PIIINP neo-epitope assay
09726674 · 2017-08-08
Assignee
Inventors
- Diana Julie Leeming (Klampenborg, DK)
- Mette Juul Nielsen (Kobenhavn S, DK)
- Morten Karsdal (Kobenhavn O, DK)
Cpc classification
C07K2317/34
CHEMISTRY; METALLURGY
G01N2800/085
PHYSICS
G01N2333/78
PHYSICS
International classification
Abstract
Provided is a monoclonal antibody specifically reactive with a C-terminal neo-epitope of PIIINP comprised in a C-terminal amino acid sequence CPTGXQNYSP-COOH (SEQ ID NO: 4) in which X is Gly or Pro, and where the monoclonal antibody does not recognize or bind an elongated version of the C-terminal amino acid sequence CPTGXQNYSPQZ-COOH (SEQ ID NO: 5), in which Z is absent or is one or more amino acids of the sequence of collagen type III. Also provided is 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, by contacting the sample with the monoclonal antibody, and determining the amount of binding of the antibody.
Claims
1. 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 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.
2. A monoclonal antibody as claimed in claim 1, wherein said 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-Gln bond between amino acids P153-Q154 in human PIIINP.
3. A monoclonal antibody as claimed in claim 1, wherein said 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-Gln bond between amino acids P154-Q155 in rodent PIIINP.
4. A monoclonal antibody as claimed in claim 1, wherein the ratio of the affinity of said antibody for amino acid sequence CPTGXQNYSP-COOH (SEQ ID NO: 4) to the affinity of said antibody for elongated amino acid sequence CPTGXQNYSPQZ-COOH (SEQ ID NO: 5) is at least 10 to 1.
5. A monoclonal antibody as claimed in claim 1, wherein said 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).
6. A monoclonal antibody as claimed in claim 1, wherein the ratio of the affinity of said antibody for amino acid sequence CPTGXQNYSP-COOH (SEQ ID NO: 4) to the affinity of said antibody for shortened amino acid sequence CPTGXQNYS-COOH (SEQ ID NO: 8) is at least 10 to 1.
7. 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 the monoclonal antibody as claimed in claim 1, and determining the amount of binding of said antibody.
8. A method as claimed in claim 7, further comprising quantifying the amount of PIIINP cleaved from intact collagen type III in biofluids.
9. A method as claimed in claim 8, wherein said biofluid is serum, plasma or amniotic fluid.
10. A method as claimed in claim 7, wherein said immunoassay is a competition assay or a sandwich assay.
11. A method as claimed in claim 7, wherein said immunoassay is a radioimmunoassay or an enzyme-linked immunosorbent assay.
12. A method as claimed in claim 8, further comprising correlating the quantity of PIIINP cleaved from intact collagen type III with standard fibrotic disease samples of known disease severity to evaluate the severity of a fibrotic disease.
13. A method as claimed in claim 12, wherein said method comprises correlating the quantity of PIIINP cleaved from intact collagen type III determined by said method with standard liver fibrosis samples of known disease severity to evaluate the severity of liver fibrosis.
14. A method as claimed in claim 12, wherein said method comprises correlating the quantity of PIIINP cleaved from intact collagen type III with MRI-determined muscle volume to evaluate muscle volume.
15. A method as claimed in claim 8 for selecting from a group of patients having a fibrotic disease which is in a deteriorating condition for pharmaceutical trial or therapy, further comprising determining severity of said fibrotic disease, and selecting from the group of patients determined to have an equivalent severity of said fibrotic disease those patients having the quantity of PIIINP cleaved from intact collagen type III above a statistical second quartile.
16. A method as claimed in claim 15, wherein determining the severity of the fibrotic disease is with an Ishak fibrosis staging scale or METAVIR scoring.
17. An assay kit for determining the quantity of PIIINP in a biological sample, comprising: the monoclonal antibody as claimed in claim 1; and at least one of: a streptavidin coated 96 well plate; a biotinylated peptide Biotin-L-PTGPQNYSP (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; and an assay visualization kit.
