations (B4) compared to patients with B2 (p=0.0026). No significant difference was demonstrated between the remaining demographics and pathological parameters.

Plasma CTX-III is Increased in Patients with Chronic Intestinal Inflammation

[0169] CD-, and UC-patients demonstrated significantly higher levels of plasma CTX-III compared to the healthy donors (p<0.0001). No statistical difference was found between patients of CD and UC (FIG. 13).

Differentiation of Luminal and Stricturing Disease Manifestations by Quantification of Plasma CTX-III

[0170] Plasma CTX-III was significantly elevated in CD patients presenting with an inactive disease and a non-stricturing and non-penetrating disease (B1) behaviour. The levels were elevated compared to patients with stricturing disease (B2) manifestations (p<0.01) (FIG. 14A). Additionally, by calculating net cross-linked fibrolysis (log(CTX-III/PC3X)) or net fibrolysis (log(CTX-III/PRO-C3)) non-stricturing and non-penetrating disease (B1) patients demonstrated higher levels of fibrolysis when compared to patients with stricturing disease (B2) (p<0.05, and p<0.01) (FIG. 14B+C).

Discussion

[0171] In this study the degree of fibrolysis in IBD patients was investigated by quantifying levels of protease degraded metabolites of cross-linked type III collagen (CTX-III) as well as the net cross-linked fibrolysis either by the CTX-III/PC3X ratio or net fibrolysis 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 when compared to the healthy donors (FIG. 13), and 2) Quantifying CTX-III or levels of cross-linked or non-cross-linked net fibrolysis (log(CTX-III/PC3X or PRO-C3)) could differentiate patients in clinical remission presenting with a luminal or structuring disease behaviour (FIG. 14).

[0172] The characteristic chronic inflammation of IBD maintaining activation of pathological wound healing is recognized as the key driver of extensive ECM remodelling. In CD patient's expression of fibrillar collagens were found significantly elevated compared to healthy individuals (70), with histological evaluation demonstrating excessive deposition in the different tissue layers of the intestines from the submucosa to the mucosa muscularis (68). Furthermore, both inflammatory cells and activated fibroblast cells produce increased amounts of MMPs resulting in increased collagen degradation, which in severe cases can result in the formation of fistulas.

[0173] Earlier studies by Haaften et al. (71) demonstrated the use of type III collagen biomarkers reflecting MMP mediated degradation and formation in differentiating CD patients based on endoscopic evaluation of disease behaviours. Patients with stricturing disease were associated with increased levels of the collagen formation markers reflecting the excessive deposition of collagens within the tissue, whereas collagen degradation was increased in patients with penetrating disease (71).

[0174] Here, increased levels of fibrolysis are demonstrated by quantifying proteolytic metabolites of cross-linked type III collagen and assessing the overall net fibrolysis in CD patients with an inactive disease presenting with a non-stricturing and non-penetrating disease behaviour (B1). These patients could be considered to present a less severe disease manifestation compared to patients with a stricturing disease (B2), and combined with an inactive disease, suggest a low degree of active inflammation as well. Enzymatic formation of intra- and inter-molecular cross-links of the fibrillar collagens deposited in the interstitial matrix of CD patients, represents the final step in collagen maturation. Thus, proteolytic degradation of cross-linked type III collagen would be associated with fibrolysis of the mature collagen fibrils. Patients with stricturing disease present with a high amount of collagen formation causing stricture formation, which combined with extensive collagen cross-linking could inhibit proteolytic degradation. This would be observed as the decreased levels of fibrolysis demonstrated in the current study. Therefore, patients with a non-stricturing and non-penetrating disease in which type III collagen deposition is increased though potentially cross-linked to a lesser extent, degradation and clearance of pathological type III collagen deposition is increased. These differences in the molecular processes of type III collagen remodelling between a non-stricturing and non-penetrating and a stricturing disease can thus be quantified by utilization of the CTX-III biomarker, and the ratio with either PC3X or PRO-C3 for net fibrolysis.

