The present invention relates to a monoclonal antibody which is specifically reactive with the C-terminal neo-epitope of PIIINP, and its use in a method of immunoassay for detecting and quantifying PIIINP.

Background of the Invention

Fibrotic diseases (including those listed in Table 1) are a leading cause of morbidity and mortality, e.g. cirrhosis with 800,000 deaths per year worldwide [1] .

Table 1. Different fibrotic diseases [2] .

NFS

Pancreas Autoimmune/hereditary causes

Intestine Crohn's disease/inflammatory bowel disease

Brain Alzheimer's disease, AIDS

Bone Cancer, ageing

marrow

Multi- Surgical complications, chemotherapeutic drug- organ induced fibrosis, radiation-induced fibrosis, fibrosis mechanical injuries

A ^x fibrotic disease' is any disease giving rise to fibrosis, whether as a main or a secondary symptom.

Fibrosis is the end result of chronic inflammatory reactions induced by a variety of stimuli including persistent

infections, autoimmune reactions, allergic responses,

chemical insults, radiation, and tissue injury. Fibrosis is characterized by the accumulation and reorganization of the extracellular matrix (ECM) . Despite having obvious

etiological and clinical distinctions, most chronic fibrotic disorders have in common a persistent irritant that sustains the production of growth factors, proteolytic enzymes, angiogenic factors, and fibrogenic cytokines, which together stimulate the deposition of connective tissue elements, especially collagens and proteoglycans, which progressively remodel and destroy normal tissue architecture [3,4].

Despite its enormous impact on human health, there are currently no approved treatments that directly target the mechanisms of fibrosis [5] . Extracellular Matrix (ECM)

The ECM is a supramolecular structure with the ability to form aggregates of proteins, thus forming a dynamic scaffold linking cells together in a three dimensional network. This scaffold controls cell-matrix interactions and cell fate through up and down regulation of proteases [6] . The ECM consists of collagens, laminins, proteoglycans, and other glycoproteins in various amounts and combinations, thereby providing a variety of biological components which can be modified by proteases to produce scaffolds with specific functions to meet the needs of the individual tissue [7] .

Collagen types I and III are the major structural proteins in the human body. Collagen type III is essential for collagen type I fibrillogenesis in the cardiovascular system and other organs [8,9]. During fibrillar assembly the N-terminal propeptide of type III procollagen (which consists of three identical -chains with a total molecular weight of 42 kDa) is cleaved off by specific N-proteases prior to incorporation of the mature collagen in the ECM. The cleaved propeptides may either be retained in the ECM or released into the circulation. However, the cleavage of the propeptide is sometimes incomplete, leaving the propeptide attached to the molecule. This results in the formation of thin fibrils with abnormal cross-links, which in turn causes the abnormal molecule to be prone to rapid metabolic turnover [10,11] . Thus, the level of the N-terminal propeptide of type III collagen (PIIINP) in a suitable sample can be a marker of formation and/or degradation of collagen type III.

Remodeling of the ECM plays an important role in the

pathogenesis of various diseases as altered components and non-coded modifications of the ECM leads to tissue stiffness and changes in the signaling potential of the intact ECM and fragments thereof. ECM remodeling is an important

prerequisite for tissue function and repair, and is tightly controlled by the enzymes responsible for the synthesis and degradation of the ECM.

During pathological events, such as fibrotic diseases, the balance between the formation and the degradation of the ECM is disturbed, leading to an altered composition of the ECM. Such an alteration results in altered tissue function

[12,13] . It has been suggested that PIIINP could be used as a biomarker for several fibrotic diseases, such as lung injury [14], viral and non-viral hepatitis [15], systemic sclerosis [16], vascular remodeling [17], and kidney diseases [18] .

Limited attention has been given to the ECM remodeling in skeletal muscle tissue. In rat models increased collagen gene expression and biosynthesis have been demonstrated in

quadriceps femoris and tibialis anterior muscles after exercise [19,20]. Additionally, increased serum levels of PIIINP have been demonstrated in clinical studies after exercise [21] . Therefore, remodeling of the skeletal muscle proteins increases the quantity of PIIINP in the circulation and may serve as a biomarker for detecting early muscle anabolism. Serum levels of PIIINP have previously been suggested as a biomarker of muscular tissue response to testosterone [22], recombinant human growth hormone [23] or the combination thereof [24,25].

In liver fibrosis the fibrillar collagens type I and III are highly up-regulated [26,27] . Type III collagen is dominant in the early stages of fibrosis, while up-regulation of type I collagen is related to the later stages of fibrosis. Fibrosis occuring in the liver results in the deposition of collagen and release of propeptides, predominantly PIIINP.

Consequently, PIIINP is one of the best studied markers for fibrogenesis [28,29,30]. Through the years, several

radioimmunoassays have been developed for the quantification of PIIINP, with a sensitivity of up to 94% and specificity of up to 81% for the detection of cirrhosis [31,32]; however none of the previous assays are neo-epitope specific.

Additionally, the current commercially available assays for quantification of PIIINP utilise polyclonal antibodies or monoclonal antibodies targeting internal sequences of the procollagen or the propeptide and do not specifically

differentiate between the formation and/or degradation of collagen type III [31,32].

Thus, to differentiate between formation and degradation of collagen type III we consider that it is necessary to

determine and detect a neo-epitopic fragment which is solely produced in the formation process (i.e. a fragment which is produced in the formation of collagen type III but not produced in the degradation of collagen type III) .

Herein is disclosed a monoclonal antibody which is specific for the C-terminal PIIINP neo-epitope comprised in the terminal amino acids of the C-terminal amino acid sequence CPTGXQNYSP-COOH (SEQ ID NO : 4 ) , wherein X can be Gly or Pro.

Brocks [31] discloses a polyclonal antibody directed to the modified Bovine C-terminal PIIINP sequence IC*QSCPTGGENYSP- COOH (SEQ ID NO:l) (C* = acetamido protected Cys; Gin

replaced with Glu (E) ) , however said antibodies are non- specific towards the terminal amino acids of the bovine

PIIINP C-terminal sequence ICQSCPTGGQNYSP-COOH (SEQ ID NO: 2) and additionally said antibodies do not recognise human

PIIINP. Bayer [33] discloses a sandwich ELISA which utilises a detector monoclonal antibody directed to the sequence ¾N- GSPGPPGICQSCPTGPQNYSP-COOH (SEQ ID NO: 3), however the binding epitope is not defined.