Description
FIGURES
(1) ) 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.
(2)
(3)
(4)
(5)
(6)
(7)
(8)
(9)
(10)
(11)
(12)
EXAMPLES
(13) Materials and General Considerations
(14) All reagents used in the experiments were high-standard chemicals from companies such as Merck (Whitehouse Station, N.J., 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
(15) Monoclonal Antibody Generation
(16) 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 α1 chain PIIINP is 100% homologues between human and rat (
(17) Clone Characterization
(18) 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, Ala., USA).
(19) Antibody Characterization
(20) 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 1× phosphate-buffered saline (PBS) and 25 μl sample was added to a microtiter plate along with 200 μl 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 (×2) and reducing agent (×10), 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 μg/ml horseradish peroxidase (HRP)-conjugated 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 4×10 minutes in TBST, incubated with 4 ml chemiluminescence detection kit (ECL), and developed using Amersham Hyperfilm.
(21) Clone Selection and Characterization
(22) The subtype was determined to be an IgG1 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 (
Example 2—PRO-C3 ELISA Using NB61N-62
(23) 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.
(24) 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 (50 mM 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 μl of peptide calibrator or sample were added to appropriate wells, followed by 100 μl of HRP-conjugated monoclonal antibody NB61N-62 dissolved in incubation buffer (50 mM PBS-BTB+10% LiquidII (Roche), pH 7.4) and the plate was incubated for 20 hours at 4° C. and washed. Finally, 100 μl 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 μl of stopping solution (1% H.sub.2SO.sub.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.
(25) Technical Evaluation
(26) 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+3×Standard Deviation (SD) of the blank from 21 determinations of standard K (i.e. buffer). Upper limit of detection (ULOD) was determined as the mean−3×SD 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.
(27) Results
(28) 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.
(29) TABLE-US-00002 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. Value Intra-assay Inter-assay Sample (ng/mL) 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
(30) Dilution recovery was performed using healthy serum and plasma samples from humans, rat and mouse. The dilution recovery was within the acceptable 100±20% recovery (Table 2). Further dilution resulted in measurements below LLOQ.
(31) TABLE-US-00003 TABLE 2 Percentage dilution recovery for the PRO-C3 assay using human-, rat-, and mouse samples. Human serum (HS), Human plasma (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 105 90 113 107 114
(32) Spiking of calibrator peptide in serum or plasma resulted in a mean recovery of 56% and 55%, respectively (Table 3).
(33) TABLE-US-00004 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 Added (n = 3) (n = 3) Added (n = 3) (n = 3) Std sRE % sRE % AF 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 56 100 Mean 55 111 sRE % sRE %
(34) 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).
(35) TABLE-US-00005 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
(36) The stability of the analyte was acceptable up to four freeze/thaw cycles with 100±20% recovery compared to 1 freeze/thaw cycle (Table 5).
(37) TABLE-US-00006 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. EDTA Heparin Citrate Serum plasma plasma plasma Freeze/thaw Mean Mean Mean Mean cycle recovery % recovery % recovery % recovery % 1 100% 100% 100% 100% 2 103 102 103 109 3 99 99 98 103 4 102 100 98 100
Example 3—Determining the Ratio of Binding Affinity
(38) 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.sub.50 values of each sequence/antibody combination. The ratio of IC.sub.50[target]/IC.sub.50[elongated or shortened] defines the ratio of binding affinity.
Example 4—Rat CCl4 Liver Fibrosis Model
(39) Serum levels of PIIINP were assessed in a CCl.sub.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 CCl.sub.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, CCl.sub.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/l) 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 CCl.sub.4 or vehicle treatment (n=13 for CCl.sub.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 CCl.sub.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 ELISA.