[0175] With the limitations of endoscopy and histological evaluation of intestinal fibrosis, inclusion of biomarkers such as CTX-III reflecting true fibrolysis, could prove beneficial in the clinical setting. By evaluating type III collagen remodelling on the molecular level by minimally invasive biomarkers, data supporting endoscopy and histology could be provided. The biomarkers could identify subclinical disease behaviours as well as providing subclinical information of treatment response. As therapeutics in fibrostenosis of CD is advancing, biomarkers such as CTX-III could be utilized for the assessment of fibrosis resolution.

Conclusion

[0176] The data presented show an increased degree of proteolytic activity releasing 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. Additionally, CD patients were stratified based on being in endoscopic and/or clinical remission (inactive), with subsequent stratification according to a Montreal classified non-stricturing and non-penetrating (B1) or stricturing disease (B2) behaviour. Here, patients in the B1 Montreal classification demonstrated the highest degree of fibrolysis compared to patients with a B2 classification.

EXAMPLE 6CANCER

Methods

[0177] Assay procedures were carried out as described above. These assays include CTX-III and PRO-C3.

[0178] The cohort included 20 patients each of pancreatic-, colorectal-, kidney-, stomach-, ovarian-, breast-, bladder-, lung-, melanoma-, head and neck- and prostate-cancer. It also included 3 liver cancer patients and 33 healthy controls. All cancer samples were obtained from Proteogenex (Los Angeles, CA, USA) and the healthy controls were obtained from BiolVT (Westbury, NY, USA).

TABLE-US-00022 TABLE 16 Cohort demographics CTX-III, Healthy Cancer Total median (N = 33) (N = 223) (N = 256) (ng/mL) Diagnosis Healthy 33 (100%) 0 (0%) 33 (12.9%) 4.66 bladder 0 (0%) 20 (9.0%) 20 (7.8%) 7.17 cancer breast 0 (0%) 20 (9.0%) 20 (7.8%) 8.93 cancer CRC 0 (0%) 20 (9.0%) 20 (7.8%) 7.90 H&N 0 (0%) 20 (9.0%) 20 (7.8%) 6.70 cancer kidney 0 (0%) 20 (9.0%) 20 (7.8%) 8.18 cancer liver 0 (0%) 3 (1.3%) 3 (1.2%) 11.96 cancer lung 0 (0%) 20 (9.0%) 20 (7.8%) 6.62 cancer melanoma 0 (0%) 20 (9.0%) 20 (7.8%) 5.68 ovarian 0 (0%) 20 (9.0%) 20 (7.8%) 5.73 cancer pancreatic 0 (0%) 20 (9.0%) 20 (7.8%) 6.49 cancer prostate 0 (0%) 20 (9.0%) 20 (7.8%) 6.48 cancer stomach 0 (0%) 20 (9.0%) 20 (7.8%) 5.97 cancer Stages I 0 (0%) 7 (3.1%) 7 (2.7%) II 0 (0%) 49 (22.0%) 49 (19.1%) III 0 (0%) 93 (41.7%) 93 (36.3%) IV 0 (0%) 74 (33.2%) 74 (28.9%) Missing 33 (100%) 0 (0%) 33 (12.9%) Age (years) Mean (SD) 57.7 (5.69) 59.3 (11.2) 59.1 (10.7) Median 57.0 49.0, 61.0 30.0, 60.0 30.0, Min, Max 69.0 87.0 87.0 Missing 0 (0%) 1 (0.4%) 1 (0.4%) Sex Male 21 (63.6%) 121 (54.3%) 142 (55.5%) Female 12 (36.4%) 102 (45.7%) 114 (44.5%) Ethnicity Black 13 (39.4%) 0 (0%) 13 (5.1%) Caucasian 11 (33.3%) 223 (100%) 234 (91.4%) Hispanic 9 (27.3%) 0 (0%) 9 (3.5%) CRC: Colorectal cancer H&N: Head & neck cancer

Results

Blood Levels of Protease Degraded Fragments of Cross-Linked Type ILL Collagen in Cancer

[0179] Serum CTX-III in healthy individuals and patients diagnosed with cancer revealed significantly elevated levels in seven out of twelve cancer types when comparing with healthy individuals. The biomarker levels were found 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 stomach cancer (p<0.05).

