Patent Description:
Chronic liver disease (CLD) is a major cause of mortality and morbidity, accounting for over <NUM> million deaths per year worldwide. Approximately <NUM> million of these CLD related deaths is due to viral hepatitis, of which hepatitis C is a leading cause<NUM>. Common amongst CLD aetiologies is the development of liver fibrosis with the eventual endpoint being liver cirrhosis. Liver fibrosis is the result of a protracted wound healing response and is characterised by the accumulation of extracellular matrix (ECM), such that the ECM composition of the liver is qualitatively and quantitatively different from that of a healthy one. Recent reports have indicated that the ECM composition of the fibrotic liver is a key determinant of patient survival, which suggests the value of tissue profiling patients. Fibrotic lesions are primarily composed of collagens, which can be broadly divided into two groups; interstitial matrix and basement membrane. The basement membrane regulates cellular function and is a reservoir of growth factors. The accumulation of basement membrane matrix is associated with a reparative tissue response. Conversely, the accumulation of interstitial matrix is associated with more severe and progressive disease.

The multiplexin type XVIII collagen is unique amongst the collagen family. Type XVIII collagen is a heparan sulphate proteoglycan located in the basement membrane, where it interacts with laminin, perlecan, nidogen, fibulins, type IV and VI collagens, playing an essential role in the flexibility of the matrix milieu<NUM>,<NUM>. Three isoforms exist with differences in their respective distribution; the short isoform is found mostly in the vasculature and skeletal muscle, whereas the intermediate and long isoforms are found predominantly in the liver where it is produced by hepatocytes<NUM>,<NUM>. All three isoforms carry a cell attachment site in the triple-helical region and the C-terminal fragments endostatin, known to have antiangiogenic and tumour growth inhibitory properties<NUM>. Nevertheless, the three isoforms do differ in their primary structure, with the most significant difference being the frizzled domain. Located in the N-terminal end on the long isoform, the frizzled (Fz) domain is a transmembrane receptor serving as a binding site for Wnt molecules with the ability to regulate cell fate, proliferation and migration. The Fz domain has been shown to regulate human cancer-cell proliferation and cell-cycle progression in both in vitro and in vivo studies<NUM>,<NUM>. In addition, Fz was found to determine adipocyte maturation and abundance in mice<NUM>. Hence the long isoform of type XVIII collagen carries a broad range of functions potentially affecting liver pathology and hepatocyte function.

The severity of liver fibrosis is typically assessed through either the use of a liver biopsy or transient elastrography. However, liver biopsy is an inherently flawed modality that is associated with high economic and labour costs; and transient elastography, although an attractive non-invasive alternative, is not widely available and does not provide information regarding the dynamics of liver fibrosis.

Consequently, there is a need in the art for novel methods of assessing the presence and/or severity of liver fibrosis in a patient. In particular, serological biomarkers that may be used clinically to identify individuals likely to progress in disease or respond to anti-fibrotic therapy would be of great utility.

The applicant has now developed an enzyme-linked immunosorbent assay (ELISA) biomarker that can accurately quantify the liver specific isoforms of type XVIII collagen, the long and intermediate isoforms, and has evaluated its clinical value in patients with mild to severe liver fibrosis.

Accordingly, the present invention provides a method of immunoassay comprising: (i) contacting a biofluid sample from a patient with a monoclonal antibody that specifically binds to the long and/or intermediate isoforms of type XVIII and that does not specifically bind to the short isoform of type XVIII; and (ii) detecting and determining the amount of binding between said monoclonal antibody and peptides in the sample.

As used herein the term "amount of binding" refers to the quantification of binding between the monoclonal antibody and peptides in the patient sample. Said quantification may for example be determined by comparing the measured values of binding in the patient sample against a calibration curve produced using measured values of binding in standard samples containing known concentrations of a peptide to which the antibody specifically binds, in order to thereby determine the quantity of peptide to which the antibody specifically binds in the patient sample. In the Examples set out below, an ELISA method is used in which spectrophotometric analysis is used to measure the amount of binding both in the patient samples and when producing the calibration curve. However, any suitable analytical method can be used.

In a preferred embodiment, the patient sample is from a patient with liver fibrosis and the method is for assessing the likelihood of the patient responding to treatment with an antifibrotic drug. The method may further comprise: (iii) correlating the amount of binding of said monoclonal antibody as determined in step (ii) with values associated with responders to treatment and/or to values associated with non-responders and/or a predetermined cut-off value. The patient sample may be from a patient with chronic liver disease. The patient sample may, for example, be from a patient with viral hepatitis, alcoholic liver disease, or non-alcoholic fatty liver disease.