(40) Results
(41) Serum levels of PIIINP determined in the PRO-C3 ELISA were statistically elevated in CCl.sub.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% increase, p=ns) (
(42) 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 ELISA 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
(43) PIIINP was measured by PRO-C3 ELISA in plasma samples from 11 young men (n=11, 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 (2 W) and after remobilization (4 W). 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.
(44) Results
(45) 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.sup.2=0.4416, P=0.0361) (
Example 6—Comparison of Competitive PRO-C3 ELISA and UniQ PIIINP Assays
(46) 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.
(47) Results
(48) 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.sup.2=0.12, p=ns) (
(49) 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)
(50) 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.
(51) 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”).
(52) Baseline Plasma Pro-C3 in Healthy Controls and CHC Patients
(53) 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) (
(54) 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 (
(55) These results demonstrate a clear ability of the Pro-C3 immunoassay to distinguish between healthy and CHC patients.
(56) Association Between Baseline Plasma Pro-C3 and Progression of Disease
(57) 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 (
(58) 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 (
(59) 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
(60) 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.
(61) Patients and Methods
(62) Patients with HBV and HCV
(63) 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.
(64) 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 μL of standard peptide (CPTGPQNYSP (SEQ ID MNO: 6)) or pre-diluted sample were added to appropriate wells, followed by 100 μL of peroxidase-conjugated specific monoclonal antibodies and incubated for 1 hour or overnight at 20° C. or 4° C., respectively. Finally, 100 μL tetramethylbenzidine (TMB) (cat.438OH, 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 μL of stopping solution (0.18 M H2SO4) and measured at 450 nm with 650 nm as the reference. A calibration curve was plotted using a 4-parametric mathematical fit model.
(65) 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:
(66) f0: no fibrosis
(67) f1: portal fibrosis without septa
(68) f2: portal fibrosis with rare septa
(69) f3: numerous septa without cirrhosis
(70) f4: cirrhosis
(71) 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).
(72) Patients with HBV Infection
(73) 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).
(74) In contrast, the degree of liver fibrosis as assessed by Metavir score did not show a significant relation to age, BMI and gender.
(75) TABLE-US-00007 TABLE 6 Demographics and PRO-C3 Results According to Metavir F-score. METAVIR F stage ANOVA HBV n 0 n 1 n 2 n 3 n 4 p Mean 39 41.0 95 40.3 35 44.5 16 37.4 4 51.7 0.080 age (9.9) (11.9) (11.1) (12.8) (15.0) (year (SD)) BMI 39 23.9 95 24.1 35 24.6 16 23.8 4 24.7 0.962 (+/−SEM) (23.3- (23.8- (23.9- (22.6- (21.8- 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 (+/−SEM) (16.8- (18.8- (23.1- (32.6- (18.0- 19.6) 20.4) 26.8) 43.8) 41.1)
(76) 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.
(77) In a graphic representation of the same data (
(78) 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 advanced 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).
(79) TABLE-US-00008 TABLE 7 PRO-C3: ROC Analysis on Metavir F score Sensi- PRO-C3 tivity Specificity P- PPV NPV Cut off HBV Group (%) (%) AUC value (%) (%) (ng/mL) Early 76.4 61.9 0.728 <0.0001 56.5 44.2 >19.21 (0-1) vs moderate- late (2-4) PPV: Positive predictive value; NPV: Negative predictive value.
Patients with HCV Infection
(80) 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).
(81) TABLE-US-00009 TABLE 8 Demographics and PRO-C3 Results According to Metavir F-score METAVIR F stage ANOVA HCV n 0 n 1 n 2 N 3 n 4 p Mean age 42 39.0 156 41.8 99 45.7 45 47.5 33 49.1 <0.001 Year (SD) (9.8) (10.4) (8.7) (9.2) (6.6) BMI 42 25.2 156 25.6 99 26.8 45 27.8 33 26.6 0.015 (+/−SEM) (24.6- (25.2- (26.3- (27.1- (26.0- 25.8) 25.9) 27.4) 28.5) 27.3) Male 23 55% 86 55% 67 68% 33 73% 21 64% 0.100 sex PRO-C3 42 17.5 156 19.9 99 24.3 45 36.4 33 41.2 <0.001 (+/−SEM) (16.7- (19.2- (23.3- (33.7- (37.8- 18.3) 20.4) 25.4) 39.3) 45.0)
(82) 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.