Thus, the aim of the present invention is to provide a neo- epitope specific antibody directed towards the C-terminal neo-epitope of PIIINP and which is specific for its terminal character for use in a method of immunoassay for evaluating the disease severity of various fibrotic diseases.

Description of the Invention In a first aspect, the invention relates to a monoclonal antibody, wherein said monoclonal antibody is specifically reactive with a C-terminal neo-epitope of PIIINP, said neo- epitope being comprised in a C-terminal amino acid sequence CPTGXQNYSP-COOH (SEQ ID NO : 4 ) , wherein X is Gly or Pro, and wherein said monoclonal antibody does not substantially recognise or bind an elongated version of said C-terminal amino acid sequence which is CPTGXQNYSPQZ-COOH (SEQ ID NO: 5) , wherein Z is absent or is one or more amino acids of the sequence of collagen type III. In a preferred embodiment of the invention, the monoclonal antibody is specifically reactive with the neo-epitope C- terminal sequence CPTGPQNYSP-COOH (SEQ ID NO: 6) in human PIIINP, which is formed by the N-protease cleavage of PIIINP from intact procollagen type III at the Pro-Gin bond between amino acids P153-Q154 in human PIIINP.

In another preferred embodiment of the invention, the

monoclonal antibody is specifically reactive with the neo- epitope C-terminal sequence CPTGGQNYSP-COOH (SEQ ID NO: 7) in rodent PIIINP, which said neo-epitope is formed by the N- protease cleavage of PIIINP from intact procollagen type III at the Pro-Gin bond between amino acids P154-Q155 in rodent PIIINP. In another preferred embodiment of the invention, the ratio of the affinity of the monoclonal antibody for amino acid sequence CPTGXQNYSP-COOH (SEQ ID NO: 4) to the affinity of said monoclonal antibody for elongated amino acid sequence CPTGXQNYSPQZ-COOH (SEQ ID NO: 5) is at least 10 to 1, preferably at least 100 to 1, more preferably at least 1,000 to 1, more preferably at least 10,000 to 1, more preferably at least 100,000 to 1, and most preferably at least 1,000,000 to 1.

In another preferred embodiment of the invention, the

monoclonal antibody does not recognise or bind a shortened version of a C-terminal neo-epitope of PIIINP, said shortened neo-epitope having the amino acid sequence CPTGXQNYS (SEQ ID NO: 8) .

In another preferred embodiment of the invention, the ratio of the affinity of the monoclonal antibody for amino acid sequence CPTGXQNYSP-COOH (SEQ ID NO: 4) to the affinity of said monoclonal antibody for shortened amino acid sequence CPTGXQNYS-COOH (SEQ ID NO: 8) is at least 10 to 1, preferably at least 100 to 1, more preferably at least 1,000 to 1, more preferably at least 10,000 to 1, more preferably at least 100,000 to 1, and most preferably at least 1,000,000 to 1.

In another aspect, the present invention relates to a method of immunoassay for detecting in a biological sample the C- terminal neo-epitope of PIIINP generated by N-protease cleavage of intact type III procollagen, said method

comprising contacting said biological sample comprising said C-terminal neo-epitope of PIIINP with a monoclonal antibody as described herein, and determining the amount of binding of said antibody.

In a preferred embodiment of the invention, the method of immunoassay is used to quantify the amount of PIIINP cleaved from intact collagen type III in a biofluid, wherein said biofluid may be, but is not limited to, serum, plasma or amniotic fluid.

In another preferred embodiment of the invention, the method of immunoassay may be, but is not limited to, a competition assay or a sandwich assay.

In another preferred embodiment of the invention, the method of immunoassay may be, but is not limited to, a

radioimmunoassay or an enzyme-linked immunosorbent assay.

In another preferred embodiment of the invention, the method of immunoassay may further comprise correlating the quantity of PIIINP cleaved from intact collagen type III determined by said method with standard fibrotic disease samples of known disease severity to evaluate the severity of a fibrotic disease . In another preferred embodiment of the invention, the method of immunoassay may be used to evaluate the severity of liver fibrosis by correlating the quantity of PIIINP cleaved from intact collagen type III determined by said method with standard liver fibrosis samples of known disease severity.

In another preferred embodiment of the invention, the method of immunoassay may be used to evaluate muscle volume by correlating the quantity of PIIINP cleaved from intact collagen type III determined by said method with MRI- determined muscle volume.

In another embodiment, the present invention relates to a method for selecting patients having a fibrotic disease which is in a deteriorating condition for pharmaceutical trial or therapy, wherein said method comprises determining the severity of said fibrotic disease, determining the quantity of PIIINP cleaved from intact collagen type III using an immunoassay as described herein, and selecting from a group of patients determined to have an equivalent severity of said fibrotic disease those patients having a quantity of PIIINP above a statistical second quartile, and preferably in a statistical upper quartile. The severity of the fibrotic disease may be determined using any suitable method,

including, but not limited to, the Ishak fibrosis staging scale or METAVIR scoring. In another aspect, the present invention provides an assay kit for determining the quantity of PIIINP in a biological sample, said assay kit comprising a monoclonal antibody as described herein and at least one of:

- a streptavidin coated 96 well plate - a biotinylated peptide Biotin-L-CPTGPQNYSP (SEQ ID NO: 9) , wherein L is an optional linker

- a biotinylated secondary antibody for use in a sandwich immunoassay

- a calibrator peptide comprising the C-terminal sequence

CPTGPQNYSP-COOH

- an antibody HRP labeling kit

- an antibody radiolabeling kit

- an assay visualization kit Definitions

As used herein the term "neo-epitope" refers to an N- or C- terminal peptide sequence at the extremity of a polypeptide, i.e. at the N- or C- terminal end of the of the polypeptide, and is not to be construed as meaning in the general

direction thereof.

The term "competitive ELISA" refers to a competitive enzyme- linked immunosorbent assay and is a technique known to the person skilled in the art.