In this context, the term "values associated with responders to treatment" means standardised quantities of binding determined by the method described supra for samples from patients known to have subsequently responded to treatment with an antifibrotic drug, e.g. in whom there was a decrease in liver fibrosis following treatment with the antifibrotic drug (as for example as determined by a decrease in one or more Ishak scores over the course of treatment); and the term "values associated with non-responders" means standardised quantities of binding determined by the method described supra for samples from patients known to have subsequently not responded to treatment with an antifibrotic drug, e.g. in whom there was an increase in liver fibrosis following treatment with the antifibrotic drug.

Also in this context, the term "predetermined cut-off value" means an amount of binding that is determined statistically to distinguish between patients that are less likely to respond to treatment with an antifibrotic drug and patients that are more likely to respond to treatment with an antifibrotic drug. For example, the predetermined cut-off value may be calculated from a previous analysis of levels on binding in samples from liver fibrosis patients who were subsequently treated with an antifibrotic drug or a placebo, wherein in patients with an amount of binding below the cut-off value there was no statistically significant difference between the placebo and antifibrotic drug treatment groups in terms of the number of patients in whom there was a decrease in liver fibrosis or the number of patients in whom there was an increase in liver fibrosis, and/or wherein in patients with a value above the cut-off value there was a statistically significant difference between the placebo and antifibrotic drug treatment groups in terms of the number of patients in whom there was a decrease in liver fibrosis and/or the number of patients in whom there was an increase in liver fibrosis.

In another embodiment, the method is for monitoring liver fibrosis in a patient, the method further comprising: (iii) correlating said amount of binding of said monoclonal antibody as determined in step (ii) with values obtained from said patient at a previous time point. The patient may be a patient with chronic liver disease. The patient may, for example, be a patient with viral hepatitis, alcoholic liver disease, or non-alcoholic fatty liver disease.

Accordingly, in this embodiment the method may comprise quantifying the amount of binding between the monoclonal antibody and peptides in at least two samples obtained from a patient at a first time point and at at least one subsequent time point.

The patient may be a patient undergoing treatment with an antifibrotic drug. In this case, the method may further be for evaluating the efficacy of a drug for treating liver fibrosis, whereby the method comprises quantifying the amount of binding between the monoclonal antibody and peptides in at least two samples obtained from a patient at a first time point and at at least one subsequent time point during a period of administration of the drug to the patient.

In another embodiment, the method is for detecting liver fibrosis in a patient. The method may further comprises: (iii) correlating said amount of binding of said monoclonal antibody as determined in step (ii) with values associated with normal healthy subjects and/or values associated with known disease severity and/or a predetermined cut-off value. In certain embodiments, the method may be for detecting chronic liver disease in a patient.

In this context, the term "values associated with normal healthy subjects" means standardised quantities of binding determined by the method described supra for samples from subjects considered to be healthy, i.e. without disease (and more specifically without liver fibrosis); and the term "values associated with known disease severity" means standardised quantities of binding determined by the method described supra for samples from patients known to have liver fibrosis of a known severity.

Also in this context, the term "predetermined cut-off value" means an amount of binding that is determined statistically to be indicative of a high likelihood of liver fibrosis in a patient, in that a measured value of binding in a patient sample that is at or above the statistical cut-off value corresponds to at least a <NUM>% probability, preferably at least an <NUM>% probability, preferably at least an <NUM>% probability, more preferably at least a <NUM>% probability, and most preferably at least a <NUM>% probability of the presence or likelihood of liver fibrosis.

In preferred embodiments, the sample is a biofluid. The biofluid may be, but is not limited to, blood, serum, plasma, urine or a supernatant from cell or tissue cultures. Preferably the biofluid is blood, serum or plasma.

The immunoassay may be, but is not limited to, a competition assay or a sandwich assay. The immunoassay may, for example, be a radioimmunoassay or an enzyme-linked immunosorbent assay (ELISA). Such assays are techniques known to the person skilled in the art.

The monoclonal antibody specifically binds to the amino acid sequence NLLNLN (SEQ ID NO. Said amino acid sequence is present at the N-terminus of the long and intermediate isoforms of type XVIII (and is absent from the short isoform of type XVIII), and accordingly said monoclonal antibodies specifically bind to the N-terminus of the long and intermediate isoforms of type XVIII.