[0180] Patients with H&N-, liver-, ovarian, prostate-cancer, and melanoma did not demonstrate significantly elevated levels of CTX-III compared to healthy individuals (p>0.05). However, median CTX-III levels of all twelve types of cancer were elevated compared to the healthy individuals, with liver cancer demonstrating the highest median level of 11.96 (Table 1).

Stage III Breast Cancer is Associated with Increased Fibrolysis

[0181] When stratifying patients with breast cancer according to cancer stage, a significantly elevated level of CTX-III in patients with stage III breast cancer compared to patients with stage II (p<0.001) was observed. Additionally, by calculating the degree of net fibrolysis utilizing the ratio of CTX-III and PRO-C3, significantly higher levels of net fibrolysis were observed in patients with stage III compared to stage II (p<0.05) (FIG. 16).

Discussion

[0182] The investigation of the CTX-III biomarkers potential in evaluating the degree of proteolytic degradation of cross-linked type III collagen as well as the net fibrolysis in various types of cancer resulted in the following: 1) Levels of CTX-III were significantly elevated in seven out of twelve types of cancer compared to the healthy individuals, and 2) Patients with stage III breast cancer presented with higher serum CTX-III and net fibrolysis in comparison with stage II patients.

[0183] Whereas a healthy individual will experience a balanced ECM remodelling in which old collagens are degraded and replaced maintaining tissue homeostasis, this process is severely skewed in the tumor stroma. In the tumor stroma, cells such as CAFs drive the formation of an increasingly stiffer ECM through the deposition and cross-linking of mainly type I collagen but also type II, III, V, and XI. A primary cause of the increased matrix stiffness is the quantity of intra- and inter-molecular cross-links within the fibrillar collagens mediated by the enzymatic actions of LOXL(L)s and TG280.

[0184] Embedded within the CTX-III neo-epitope is a Lys suggested to be involved in LOX(L) mediated cross-linking (81), thereby allowing for specific quantification of cross-linked fragments released following proteolytic degradation of type III collagen. As such, increased release of MMPs, deposition of type III collagen and LOX(L) mediated cross-linking characterizing the tumor stroma would be indicated by increased levels of the CTX-III biomarker.

[0185] In agreement with this theory, increased levels of the CTX-III biomarker were observed in the current study in which the biomarker could differentiate between healthy individuals and cancer patients, identifying individuals with an underlying pathological degradation and cross-linking of type III collagen. However, of the twelve types of cancer investigated, H&N-, liver-, ovarian, prostate-cancer, and melanoma did not demonstrate a level of fibrolysis significantly higher than that of the healthy individuals. Though the levels in the current study were not statistically different from the CTX-III level of the healthy individuals, an increase was observed on median CTX-III of cancer patients. This indicates an overall elevated degree of proteolytic degradation of cross-linked type III collagen in these patients. The lack of statistical differentiation could be caused by a limited sample size, which is especially true observing the liver cancer patients consisting of only three patients.

[0186] Furthermore, both the CTX-III biomarker levels and the overall degree of net fibrolysis (CTX-III/PRO-C3) were elevated in the later stages of breast cancer. These data indicate the use of the CTX-III biomarker in diagnosing cancer patients, and potentially the biomarkers ability to stratify patients according to the severity of their disease. A significant differentiation was observed between stage III and stage IV breast cancer patients in terms of fibrolysis.