The monoclonal antibody NB61N-62 refers to a neo-epitope specific antibody directed towards the C-terminal neo-epitope of PIIINP, said neo-epitope comprising the C-terminal sequence CPTGXQNYSP-COOH (SEQ ID NO : 4 ) , wherein X is Gly or Pro .

The term "PRO-C3" is used to distinguish the herein described PIIINP assay from the PIIINP assays known in the art which are not based on the specific binding of neo-epitopes originating from PIIINP. Figures

Figure 1: Alignment of the targeted PIIINP al chain sequence in human and rat species (highlighted by the box) . Position of the corresponding human (—) and rat (—) sequences within the alpha 1 chain of the N-terminal pro-peptide of type III collagen. The alignment was performed using the NLP CLUSTALW software .

Figure 2: Western Blot showing the specific bands of N- terminal propeptide of type III collagen in Amniotic fluid from a) rat and b) human recognized by the monoclonal antibody NB61N62 (lane 1 and 3) and NB61N62 + selection peptide (lane 2+4) . Two bands around 52-60 kDA was observed for the rat, whereas one band was observed for human.

Addition of selection peptide resulted in weakness of band intensity for both rat and human.

Figure 3: PRO-C3 ELISA runs showing typical calibration curves and native reactivity against human, rodent, and mouse material. A) Calibration curve and inhibition of the

competitive PRO-C3 ELISA using healthy human serum, plasma, and amniotic fluid (AF) . The calibrator curve was diluted in 2-fold from 76.31 ng/mL, whereas native material was run diluted 1:2 to 1:16 as indicated (--) , B) Calibration curve and inhibition of the competitive PRO-C3 ELISA using healthy rat serum, plasma, and AF. The calibrator curve was diluted in 2-fold from 200 ng/mL, whereas native material was run undiluted to 1:8 as indicated (--) , C) Calibration curve and inhibition of the competitive PRO-C3 ELISA using healthy mouse serum and plasma. The calibrator curve was diluted in 2-fold from 200 ng/mL, whereas native material was run undiluted to 1:4 as indicated (--) , D) Neo-epitope specificity of the PIIINP neo-epitope specific antibody using elongated peptide, i.e. peptide sequence of calibration peptide with one additional amino acid in the C-terminal end. The calibration curve, elongated peptide, and non-sense peptide were diluted in 2-fold from 76.31 ng/mL. The signal is seen as the optical density at 450 nm, subtracting the background at 650nm, as a function of peptide concentration.

Figure 4: PIIINP levels measured in the CCI ₄ inhalation rat model: A) Serum levels of PIIINP was assessed using the PRO- C3 ELISA in samples from vehicle treated rats at termination (controls) as well as in CCI ₄ treated rats at termination (CCI ₄ ) at week 8, 12, 16, and 20. Results shown are mean ± standard error of the mean (SEM) ; B) Serum levels of PIIINP using the PRO-C3 ELISA in samples from vehicle treated rats and CCI ₄ rats stratified in quartiles according to total collagen in the liver; C-D) Correlation between PIIINP measured by PRO-C3 ELISA and Sirius red in samples from CCI ₄ treated rats and in vehicle rats. Asterisks indicate

statistical significance as indicated by bars. (*=p<0.05;

**=p<0.01; ***= p<0.001, ns = non-significant difference).

Figure 5: Serum PIIINP titers measured in the PRO-C3 ELISA at baseline correlate significantly with quad muscle mass measured by thigh MRI using Pearson correlation. Data have not been transformed. Figure 6: Comparison between PIIINP measured in the PRO-C3 ELISA and UniQ PIIINP serum levels in 20 randomly selected serum samples from healthy human individuals; ns = nonsignificant difference. Figure 7: Baseline levels of Plasma PIIINP in CHC patients stratified by Ishak stage. Pro-C3: Controls: 10.9 ng/mL

(n=22); Ishak stage 2: 15.8 ng/mL (n=78); Ishak stage 3: 17.4 ng/mL (n=88) and Ishak stage 4: 25.5 ng/mL (n=28) . The dotted horizontal line represents the level of controls. Data are presented as geometric mean ± standard error of the mean (SEM) . Asterisks indicate statistical significance indicated by bars : *p<0.05.

Figure 8: Diagnostic performance of Pro-C3. A) Receiver operating characteristic curve (ROC) analysis comparing controls (n=22) and mild fibrosis patients (Ishak stage 2 and 3, n=167) . B) ROC analysis comparing controls (n=22) and moderate fibrosis patients (Ishak stage 4, n=28) . C) ROC analysis comparing patients with mild fibrosis (n=167) and moderate fibrosis (n=28) . D) Odds ratio for selecting CHC patients. Patients and controls (n=222) were divided into quartiles according to Pro-C3 plasma level. The broken top- line in Q4 represents a value above 100. Asterisks indicate statistical significance compared to Ql : **p<0.001;

***p<0.0001.

Figure 9: Baseline levels of Plasma Pro-C3 in CHC patients stratified according to changes in Ishak stage after 52 weeks. Group -1: decrease of 1 in Ishak stage; Group 0: no change in Ishak stage; Group 1: increase of 1 in Ishak stage; and Group 2: increase of 2 Ishak stages. There is a

significant increase in Pro-C3 in patients in Group 1

compared to Group -1, and in patients in Group 2 compared to both Group 0 and Group -1. Data are shown as geometric mean ± SEM. Asterisks indicate statistical significance indicated by bars: *p<0.05. Figure 10: Prognostic performance of Pro-C3. A) ROC analysis comparing stable (n=103) and progressing fibrosis patients (n=36) yielded an AUC of 0.63 (p=0.023). B) Odds ratio of disease progression. Stable and progressing patients (n=139) were assigned into quartiles according to Pro-C3 plasma level. Q4 was associated with significant OR for disease progression (p=0.015), indicated by asterisk.

Figure 11: Concentration of PRO-C3 (geometric mean with SEM) in Hepatitis B Virus (HBV) patients classified according to Metavir score.

Figure 12: Concentration of PRO-C3 (geometric mean with SEM) in Hepatitis C Virus (HCV) patients classified according to Metavir score.