(83) In a graphic representation of the data (
(84) 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).
(85) TABLE-US-00010 TABLE 9 PRO-C3: ROC Analysis on Metavir F score Sensi- PRO-C3 tivity Specificity P- PPV NPV Cut off HCV Group (%) (%) AUC value (%) (%) (ng/mL) Early 68.4 72.7 0.758 <0.0001 49.4 51.4 >22.21 (0-1) vs moderate- late (2-4) PPV: Positive predictive value; NPV: Negative predictive value.
(86) 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.
REFERENCES
(87) [1] World Health Organization. Reducing Risks, Promoting Healthy Life. Peducing Risks, Promoting Healthy Life, Geneva: WHO, 2002:1-230. [2] Wynn T A. Cellular and molecular mechanisms of fibrosis. J Pathol 2008; 214:199-210. [3] Friedman S L. Mechanisms of disease: Mechanisms of hepatic fibrosis and therapeutic implications. Nat Clin Pract Gastroenterol Hepatol 2004; 1:98-105. [4] Tomasek J J, Gabbiani G, Hinz B, Chaponnier C, Brown R A. Myofibroblasts and mechano-regulation of connective tissue remodelling. Nat Rev Mol Cell Biol 2002; 3:349-363. [5] Wynn T A. Common and unique mechanisms regulate fibrosis in various fibroproliferative diseases. J Clin Invest 2007; 117:524-529. [6] Bosman, F. T., and Stamenkovic, I. 2003. Functional structure and composition of the extracellular matrix. J. Pathol. 200:423-428. [7] Bruckner, P. 2010. Suprastructures of extracellular matrices: paradigms of functions controlled by aggregates rather than molecules. Cell Tissue Res. 339:7-18. [8] Bao X, Zeng Y, Wei S, Wang G, Liu C, Sun Y, Chen Q, and Li H. Developmental changes of Col3a1 mRNA expression in muscle and their association with intramuscular collagen in pigs. J Genet Genomics 2007; 34(3): 223-228. [9] Jensen L T and Host N B. Collagen: scaffold for repair or execution. Cardiovasc Res 1997; 33(3): 535-539. [10] Niemela O, Risteli L, Parkkinen J, and Risteli J. Purification and characterization of the N-terminal propeptide of human type III procollagen. Biochem J 1985; 232(1): 145-150. [11] Wang W M, Ge G, Lim N H, Nagase H, and Greenspan D S. TIMP-3 inhibits the procollagen N-proteinase ADAMTS-2. Biochem J 2006; 398(3): 515-519. [12] Van den Steen P E, Opdenakker G, Wormald M R, Dwek R A, and Rudd P M. Matrix remodelling enzymes, the protease cascade and glycosylation. Biochim Biophys Acta 2001; 1528(2-3): 61-73. [13] Cuzner M L and Opdenakker G. Plasminogen activators and matrix metalloproteases, mediators of extracellular proteolysis in inflammatory demyelination of the central nervous system. J Neuroimmunol 1999; 94(1-2): 1-14. [14] Meduri G U, Tolley E A, Chinn A, Stentz F, and Postlethwaite A. Procollagen types I and III aminoterminal propeptide levels during acute respiratory distress syndrome and in response to methylprednisolone treatment. Am J Respir Crit Care Med 1998; 158(5 Pt 1): 1432-1441. [15] Teare J P, Sherman D, Greenfield S M, Simpson J, Bray G, Catterall A P, Murray-Lyon I M, Peters T J, Williams R, and Thompson R P. Comparison of serum procollagen III peptide concentrations and PGA index for assessment of hepatic fibrosis. Lancet 1993; 342(8876): 895-898. [16] Scheja A, Akesson A, and Horslev-Petersen K. Serum levels of aminoterminal type III procollagen peptide