[0187] Targeting cross-linked collagen fragments for the assessment of pathological collagen remodelling in cancer patients was recently indicated in a study by Christina Jensen et al. Here, the researchers investigated the blood-based biomarker PC3X in a study of hepatocellular carcinoma together with the PRO-C3 biomarker 82. While PRO-C3 quantifies cross-linked and non-cross-linked N-terminal propeptides of type III collagen, reflecting type III collagen formation, the PC3X biomarker specifically targets cross-linked N-terminal propeptides. PC3X levels in hepatocellular carcinoma patients compared to PRO-C3 indicated increased levels of cross-linked type III collagen, supporting the increased quantity of collagen cross-linking in the tumor stroma.

[0188] In recent years the enzymatic cross-linking of collagens has been gaining interest within the therapeutic field of cancer83. As mentioned, enzymes such as LOX(L)s and TG2 drive the increasing quantity of cross-links, but the biochemical nature of the cross-links is also of critical importance. The enzymatic actions of especially intra- and extra-cellularly expressed lysyl hydroxylase 2 have shown to promote metastasis and reduce survival. LH2 mediates the hydroxylation of specific Lys within the collagen -chains resulting in a higher degree of Lys hydroxylated derived cross-links. Due to the important mechanistic roles in governing matrix stiffness and thereby enhancing tumor progression, LOXL2 and LH2 has been identified as targets for future therapeutic options (84).

[0189] Thus, utilizing blood-based biomarkers specifically targeting metabolites of cross-linked collagens could provide a quantitative measure reflecting CAF activity and the enzymatic activity of cross-linking enzymes. In a clinical setting, the biomarkers could potentially be utilized for diagnostic and prognostic purposes, separating patients based on the degree of fibrolysis and identify patients in which therapeutic options targeting collagen cross-linking would be beneficial.

Conclusion

[0190] Proteolytic fragments of cross-linked type III collagen reflecting fibrolysis were demonstrated to be released and quantifiable in twelve types of cancer, with median levels elevated in all types of cancer compared to healthy individuals. Though elevated in cancer patients, the CTX-III levels were only found significantly elevated in seven types of cancer. Furthermore, quantification of cross-linked type III collagen fibrolysis allowed differentiation between breast cancer patients in either stage II or stage III, with elevated levels associated with late-stage breast cancer.

[0191] It can be concluded that the CTX-III biomarker can be used for the quantification of cross-linked type III collagen fragments released into the circulation following proteolytic degradation, and thereby its use in a clinical setting of cancer patients.

Summary

[0192] The development and validation of a highly neo-epitope specific ELISA capable of measuring cross-linked fragments of type III collagen has been demonstrated. The assay was able to distinguish between HDs and obese patients suffering from NAFLD, HDs and patients with liver fibrosis, HDs and patients with EoE, HDs and patients with chronic intestinal disease, and HDs and cancer patients illustrating the CTX-III marker's relevance as a disease marker in pathologies with known accumulation of type III collagen and increased levels of cross-linking enzymes.

[0193] Furthermore, the CTX-III biomarker and calculation of the net fibrolysis ratio (CTX-III/PRO-C3) using the PRO-C3 biomarker demonstrated elevated levels in HCV related liver fibrosis with the ability to differentiate patients according to their spontaneous fibrotic phenotype. Calculation of net fibrolysis was also able to differentiate patients according to their disease severity in chronic intestinal diseases, in particular Crohn's disease, and cancer, such as breast cancer. Thus, the CTX-III biomarker and the related net fibrolysis ratio was not only capable of identifying patients with HCV related liver fibrosis, chronic intestinal disease or cancer but could also be applied as a prognostic biomarker potentially predicting response at screening.

[0194] In this specification, unless expressly otherwise indicated, the word or is used in the sense of an operator that returns a true value when either or both of the stated conditions is met, as opposed to the operator exclusive or which requires that only one of the conditions is met. The word comprising is used in the sense of including rather than in to mean consisting of. All prior teachings acknowledged above are hereby incorporated by reference. No acknowledgement of any prior published document herein should be taken to be an admission or representation that the teaching thereof was common general knowledge in Australia or elsewhere at the date hereof.

[0195] The following references are cited herein:

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