Examples Materials and general considerations

All reagents used in the experiments were high-standard 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 validation were 1) Immunogenic peptide: Ovalbumine (OVA) - CGG-CPTGPQNYSP (SEQ ID NO: 10), 2) Screening peptide: Biotin- CGG-CPTGPQNYSP (SEQ ID NO: 11), and 3) Selection peptide: CPTGPQNYSP (SEQ ID NO 6) . All synthetic peptides were

purchased from the Chinese Peptide Company, Beijing, China. Example 1 - Monoclonal antibody NB61-N62 Monoclonal antibody generation

The sequence for the N-terminal propeptide of type III collagen was aligned between human, rat and mouse species and selected from homology between the species and uniqueness among other ECM proteins by protein blasting. The amino acid sequence 145' -CPTGPQNYSP-'153 (SEQ ID NO: 6) in the l chain PIIINP is 100% homologues between human and rat (figure 1) . Generation of monoclonal antibodies was initiated by

subcutaneous immunization of 4-5 week old Balb/C mice with 200 μΐ emulsified antigen and 50 yg PIIINP neo-epitope C- terminal sequence (OVA-CGG-CPTGPQNYSP (SEQ ID NO: 10)) using Freund' s incomplete adjuvant. The immunizations were repeated every 2 weeks until stable serum titer levels were reached. The mouse with the highest serum titer was selected for fusion. The mouse was rested for a month and then boosted intravenously with 50 yg PIIINP neo-epitope C-terminal sequence in 100 μΐ 0.9% NaCl solution three days before isolation of the spleen. The spleen cells were fused with SP2/0 myeloma cells to produce hybridoma as described by

[34], and cloned in culture dishes using the semi-medium method. The clones were plated into 96-well microtiter plates for further growth employing the limited dilution method to secure monoclonal growth. The supernatants were screened for reactivity against calibrator peptide and native material in an indirect ELISA using streptavidin-coated plates. Biotin- CGG-CPTGPQNYSP (SEQ ID NO: 11) was used as screening peptide, while the free peptide CPTGPQNYSP (SEQ ID NO: 6) was used as calibrator to test for further specificity of clones. Clone characterization

Native reactivity and affinity of the peptide were assessed using different biological materials such as urine, serum, and amniotic fluid (AF) from both humans and rats in a preliminary ELISA using 2 ng/ml biotinylated peptide on streptavidin-coated microtiter plates and the supernatants from growing monoclonal hybridoma cells. Human AF was

obtained from 30 women undergoing elective lower segment Caesarean sections at the Beijing Obstetrics Gynecology

Hospital over a 2 month period. 100-200 ml AF was collected directly after incision and the fluid was stored at -20°C until use. The local ethical board had approved the study and all women provided written consent prior to collection. Rat AF was drawn from the uterus of pregnant Wistar rats two days prior to expected birth. Antibody specificity was tested in a preliminary assay using deselection and elongated peptides (i.e. calibrator peptide with ten amino acid substitutions and calibrator peptide with one additional amino acid at the cleavage site, respectively) . The isotype of the monoclonal antibodies was determined using the Clonotyping System-HRP kit, cat. 5300-05 (Southern Biotech, Birmingham, AL, USA) .

Antibody characterization

Prior to Western Blotting, the total protein concentration of human and rat AF was measured using Bicinchoninic acid (BCA) Protein Assay according to manufacturer's instruction.

Briefly, BCA was diluted 2-fold in PBS from 2 mg/ml to produce a standard row for calculation of the samples.

Samples were diluted 1:4 in lx phosphate-buffered saline (PBS) and 25 μΐ sample was added to a microtiter plate along with 200 μΐ working reagent (Reagent A and B mixed in the ratio 50:1) . The content was mixed on a plate shaker for 30 seconds followed by incubation for 30 minutes at 37°C. After ended incubation the plate was cooled to room temperature and the absorbance was measured in the ELISA reader at 562 nm (Molecular Devices, SpectraMax M, CA, USA) . Hereafter, rat or human AF was mixed with sample buffer (x2) and reducing agent (xlO), heated at 70°C for 10 minutes, loaded on a 4-20% tris- glycein sodium dodecyl sulphate-polyacrylamide gel

electrophoresis (SDS-page) , and run for 1 hour at 180V.

Protein bands were blotted onto a nitrocellulose membrane using the Invitrogen i-Blot gel transfer system according to manufacturer's instruction. The membrane was blocked in blocking buffer (5% skimmed milk in Tris-buffered saline with Tween (TBST) overnight at 4°C and incubated with 1 yg/ml horseradish peroxidase (HRP) -conj ugated PIIINP neo-epitope specific monoclonal antibody NB61N-62 for 2 hours.

Specificity of the PIIINP neo-epitope specific monoclonal antibody was investigated by addition of excess PIIINP neo- epitope calibrator peptide and antibody in the ratio 10:1 and allowed to pre-incubate for 1 hour before it was added to the membrane for overnight incubation. After incubation the membranes was washed 4x10 minutes in TBST, incubated with 4 ml chemiluminescence detection kit (ECL) , and developed using Amersham Hyperfilm. Clone selection and characterization

The subtype was determined to be an IgGl subtype. From the Western Blot analysis it was seen that the PIIINP neo-epitope specific monoclonal antibody NB61N-62 recognized two bands with molecular sizes around 52-60 kDa in rat amniotic fluid, while only one band around 52 kDa was detected in human amniotic fluid. In addition, the signal could be partly inhibited by the selection peptide in the rat, and inhibited in human (Figure 2) . Native reactivity was observed using the NB61N-62 antibody in the ELISA. Native reactivity was seen towards human serum, plasma, and AF as well as against rodent serum, plasma, and AF (Figure 3A-C) . The signal was slightly less inhibited against mouse serum and plasma. The signal of the competitive ELISA was inhibited using from 1:2 to 1:16, undiluted to 1:8, or undiluted to 1:4 in human, rodent, and mouse native material, respectively. Dilution of the native material approximately followed the same dilution pattern as the calibrator curve for all three species. Human AF

inhibited the signal up to 100%; 80% for rat AF; 70% for human serum and plasma and rat serum; 44% for rat plasma, and 35% for mouse serum and plasma. Zero inhibition was observed using the elongated peptide (CPTGPQNYSPQ (SEQ ID NO: 6)) and non-sense peptide (GSPGKDGVRG (SEQ ID NO: 12)) (Figure 3D).