In certain embodiments, the monoclonal antibody is a monoclonal antibody raised against a synthetic peptide having the amino acid sequence NLLNLNKDKE (SEQ ID NO.

As used herein the term "monoclonal antibody" refers to both whole antibodies and to fragments thereof that retain the binding specificity of the whole antibody, such as for example a Fab fragment, F(ab')<NUM> fragment, single chain Fv fragment, or other such fragments known to those skilled in the art. As is well known, whole antibodies typically have a "Y-shaped" structure of two identical pairs of polypeptide chains, each pair made up of one "light" and one "heavy" chain. The N-terminal regions of each light chain and heavy chain contain the variable region, while the C-terminal portions of each of the heavy and light chains make up the constant region. The variable region comprises three complementarity determining regions (CDRs), which are primarily responsible for antigen recognition. The constant region allows the antibody to recruit cells and molecules of the immune system. Antibody fragments retaining binding specificity comprise at least the CDRs and sufficient parts of the rest of the variable region to retain said binding specificity.

In the methods of the present invention, a monoclonal antibody comprising any constant region known in the art can be used. Human constant light chains are classified as kappa and lambda light chains. Heavy constant chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. The IgG isotype has several subclasses, including, but not limited to IgGl, IgG2, IgG3, and IgG4. The monoclonal antibody may preferably be of the IgG isotype, including any one of IgGl, IgG2, IgG3 or IgG4.

The CDR of an antibody can be determined using methods known in the art such as that described by Kabat et al. Antibodies can be generated from B cell clones as described in the examples. The isotype of the antibody can be determined by ELISA specific for human IgM, IgG or IgA isotype, or human IgG1, IgG2, IgG3 or IgG4 subclasses. The amino acid sequence of the antibodies generated can be determined using standard techniques. For example, RNA can be isolated from the cells, and used to generate cDNA by reverse transcription. The cDNA is then subjected to PCR using primers which amplify the heavy and light chains of the antibody. For example primers specific for the leader sequence for all VH (variable heavy chain) sequences can be used together with primers that bind to a sequence located in the constant region of the isotype which has been previously determined. The light chain can be amplified using primers which bind to the <NUM>' end of the Kappa or Lamda chain together with primers which anneal to the V kappa or V lambda leader sequence. The full length heavy and light chains can be generated and sequenced.

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

Preferably the antibody or fragment thereof comprises at least <NUM>,<NUM>,<NUM>,<NUM> or <NUM> of the above listed CDR sequences.

Preferably the monoclonal antibody or fragment thereof has a light chain variable region comprising the CDR sequences.

Preferably the monoclonal antibody or fragment thereof has a light chain that comprises framework sequences between the CDRs, wherein said framework sequences are substantially identical or substantially similar to the framework sequences between the CDRs in the light chain sequence below (in which the CDRs are shown in bold and underlined, and the framework sequences are shown in italics)
<IMG>.

Preferably the monoclonal antibody or fragment thereof has a heavy chain variable region comprising the CDR sequences.

Preferably the monoclonal antibody or fragment thereof has a heavy chain that comprises framework sequences between the CDRs, wherein said framework sequences are substantially identical or substantially similar to the framework sequences between the CDRs in the heavy chain sequence below (in which the CDRs are shown in bold and underlined, and the framework sequences are shown in italics)
<IMG>.

As used herein, the framework amino acid sequences between the CDRs of an antibody are substantially identical or substantially similar to the framework amino acid sequences between the CDRs of another antibody if they have at least <NUM>%, <NUM>%, <NUM>% or at least <NUM>% similarity or identity. The similar or identical amino acids may be contiguous or non-contiguous.

The framework sequences may contain one or more amino acid substitutions, insertions and/or deletions. Amino acid substitutions may be conservative, by which it is meant the substituted amino acid has similar chemical properties to the original amino acid. A skilled person would understand which amino acids share similar chemical properties. For example, the following groups of amino acids share similar chemical properties such as size, charge and polarity: Group <NUM> Ala, Ser, Thr, Pro, Gly; Group <NUM> Asp, Asn, Glu, Gln; Group <NUM> His, Arg, Lys; Group <NUM> Met, Leu, Ile, Val, Cys; Group <NUM> Phe Thy Trp.

A program such as the CLUSTAL program to can be used to compare amino acid sequences. This program compares amino acid sequences and finds the optimal alignment by inserting spaces in either sequence as appropriate. It is possible to calculate amino acid identity or similarity (identity plus conservation of amino acid type) for an optimal alignment. A program like BLASTx will align the longest stretch of similar sequences and assign a value to the fit. It is thus possible to obtain a comparison where several regions of similarity are found, each having a different score. Both types of analysis are contemplated in the present invention. Identity or similarity is preferably calculated over the entire length of the framework sequences.