and hyaluronan predict mortality in systemic sclerosis. Scand J Rheumatol 1992; 21(1): 5-9. [17] Lin Y H, Ho Y L, Wang T D, Liu C P, Kao H L, Chao C L, Chien K L, Hung C S, Wu V C, Tsai I J, Yen R F, Shiau Y C, and Chen W J. The relation of amino-terminal propeptide of type III procollagen and severity of coronary artery disease in patients without myocardial infarction or hibernation. Clin Biochem 2006; 39(9): 861-866. [18] Teppo A M, Tornroth T, Honkanen E, and Gronhagen-Riska C. Urinary amino-terminal propeptide of type III procollagen (PIIINP) as a marker of interstitial fibrosis in renal transplant recipients. Transplantation 2003; 75(12): 2113-2119. [19] Han X Y, Wang W, Komulainen J, Koskinen S O, Kovanen V, Vihko V, Trackman P C, and Takala T E. Increased mRNAs for procollagens and key regulating enzymes in rat skeletal muscle following downhill running. Pflugers Arch 1999; 437(6): 857-864. [20] Koskinen S O, Ahtikoski A M, Komulainen J, Hesselink M K, Drost M R, and Takala T E. Short-term effects of forced eccentric contractions on collagen synthesis and degradation in rat skeletal muscle. Pflugers Arch 2002; 444(1-2): 59-72. [21] Crameri R M, Langberg H, Teisner B, Magnusson P, Schroder H D, Olesen J L, Jensen C H, Koskinen S, Suetta C, and Kjaer M. Enhanced procollagen processing in skeletal muscle after a single bout of eccentric loading in humans. Matrix Biol 2004; 23(4): 259-264. [22] Chen F, Lam R, Shaywitz D, Hendrickson R C, Opiteck G J, Wishengrad D, Liaw A, Song Q, Stewart A J, Cummings C E, Beals C, Yarasheski K E, Reicin A, Ruddy M, Hu X, Yates N A, Menetski J, and Herman G A. Evaluation of early biomarkers of muscle anabolic response to testosterone. J Cachexia Sarcopenia Muscle 2011; 2(1): 45-56. [23] Longobardi S, Keay N, Ehrnborg C, Cittadini A, Rosen T, Dall R, Boroujerdi M A, Bassett E E, Healy M L, Pentecost C, Wallace J D, Powrie J, Jorgensen J O, and Sacca L. Growth hormone (GH) effects on bone and collagen turnover in healthy adults and its potential as a marker of GH abuse in sports: a double blind, placebo-controlled study. The GH-2000 Study Group. J Clin Endocrinol Metab 2000; 85(4): 1505-1512. [24] Bhasin S, He E J, Kawakubo M, Schroeder E T, Yarasheski K, Opiteck G J, Reicin A, Chen F, Lam R, Tsou J A, Castaneda-Sceppa C, Binder E F, Azen S P, and Sattler F R. N-terminal propeptide of type III procollagen as a biomarker of anabolic response to recombinant human GH and testosterone. J Clin Endocrinol Metab 2009; 94(11): 4224-4233. [25] Nelson A E, Meinhardt U, Hansen J L, Walker I H, Stone G, Howe C J, Leung K C, Seibel M J, Baxter R C, Handelsman D J, Kazlauskas R, and Ho K K. Pharmacodynamics of growth hormone abuse biomarkers and the influence of gender and testosterone: a randomized double-blind placebo-controlled study in young recreational athletes. J Clin Endocrinol Metab 2008; 93(6): 2213-2222. [26] Zachariae H, Heickendorff L, and Sogaard H. The value of amino-terminal propeptide of type III procollagen in routine screening for methotrexate-induced liver fibrosis: a 10-year follow-up. Br J Dermatol 2001; 144(1): 100-103. [27] Gressner A M and Weiskirchen R. Modern pathogenetic concepts of liver fibrosis