Example 2 - PRO-C3 ELISA using NB61N-62

Supernatant from the antibody producing hybridoma was

collected and the monoclonal antibody was purified using

HiTrap affinity columns (GE Healthcare Life Science, Little Chalfont, Buckinghamshire, UK) and labeled with HRP using Lightning-Link™ HRP Conjugation Kit (Innova Biosciences, Babraham, Cambridge, UK), according to the manufacturer's instructions.

The PRO-C3 competitive ELISA procedure was as follows: A 96- well streptavidin-coated ELISA plate from Roche,

cat .11940279, was coated with the biotinylated peptide

Biotin-CGG-CPTGPQNYSP (SEQ ID NO: 11) dissolved in coater buffer (50mM PBS-BTE + 10% sorbitol, pH 7.4), incubated for 30 min at 20°C in the dark and subsequently washed in washing buffer (20 mM Tris, 50 mM NaCl, pH 7.2). Thereafter 20 μΐ of peptide calibrator or sample were added to appropriate wells, followed by 100 μΐ of HRP-conj ugated monoclonal antibody NB61N-62 dissolved in incubation buffer (50 mM PBS-BTB + 10% Liquidll (Roche), pH 7.4) and the plate was incubated for 20 hours at 4°C and washed. Finally, 100 μΐ

tetramethylbenzinidine (TMB) (Kem-En-Tec cat.: 4380H) was added, the plate was incubated for 15 min at 20 °C in the dark and in order to stop the reaction, 100 μΐ of stopping

solution (1% H ₂ SO ₄ ) was added and the plate was analyzed in the ELISA reader at 450 nm with 650 nm as the reference

(Molecular Devices, SpectraMax M, CA, USA) . A calibration curve was plotted using a 4-parametric mathematical fit model.

Technical Evaluation

A 2-fold dilution of healthy serum and plasma samples from human and rats were used to determine linearity and

calculated as percentage of recovery of the 100% sample.

Antibody specificity was calculated as percentage of recovery of the 100% calibrator peptide (CPTGPQNYSP (SEQ ID NO: 6)), elongated peptide (CPTGPQNYSPQ (SEQ ID NO: 13)), and non ^¬ sense peptide (GSPGKDGVRG (SEQ ID NO: 12)). Lower limit of detection (LLOD) was calculated as the mean + 3xStandard Deviation (SD) of the blank from 21 determinations of

standard K (i.e. buffer) . Upper limit of detection (ULOD) was determined as the mean - 3xSD of 10 measurements of Standard A. Lower limit of quantification (LLOQ) was determined as the lowest concentration reproducibly measured with a precision lower than 30%. The intra- and inter-assay variation was determined by 10 independent runs of 8 QC samples, with each run consisting of double determination of the samples.

Accuracy of the samples was measured in healthy human serum samples spiked with standard curve or human amniotic fluid at significant concentrations and calculated as the percentage recovery of the theoretical amount of serum. Interference was measured in healthy human serum spiked with hemoglobin, lipemia, and biotin at significant concentrations and

calculated as the percentage recovery of the theoretical amount of serum.

Results

The measurement range of the human PRO-C3 ELISA was

determined by calculating ULOD and LLOQ providing a range from 0.867-60.1 ng/ml with a LLOD of 0.606 ng/ml . The

technical performance of the PRO-C3 ELISA showed acceptable inter- and intra assay variation of mean 11.03% and 4.11% (Table 1), with acceptance range below 15% and 10%,

respectively .

Table 1: Inter- and intra-assay variation for the PRO-C3 assay using human serum quality control samples # 1-8 (HS1- HS8) . The variation was calculated as the mean variation between 10 individual determinations of each sample.

Sample Value Intra-assay Inter-assay

(ng/itiL) variability % variability %

HS1 24.24 2.28 5.94

HS2 11.62 2.90 6.45

HS3 8.40 5.31 11.99 HS4 6.54 4.46 11.31

HS5 6.36 3.88 13.09

HS6 5.23 3.98 12.31

HS7 4.29 3.53 12.94

HS8 2.98 4.66 18.56

Mean 4.11 11.03

Dilution recovery was performed using healthy serum and plasma samples from humans, rat and mouse. The dilution recovery was within the acceptable 100120% recovery (Table 2) . Further dilution resulted in measurements below LLOQ.

Table 2: Percentage dilution recovery for the PRO-C3 assay using human-, rat-, and mouse samples. Human serum (HS) ,

Human piasma (HP) , Rat serum (RS) , Mouse serum (MS) , Mouse plasma (MP) .

PIIINP HS HP RS MS MP

ng/ml (n=2) (n=3) (n=10) (n=2) (n=2)

Undiluted 100% ⁼ 100% 100% ⁼

Dilution 98 100% 116 96 100%

1:2

Dilution 103 91 110 118 114

1:4

Dilution 114 87 - - - 1 : 8

Dilution - 92 - - -

1 : 16

Mean Ϊ05 90 ΪΪ3 Ϊ07 ΪΪ4

Spiking of calibrator peptide in serum or plasma resulted in a mean recovery of 56% and 55%, respectively (Table 3) .

Table 3: Spiking recovery of calibrator peptide in human serum or plasma, and human AF in human serum or plasma. The recovery was calculated as percent recovery of calculated peptide/AF in serum/plasma compared to pure serum/plasma. Concentration of calibrator peptide were 38.16 ng/ml (StdB) , 19.08 ng/ml (StdC) , 9.54 ng/ml (StdD) , 4.77 ng/ml (StdE) , 2.39 ng/ml (StdF) and 1.19 ng/ml (StdG) . AF was added in 2- fold dilution starting from 1:2.

Serum Plasma Serum Plasma (n=3) (n=3) (n=3) (n=3)

Added Added AF

Std sRE% sRE% sRE% sRE%

StdB 16 15 2x 101 103

StdC 29 25 4x 103 108

StdD 42 38 8x 106 113

StdE 58 54 16x 103 112

StdF 70 69 32x 104 115

StdG 82 83 64x 103 110 Buffer 92 100 Buffer 99 104

Mean Mean

56 100 55 111 sRE% sRE%

However, spiking of human AF in 2-fold dilution starting from 1:2 into healthy human serum or plasma resulted in mean recovery of 100% and 111%, respectively. No interference was observed in serum spiked with different concentrations of hemoglobin, biotin, and lipemia (Table 4) .