In certain preferred embodiments, the monoclonal antibody or fragment thereof may comprise the light chain variable region sequence:
<IMG>
and/or the heavy chain variable region sequence:
<IMG>
(CDRs bold and underlined; Framework sequences in italics) Provided herein is a monoclonal antibody that specifically binds to the long and/or intermediate isoforms of type XVIII and that does not specifically bind to the short isoform of type XVIII.

The monoclonal antibody specifically binds to the amino acid sequence NLLNLN (SEQ ID NO.

The monoclonal antibody may be a monoclonal antibody raised against a synthetic peptide having the amino acid sequence NLLNLNKDKE (SEQ ID NO.

Further preferred embodiments and features of the monoclonal antibody provided herein will be apparent from the above discussion of the preferred monoclonal antibodies for use in the method of the invention. In particular, the monoclonal antibody may preferably comprise one or more complementarity-determining regions (CDRs), framework sequences and/or variable region sequences as described above.

Also provided herein is an immunoassay kit comprising: (a) a monoclonal antibody that specifically binds to the long and/or intermediate isoforms of type XVIII and that does not specifically bind to the short isoform of type XVIII; and (b) at least one of:.

In a preferred embodiment, the monoclonal antibody specifically binds to the amino acid sequence NLLNLN (SEQ ID NO. The monoclonal antibody may be raised against a synthetic peptide having the amino acid sequence NLLNLNKDKE (SEQ ID NO. In particular, the monoclonal antibody is preferably a monoclonal antibody as described above.

In certain embodiments the calibrator protein may be a peptide comprising the amino acid sequence NLLNLNKDKE (SEQ ID NO. In certain embodiments the biotinylated peptide may comprise NLLNLNKDKE-L-Biotin (SEQ ID NO. <NUM>), wherein L is an optional linker.

The presently disclosed embodiments are described in the following Examples, which are set forth to aid in the understanding of the disclosure, and should not be construed to limit in any way the scope of the disclosure as defined in the claims which follow thereafter. The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the described embodiments, and are not intended to limit the scope of the present disclosure nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

In the following examples, the following materials and methods were employed.

All reagents used for the experiment were high-quality chemicals from Merck (Whitehouse Station, NJ, USA) and Sigma Aldrich (St. Louis, MO, USA) and all synthetic peptides purchased from GenScript (Piscataway, NJ, USA). Sequences defining the peptides for antibody production and validation were as follows:.

Six to seven weeks old Balb/C mice were immunized subcutaneously with <NUM>µg emulsified immunogen consisting of PRO-C18L keyhole limpet hemocyanin (KLH)-conjugated antigenic peptide (NLLNLNKDKEKK-GGC-KLH (SEQ ID NO. <NUM>), the immunogenic peptide) mixed with Stimune Immunogenic Adjuvant (SPECOL, cat #<NUM>, Invitrogen). Immunization was repeated every second week until serum titer was found stable. The mouse with the highest serum titer and best peptide inhibition was selected for fusion. Thus, after resting at least three weeks, the selected mouse was given a final boost of <NUM>-<NUM>µg emulsified immunogen (depending on antibody titer) mixed with <NUM>µl <NUM>% NaCl solution intraperitoneally. Three days following the final boost, the mouse were sacrificed and spleen cells isolated and fused with SP2/<NUM> myeloma cells to generate hybridomas for further culturing, this procedure have previously been described<NUM>. Hybridomas were plated in <NUM>-well microtiter plates, using semi-medium method and employing limited dilution to ensure monoclonality. In addition, the subcloning were repeated a minimum of two times to further ensure monoclonality. Supernatant was screened for reactivity and specificity against the standard peptide, a non-sense peptide (LHDSNPYPRR (SEQ ID NO. <NUM>)) and a deselection peptide (NALNLNWLWF (SEQ ID NO. <NUM>)) in a direct competitive ELISA using streptavidin-coated plates to confirm binding to the standard peptide and lack of binding to the non-sense peptide and deselection peptide. To confirm an IgG isotype the clone was further tested in a Clonotyping System-HRP kit, cat. No. <NUM>-<NUM> (Southern Biotech, Birmingham, AL, USA).

The antibodies generated were sequenced and the CDRs determined.

The sequence of the chains are as follows:.