suggest stellate cells and TGF-beta as major players and therapeutic targets. J Cell Mol Med 2006; 10(1): 76-99. [28] Jarcuska P, Janicko M, Veseliny E, Jarcuska P, and Skladany L. Circulating markers of liver fibrosis progression. Clin Chim Acta 2010; 411(15-16): 1009-1017. [29] Frei A, Zimmermann A, and Weigand K. The N-terminal propeptide of collagen type III in serum reflects activity and degree of fibrosis in patients with chronic liver disease. Hepatology 1984; 4(5): 830-834. [30] Fabris P, Marranconi F, Bozzola L, Biasin M R, De Lazzari F, Plebani M, Benedetti P, Tositti G, Pellizzer G, Stecca C, and de L F. Fibrogenesis serum markers in patients with chronic hepatitis C treated with alpha-IFN. J Gastroenterol 1999; 34(3): 345-350. [31] Brocks D G, Steinert C, Gerl M, Knolle J, Neubauer H P, and Gunzler V. A radioimmunoassay for the N-terminal propeptide of rat procollagen type III. Application to the study of the uptake of the N-terminal propeptide of procollagen type III in isolated perfused rat liver. Matrix 1993; 13(5): 381-387. [32] Rohde H, Vargas L, Hahn E, Kalbfleisch H, Bruguera M, and Timpl R. Radioimmunoassay for type III procollagen peptide and its application to human liver disease. Eur J Clin Invest 1979; 9(6): 451-459. [33] Bayer Aktiengesellschaft. (1999) Monoclonal antibody and assay for detecting PIIINP. Patent Cooperation Treaty Appn. WO 99/61477. [34] Warming L, Hassager C, and Christiansen C. Changes in bone mineral density with age in men and women: a longitudinal study. Osteoporos Int 2002; 13(2): 105-112. [35] Segovia-Silvestre T, Reichenbach V, Fernandez-Varo G, Vassiliadis E, Barascuk N, Morales-Ruiz M, Karsdal M A, and Jimenez W. Circulating CO3-610, a degradation product of collagen III, closely reflects liver collagen and portal pressure in rats with fibrosis. Fibrogenesis Tissue Repair 2011; 4: 19 [36] Clária J and Jiménez W. Experimental Models of Cirrhosis and Ascites. 2005; Second edition (17) [37] Schuppan D, Ruehl M, Somasundaram R, and Hahn E G. Matrix as a modulator of hepatic fibrogenesis. Semin Liver Dis 2001; 21 (3): 351-372. [38] Suetta C, Hvid L G, Justesen L, Christensen U, Neergaard K, Simonsen L, Ortenblad N, Magnusson S P, Kjaer M, and Aagaard P. Effects of aging on human skeletal muscle after immobilization and retraining. J Appl Physiol 2009; 107(4): 1172-1180. [39] McHutchison J, Goodman Z, Patel K, Makhlouf H, Rodriguez-Torres M, Shiffman M, et al. Farglitazar lacks antifibrotic activity in patients with chronic hepatitis C infection. Gastroenterology 2010 April; 138(4):1365-73, 1373. [40] Ishak K, Baptista A, Bianchi L, Callea F, De G J, Gudat F, et al. Histological grading and staging of chronic hepatitis. J Hepatol 1995 June; 22(6):696-699. [41] Warming L, Hassager C, Christiansen C. Changes in bone mineral density with age in men and women: a longitudinal study. Osteoporos Int 2002; 13(2):105-112. [42] Mouritzen U, Christgau S, Lehmann H J, Tanko L B, Christiansen C. Cartilage turnover assessed with a newly developed assay measuring collagen type II degradation products: influence of age, sex, menopause, hormone replacement therapy, and body mass index. Ann Rheum Dis 2003 April; 62(4):332-336.