Table 4: Interference of hemoglobin, lipemia and biotin in human serum added in various concentrations. All data are shown as percent recovery compared to pure serum.

Hemoglobin Lipemia Biotin mmol/L RE% mmol/L RE% ng/L RE%

0.5 68 0.56 101 160, 000 134

0.25 74 0.28 103 80,000 113

0.13 81 0.14 99 40,000 96

0.063 81 0.07 104 20,000 97

0.031 82 0.04 101 10,000 94

0.016 86 0.00 100 5, 000 87

0.008 95 2,500 100

0.000 100 0 100 Mean 83 101 103

The stability of the analyte was acceptable up to four freeze/thaw cycles with 100120% recovery compared to 1 freeze/thaw cycle (Table 5) .

Table 5: Analyte stability in three human serum and plasma samples in four freeze/thaw cycles. All data are shown as mean percent recovery compared to 1 freeze/thaw cycle.

Freeze/thaw Serum EDTA Heparin Citrate cycle plasma plasma plasma

Mean

recovery % Mean Mean Mean

recovery % recovery % recovery

1 100 ^! 100 ^! 100 ^! 100 ^;

103 102 103 109

3 99 99 9^ 103 4 102 100 9^ 100

Example 3 - Determining the ratio of binding affinity

To determine the ratio of the binding affinity of the

monoclonal antibody for the target sequence to the binding affinity of the monoclonal antibody for the elongated or shortened sequence, each of the sequences are synthesized and used as calibrator peptides in the PRO-C3 ELISA as described in example 2. The resultant calibration curves are used to determine the IC ₅₀ values of each sequence/antibody combination. The ratio of IC ₅ o [target] / IC ₅ o [elongated or shortened] defines the ratio of binding affinity.

Example 4 - Rat CC1 ₄ liver fibrosis model

Serum levels of PIIINP were assessed in a CCI ₄ inhalation rat model of liver fibrosis. Complete details of the study are described elsewhere [35] . The study included 52 male Wistar rats treated with CCI ₄ and 28 male Wistar vehicle rats

(Charles-River, Saint Aubin les Elseuf, France) . Induction of liver fibrosis was performed as previously described by others [36] . Briefly, CCI ₄ was administered by inhalation twice a week, starting with 0.5 minutes per exposure. The duration of exposure was increased by one minute after every three session until it reached five minutes, which was used until the end of the investigation. Phenobarbital (0.3 g/1) was added to the drinking water and vehicle rats received phenobarbital only. Animals were stratified into groups receiving 8, 12, 16, or 20 weeks of CCI ₄ or vehicle treatment (n=13 for CCI ₄ ; n=7 vehicle for each group) . The study was performed according to the criteria of the Investigation and Ethics Committee of the Hospital Clinic Universitari

(Barcelona, Spain), approval #B-NNP-233/09. Four animals from the CCI ₄ groups died during the study. Blood was collected at termination and allowed to stand at room temperature for 20 min to clot before centrifugation at 2500 rpm for ten

minutes. Samples were stored at -80°C prior to biomarker assessment in the PRO-C3 ELI SA .

Results

Serum levels of PIIINP determined in the PRO-C3 ELI SA were statistically elevated in CCI ₄ treated rats compared to vehicle rats at week 8 (+30.17% increase, p<0.001), week 12 (+26.58% increase, p<0.05), and week 16 (+44.15% increase, p<0.05), however not in week 20 (+6.24 ~6 increase, p—ns) (Figure 4A) . When rats were stratified into quartiles

according to total amount of collagen in the liver assessed by Sirius Red staining, PIIINP was statistically elevated in all four quartiles compared to vehicle animals (quartile 1 (Ql) +21% increase, p<0.01; Q2 +21% increase, p<0.05; Q3 +33% increase, p<0.001; Q4 +56% increase, p<0.001) (Figure 4B) . Furthermore, serum levels of PIIINP were correlated to the total collagen in the liver. PIIINP correlated significantly to collagen in CCI ₄ rats (r=0.46, p<0.01), however not in vehicle rats (r=0.07, p=ns) (Figure 4C-D) .

During liver fibrosis the amount of ECM components are known to be highly increased, up to 6 fold [37], including type III collagen, and it is well known that PIIINP is a marker for describing liver fibrosis [34,35,36,37]. The PRO-C3 ELI SA described herein was used to evaluate the quantity of PIIINP in a rat model of liver fibrosis. It was found that serum PIIINP was significantly elevated at termination after 8, 12 and 16 weeks and when stratified into quartiles of the total amount of collagen compared to controls. At the 20 week termination point serum PIIINP had regressed back to control levels. These data indicate that this marker reflects

fibrogenesis rather than degradation since the serum PIIINP determined by the PRO-C3 ELISA were initially high in this model .

Example 5 - Muscle loss

PIIINP was measured by PRO-C3 ELISA in plasma samples from young men (n=ll, age: 24.4 ± 0.5 y, height: 181.4 ± 1.8 cm, weight: 72.2 ± 2.3 kg) that were subjected to two weeks of unilateral leg immobilization (through full leg casting) followed by four weeks of resistance training remobilization . Subjects were sampled for venous blood, leg muscle volume and strength at baseline (PRE) , after immobilization (2W) and after remobilization (4W) . During immobilization the subjects lost approximately 9 and 20%, of muscle size (MRI-determined quadriceps muscle volume) and strength (knee extensor force measured by maximal voluntary contraction in KinCom device) respectively, as previous reported [38]. Subjects were not fasted or placed on custom diets prior to testing and

sampling. Samples were stored at -80°C prior to biomarker assessment in the PRO-C3 ELISA.

Results Levels of PIIINP did not differ significantly between

intervention time points when correlated against muscle mass at baseline, however a significant positive correlation was observed between PIIINP and muscle volume (R ² =0.4416,

P=0.0361) (Figure 5), meaning that higher PIIINP levels strongly predict higher muscle mass in healthy individuals not exposed to any intervention.