Selected clones were purified using HiTrap affinity columns (GE Healthcare Life Science, Little Chalfont, Buckinghamshire, UK) and labelled with horseradish peroxidase (HRP) using Roche Peroxidase Labelling Kit, cat. <NUM> (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer instructions. Native reactivity and affinity of the clones were assessed using different human biological material such as serum or plasma with EDTA, heparin or citrate.

To measure the serological levels of PRO-C18L in serum or plasma samples, the following procedure is used:.

The standard curve is made by a <NUM>-fold dilution of the standard peptide.

To determine linearity of the measurements, recovery percentage of the <NUM>% sample were determined in <NUM>-fold dilutions of serum and plasma samples from human. Likewise, antibody specificity was calculated as recovery percentage of the <NUM>% calibrator peptide (the standard peptide, NLLNLNKDKE (SEQ ID NO. <NUM>)), the nonsense peptide (LHDSNPYPRR (SEQ ID NO. <NUM>)) and the deselection peptide (NALNLNWLWF (SEQ ID NO. Lower limit of detection (LLOD) was determined as the mean +3x standard deviations (SD) of <NUM> blanks (buffer). Upper limit of detection (ULOD) was determined as the mean -3x SD of <NUM> measurements of Standard A. To assess the intra- and inter-assay variations, <NUM> independent runs of <NUM> quality control (QC) samples were measured in doublet determinations and calculated as mean coefficient of variance (CV%). Accuracy of the measurements was evaluated by measuring a healthy human serum sample spiked with standard peptide and calculated as the percentage recovery of the theoretical collected amount of analyte. Interference was measured as the percentage recovery of human serum sample spiked with haemoglobin, lipemia and biotin at significant concentrations. Acceptable range for linearity, accuracy and interference were ±<NUM>%.

Three serum and plasma human samples were subjected to five freeze-thaw cycles to determine stability of the analyte, with the first cycle serving as reference. In addition, the analyte stability was tested by incubating three serum and three plasma samples at <NUM> and <NUM> for <NUM>-, <NUM>-, <NUM>- and <NUM>-hours and tested against the non-stressed sample reference. All sample teste was run as doublet determinations. Reagent stability was tested by incubating the HRP labelled antibody at <NUM> and <NUM> for seven days and tested against a none-stressed reagent. Acceptable range for analyte and reagent stability tests were ±<NUM>%.

PRO-C18L ELISA was tested in a cohort from a multicentre, phase II clinical study investigating the effectiveness of farglitazar, a peroxisome proliferator-activated receptor-gamma agonist, a potential antifibrotic compound for hepatitis C affected patients (NCT00244751) as described previously<NUM>.

Plasma samples were available in a subpopulation of <NUM> patients receiving either daily doses of farglitazar or matching placebo for <NUM> weeks. Liver biopsies were available at baseline and after <NUM> weeks and viewed by a central experienced histopathologist using Ishak modified histological activity index for grading and staging. The study was conducted in compliance with the Declaration of Helsinki and followed ethical guidelines for all countries involved. All patients provided written informed consent. Baseline patient characteristics are shown in table <NUM> below.

The cohort were selected for having chronic hepatitis C and difficult to treat with previous antiviral therapies failing. The initial study concluded that there were no effects of farglitazar, as the proportion of subjects with a progression or regression of disease, based on Ishak scoring, were the same from baseline to end of study<NUM>. However, in a later publication on the same cohort a marker of type III collagen formation was found to identify responders to treatment, showing that serological measurements of fibrosis provides a more dynamic quantification of the disease activity<NUM>.

PRO-C18L was measured in duplicates in EDTA plasma from the patients described above. The ELISA and measurements were developed and measured at Nordic Bioscience (Herlev, Denmark), as described above.

Graphs and statistical analysis were performed using MedCalc version <NUM> (MedCalc Software Ltd, Ostend, Belgium) and GraphPad Prism version <NUM> (GraphPad Software, La Jolla, CA, USA). Data is shown as Tukey box plots. A p-value of p<<NUM> is considered statistically significant.

To select the best antibody from the produced clones: native reactivity, specificity and stability was evaluated. The antibody chosen for the ELISA development was named NBH133A1-2B4 and determined to be an IgG2a subtype. To ensure antibody specificity, its reactivity was tested against the standard peptide, a non-sense peptide (LHDSNPYPRR (SEQ ID NO. <NUM>)) and a deselection peptide (NALNLNWLWF (SEQ ID NO. <NUM>)) using the competitive ELISA procedure described above. The standard peptide inhibited the signal in a dose-dependent manner (<FIG>), whereas there was no reactivity against the non-sense peptide (data not shown) or deselection peptide (<FIG>). This data shows that the selected monoclonal antibody has high specificity towards the PRO-C18L N-terminus epitope.