Example 6 - Comparison of competitive PRO-C3 ELISA and UniQ PIIINP assays

20 randomly selected healthy human serum samples were

evaluated for PIIINP using the competitive PRO-C3 ELISA and the results compared with the results obtained by measuring the level of PIIINP using the UniQ PIIINP RIA (Orion

Diagnostica, Espoo, Finland) according to the manufacturer's instructions . Results

Serum levels of PIIINP as determined by the competitive PRO- C3 ELISA did not correlate significantly to serum levels of PIIINP as determined by the UniQ PIIINP RIA (R ² =0.12, p=ns) (Figure 6) .

The competitive PRO-C3 ELISA described herein quantifies the formation of type III collagen and not degradation. The lack of correlation between the results from said competitive PRO- C3 ELISA and the UniQ PIIINP RIA is further evidence that the commercially available immunoassays for PIIINP detection and/or quantification do not differentiate between PIIINP formed by collagen type III formation and PIIINP formed by collagen type III degradation.

Example 7 - PIIINP assessments in plasma samples from

patients with Chronic Hepatitis C (CHC)

The study cohort was from a multicenter, phase II clinical study to assess the effectiveness of farglitizar, a

peroxisome proliferator-activated receptor-gamma agonist, as a potential antifibrotic compound for adult CHC patients (NCT00244751) as described previously [39] . This study subsequently found no significant effect of this compound on fibrosis or stellate cell activation after 12 months. Plasma samples were available from a subpopulation of 194 patients with CHC genotype 1 infection and compensated liver disease with Ishak fibrosis stage 2-4. 131 patients received 0.5 or 1.0 mg farglitazar twice a day and 63 received matching placebo for 52 weeks. Liver biopsies at baseline and 52 weeks were reviewed by a single experienced histopathologist using the Ishak modified histologic activity index (HAI) for grading and staging [40] . These methods and specimen quality measures have been described in detail previously [39] . The controls were derived from a previously described study [41, 42] . This study was approved by the Duke University

Institutional Review Board.

Type III collagen formation was assessed in all baseline plasma samples from CHC patients and healthy controls using the herein described competitive ELISA for PIIINP ("Pro-C3") .

Baseline plasma Pro-C3 in healthy controls and CHC patients No difference between treated patients and placebo was observed for Pro-C3 (p=0.299) (data not shown) . Pro-C3 levels showed an overall significant difference in all group

comparisons for Ishak stage 2, 3 and 4 (p<0.001) (Figure 7) . Pro-C3 levels were increased in Ishak stage 4 compared to stage 2 (p<0.05) or stage 3 (p<0.05).

The diagnostic value of Pro-C3 for separation of healthy and CHC patients was performed using ROC analysis. The AUC was 0.82 (p<0.001) and 0.91 (p<0.001) for distinguishing controls from patients with mild (Ishak stage 2 and 3) and moderate (stage 4) fibrosis, respectively (Figure 8A-B) . AUC for differentiating mild from moderate fibrosis stages was 0.72 (p<0.001) for Pro-C3 (Figure 8C) . Controls and CHC patients were further divided into quartiles according to their Pro-C3 plasma level. The odds ratios (OR) for differentiating controls from CHC patients was calculated for each quartile

(Q) (Fig. 8D) : Ql was set to an OR=l, Q2 : OR=4.1 [95% CI 1.4- 12.1], Q3: OR=21.6 [95% CI 2.7-169.7], and Q4 included only CHC patients. These results demonstrate a clear ability of the Pro-C3 immunoassay to distinguish between healthy and CHC patients.

Association between baseline plasma Pro-C3 and progression of disease The association between baseline biomarker levels and

regression or progression of disease after 52 weeks was investigated by comparing the patients with a decrease of 1 in Ishak stage (group -1, n=20), the stable patients (group 0, n=103) , the patients with an increase of 1 (group 1, n=30) and with an increase of 2 in Ishak stage (group 2, n=6) .

There was an overall difference in baseline Pro-C3 mean levels (p=0.005) between the four groups (Figure 9) . Pro-C3 levels were significantly higher in group 1 compared to group -1 (p=0.049) and for group 2 compared to both -1 (p=0.012) and 0 (p=0.037) (Figure 9).

The prognostic value of Pro-C3 for disease progression was investigated in the different baseline Ishak stages 2, 3 and 4. Patients in each stage were classified as "progressors" (n=36) or "stable" (n=103) and means were calculated. Plasma Pro-C3 levels were significantly elevated for progressors compared to stable CHC patients in Ishak 2 (p=0.014) and Ishak 3 (p=0.020) . There were no significant differences for Ishak 4 (Data not shown) . ROC analysis of the Pro-C3

prognostic value was performed (Figure 10A) , yielding an AUC of 0.63 (p=0.030), when stable patients were compared to progressors. For differentiating progressors from stable patients the OR was calculated in the Pro-C3 quartiles (Q) : Ql was set to OR=l, Q2 : OR=l .0 [95% CI 0.28-3.63], Q3 : OR=1.5 [95% CI 0.48-4.72], and Q4 : OR=3.4 [95% CI 1.21-9.69]

(p=0.015) (Figure 10B) . These results demonstrate the prognostic ability of the Pro- C3 immunoassay for detecting CHC patients whose condition is deteriorating (i.e. liver fibrosis is increasing), or likely to deteriorate, particularly for patients with an Ishak score of 2 or 3. Specifically, patients of a given Ishak score that have a Pro-C3 value of above the statistical second quartile (>Q2) for that Ishak score will likely have a deteriorating condition. This is particularly useful

information when selecting patients for pharmaceutical trials and/or when prescribing drug therapy as the level of false results based on patients "responding" to preventative treatment may be reduced by eliminating patients who would not have deteriorated without treatment.

Example 8 - PRO-C3 ELISA for assessment of Liver Fibrosis

The clinical utility, i.e. sensitivity and specificity, of the PRO-C3 ELISA in patients with liver fibrosis was investigated in two study populations, i.e. patients with chronic hepatitis B (HBV) infection and another group of patients with chronic hepatitis C (HCV) infection. Patients and methods

Patients with HBV and HCV

A cross-sectional study in 189 patients with chronic HBV infection and 375 patients with chronic HCV infection was conducted. Presence and severity of liver fibrosis was evaluated using liver biopsies as described below.

Briefly, 96-well pre-coated streptavidin plates (Roche Diagnostics, Mannheim, DE) were coated with the appropriate biotinylated synthetic peptides (biotin-CGG-CPTGPQNYSP (SEQ ID NO: 11)) and incubated for 30 minutes at 20°C. 20 yL of standard peptide (CPTGPQNYSP (SEQ ID MNO: 6)) or pre-diluted sample were added to appropriate wells, followed by 100 yL of peroxidase-conj ugated specific monoclonal antibodies and incubated for 1 hour or overnight at 20°C or 4°C, respectively. Finally, 100 yL tetramethylbenzidine (TMB) (cat.4380H, Kem-En-Tec Diagnostics, Taastrup, Denmark) was added, and the plates were incubated for 15 minutes at 20°C in the dark. All the above incubation steps included shaking at 300 rpm. After each incubation step, the plate was washed five times in washing buffer (20 mM Tris, 50 mM NaCl, pH 7.2) . The TMB reaction was stopped by adding 100 yL of stopping solution (0.18 M H2S04) and measured at 450 nm with 650 nm as the reference. A calibration curve was plotted using a 4-parametric mathematical fit model.

The stained liver biopsies were examined by experienced pathologists and scored according to the Metavir scoring system for the stage of fibrosis (f0-f4) . This system assesses histologic lesions in the liver and the scores are defined as follows:

f0 : no fibrosis

f1 : portal fibrosis without septa

f2 : portal fibrosis with rare septa

f3: numerous septa without cirrhosis

f4 : cirrhosis Data were logarithmically transformed to obtain normality and symmetry of variance. Comparisons between the mean marker levels stratified according to F-score were performed using one-way Analysis of Variance (ANOVA) test with Tukey' s multiple comparisons test using each group as fixed factor. Correlations were calculated as the Pearson Rho coefficient. Data are shown as geometric mean ± standard error of the mean (SEM) . P-values less than 5% were considered significant. All statistical analyses were calculated in MedCalc® version 12 (MedCalc Software, Ostend, Belgium) and graphs were designed using GraphPad Prism® version 5 (GraphPad Software, Inc., CA, USA) .

Patients with HBV infection

First, the demographic data and the PRO-C3 data were

summarized according to Metavir F score (table 6) .

By ANOVA statistics it was demonstrated that PRO-C3 test results classified according to Metavir score differed significantly with liver fibrosis (p<0.001). After

adjustment for the co-variables age and BMI this relationship remained significant (data not shown) . In contrast, the degree of liver fibrosis as assessed by

Metavir score did not show a significant relation to age, BMI and gender.

Table 6. Demographics and PRO-C3 Results According to

Metavir F-score.

HBV METAVIR F stage ANOVA n 0 n 1 n 2 n 3 n 4 P

Mean

age 41.0 40.3 44.5 37.4 51.7

39 95 35 16 4 0.080

(year (9.9) (11.9) (11.1) (12.8) (15.0)

(SD))

BMI 39 23.9 95 24.1 35 24.6 16 23.8 4 24.7 0.962

(+/- (23.3- (23.8- (23.9- (22.6- (21.8- SEM) 24.6) 24.5) 25.3) 25.1) 28.0)

Male 20 51% 58 61% 24 69% 13 81% 3 75% 0.253 (%)

PRO-C3 39 18.2 95 19.6 35 24.9 16 37.8 4 27.2 <0.001

(+/- (16.8- (18.8- (23.1- (32.6- (18.0- SEM) 19.6) 20.4) 26.8) 43.8) 41.1) Age followed a normal distribution and is reported as geometric mean with standard deviation. BMI and PRO-C3, however, did not follow a normal distribution and are

reported as geometric mean ±SEM.

In a graphic representation of the same data (Figure 11), the association of the liver fibrosis score with the PRO-C3 assessment in EDTA plasma was clearly observed. Due to the low number of patients with Metavir F score 4 (n=4), this group was merged with Metavir F score 3.

Clinically, the most important decision point is the ability to detect liver fibrosis in its early stages. Therefore, the ability of the PRO-C3 to distinguish patients with Metavir F score 0-1 from the more advanged stages (score 2-4), was investigated. A ROC analysis demonstrated that using a cut off of 19.21 ng/mL for PRO-C3, the test had a sensitivity and specificity of 76.4 and 61.7%, respectively (Table 7) . The ability to distinguish early from moderate to late liver fibrosis was statistically highly significant (p<0.0001). Positive and negative predicted values for PRO-C3 were in the range 44-57% (Table 7) .

Table 7. PRO-C3 : ROC Analysis on Metavir F score

PPV: Positive predictive value; NPV: Negative predict value . Patients with HCV infection

Demographic and PRO-C3 data are summarized according to

Metavir F score in the table below (Table 8) . By ANOVA statistics, it was demonstrated that PRO-C3 test results differed significantly with liver fibrosis as assessed by

Metavir F score. In this study population, severity of liver fibrosis was associated with both older age and increased BMI, however, the association between PRO-C3 and the Metavir score remained significant even after adjustment with the co- variables age and BMI (data not shown) .

Table 8. Demographics and PRO-C3 Results According to

Metavir F-score

Age followed a normal distribution and is reported as

geometric mean with standard deviation. BMI and PRO-C3, however, did not follow a normal distribution and are

reported as geometric mean ±SEM.

In a graphic representation of the data (Fig. 12), the association of the liver fibrosis score with the PRO-C3 assessments in plasma was clearly observed. ROC analysis demonstrated that using a cut-off of 22.21 ng/mL for PRO-C3, the test had a sensitivity and specificity of 68.4 and 72.6%, respectively (Table 4). The ability to distinguish early from moderate to late liver fibrosis was statistically highly significant (p<0.0001). Positive and negative predicted values for PRO-C3 were in the range 49-51% (Table 9) .

Table 9. PRO-C3 : ROC Analysis on Metavir F score

PPV: Positive predictive value; NPV: Negative predict value .

In this specification, unless expressly otherwise indicated, the word ^x 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. References

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