Technical evaluation of the PRO-C18L ELISA was assessed through the different validation steps described in the methods section above, and the results are summarized below in table <NUM>.

The IC50 was <NUM> ng/ml. Lower and upper limit of detection was <NUM> ng/ml and <NUM> ng/ml. Intra- and inter-assay variations were <NUM>% and <NUM>%, which is below the accepted criteria limits of <NUM>% and <NUM>%, respectively. Dilution recovery in both serum and plasma, from undiluted to <NUM>:<NUM> dilution, was found to be within the accepted criteria of <NUM>±<NUM>%. In a spiking test of calibration peptide (the standard peptide) in serum the mean recovery was found to be <NUM>%; the binding affinity for biological fragment versus synthetic standard was the same, confirming N-terminus binding and high assay specificity. After testing the analyte with four freeze/thaw cycles the analyte recovery was <NUM>% and <NUM>% in serum and EDTA plasma, respectively. In addition, storage of serum, heparin plasma and EDTA plasma at <NUM> and <NUM> for <NUM> hours did not affect the analyte recoveries of <NUM>% and <NUM>% for serum, <NUM>% and <NUM>% for heparin plasma and <NUM>% and <NUM>% for EDTA plasma. No interference was detected from either low or high levels of biotin, lipemia or haemoglobin in serum and plasma with recoveries ranging from <NUM>% - <NUM>%. These results all together argue that PRO-C18L is a technically robust assay.

To test the potential of PRO-C18L as a biomarker of fibrosis, a longitudinal study of <NUM> HCV patients treated with farglitazar or placebo were measured. Demographics as tested in table <NUM> showed no significant difference in distribution between the treated and placebo group.

First the change in PRO-C18L from baseline (BL) to week <NUM> (W52) was assessed in placebo versus farglitazar treated patients, finding that placebo increased while treated patients decreased in PRO-C18L levels (p=<NUM>) as shown in <FIG>. To further test if higher baseline levels (above median) of PRO-C18L were consistent with more treatment response, above and below median of PRO-C18L (<NUM> ng/ml) were tested in a similar manner: delta PRO-C18L from BL to W52 in treated vs placebo patients. Although, above median levels of baseline PRO-C18L visually decreased more with treatment than the below median treated patients (<FIG>), indicating modulations to the mean, the result was not significant. However, these results indicate that PRO-C18L is modulated by therapy.

To further evaluate PRO-C18L as a biomarker of liver fibrosis, patients were divided into regressors, stable or progressors based on delta Ishak from BL to W52. Regressors and progressors were characterized as having a decrease or increase, respectively, of one or more Ishak scores from BL to W52 while stable had no change in Ishak. To assess the predictive value of PRO-C18L, the placebo and farglitazar treatment groups were further divided into tertiles based on PRO-C18L levels at baseline. In the group of patients in tertile <NUM> (the tertile consisting of the patients with the highest PRO-C18L baseline levels) the farglitazar treatment group showed a significant difference in the proportion of regressors and progressors when compared to the placebo group (<FIG>), whereas this difference was not found in tertile <NUM> patient group (the tertile consisting of the patients with the lowest PRO-C18L baseline levels). , suggesting that patients with high PRO-C18L levels at baseline are more likely to respond to treatment.

Finally, to assess if PRO-C18L is different from PRO-C3 (found to predict responders to treatment in an earlier study of the same patients) the two markers were correlated on their preliminary cut off levels (<NUM> ng/ml for PRO-C3 and <NUM> ng/ml for PRO-C18L) (<FIG>). No correlation was found, suggesting that PRO-C18L identifies additional responders to treatment in the same population.

Claim 1:
A method of immunoassay for detecting or monitoring liver fibrosis in a patient or for assessing, in a patient with liver fibrosis, the likelihood of the patient responding to treatment with an antifibrotic drug, the method of immunoassay comprising:
(i) contacting a patient sample with a monoclonal antibody that specifically binds to the long and/or intermediate isoforms of type XVIII collagen and that does not specifically bind to the short isoform of type XVIII collagen, wherein the monoclonal antibody specifically binds to the amino acid sequence NLLNLN (SEQ ID NO. <NUM>); and
(ii) detecting and determining the amount of binding between said monoclonal antibody and peptides in the sample.