Immunoassay for collagen type VI
11634479 · 2023-04-25
Assignee
Inventors
- Anders Nedergaard (Copenhagen, DK)
- Jannie Marie Sand (Malov, DK)
- Shu Sun (Farum, DK)
- Diana Julie Oersnes-Leeming (Klampenborg, DK)
- Kim Henriksen (Hillerod, DK)
Cpc classification
C07K2317/32
CHEMISTRY; METALLURGY
C07K2317/34
CHEMISTRY; METALLURGY
G01N2333/78
PHYSICS
International classification
Abstract
The present invention provides an immunological binding partner reactive with a C-terminal epitope of the C5 domain of the α3 chain of collagen Type 6, and a method of immunoassay using the immunological binding partner for detecting and quantifying the C-terminal epitope. The invention also provides a method of investigating the rate of formation of extracellular matrix and a method for identifying a subject suitable for treatment with an insulin sensitizer.
Claims
1. An isolated monoclonal antibody that specifically binds a C-terminal epitope of the C5 domain of the a3 chain of collagen Type 6, wherein the antibody comprises (a) a variable heavy chain region comprising CDRH1 comprising the amino acid sequence of SEQ ID NO.: 6, CDRH2 comprising the amino acid sequence of SEQ ID NO.: 7, and CDRH3 comprising the amino acid sequence of SEQ ID NO.: 8; and (b) a variable light chain region comprising CDRL1 comprising the amino acid sequence of SEQ ID NO.: 10, CDRL2 comprising the amino acid sequence of SEQ ID NO.: 11, and CDRL3 comprising the amino acid sequence of SEQ ID NO.: 12, wherein the antibody specifically binds the amino acid sequence consisting of SEQ ID NO:1.
2. The monoclonal antibody of claim 1, wherein said antibody does not specifically bind an elongated version of said C-terminal amino acid sequence consisting of SEQ ID. NO:2.
3. The monoclonal antibody of claim 1, wherein the ratio of the affinity of the antibody for amino acid sequence of SEQ ID. NO:1 to the affinity of said antibody for elongated amino acid sequence of SEQ ID. NO:2 is greater than 10 to 1.
4. The monoclonal antibody of claim 1, wherein said antibody does not specifically bind a truncated version of said C-terminal amino acid sequence consisting of SEQ ID. NO:3.
5. The monoclonal antibody of claim 1, wherein the ratio of the antibody for amino acid sequence of SEQ ID. NO:1 to the affinity of said antibody for truncated amino acid sequence of SEQ ID. NO:3 is greater than 10 to 1.
6. An immunoassay method for detecting in a sample a C-terminal epitope of the C5 domain of the a3 chain of collagen type VI, wherein said method comprises performing an immunoassay comprising: contacting a sample comprising said C-terminal epitope of the a3 chain of collagen type VI with the isolated monoclonal antibody as claimed in claim 1; and determining binding of said monoclonal antibody to the terminal epitope of the C5 domain of the a3 chain of collagen type VI in the sample.
7. The method as claimed in claim 6, wherein said method further comprises quantifying the amount of said C-terminal epitope of the a3 chain of collagen type VI in the sample, wherein the sample is a biofluid.
8. The method as claimed in claim 7, wherein said biofluid is serum, plasma, urine or amniotic fluid.
9. The method as claimed in claim 6, wherein said immunoassay is a competition assay or a sandwich assay.
10. The method as claimed in claim 9, wherein said immunoassay is a radioimmunoassay or an enzyme-linked immunosorbent assay.
11. An immunoassay assay kit for determining the quantity of a C-terminal epitope of the C5 domain of the a3 chain of collagen Type VI in a biological sample, comprising: the isolated monoclonal antibody of claim 1 and at least one of: a streptavidin coated 96 well plate, a biotinylated peptide wherein the peptide has an amino acid sequence as set forth in SEQ ID. NO:1, an optionally biotinylated secondary antibody for use in a sandwich immunoassay; a calibrator peptide comprising an amino acid sequence as set forth in SEQ ID. NO:1, an antibody HRP labeling kit, an antibody radiolabeling kit; and an assay visualization kit.
Description
FIGURES
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EXAMPLES
Example 1: Antibody Development for Pro-C6
(14) We used the last 10 amino acids of the type VI collagen α3 chain (.sup.3168‘KPGVISVMGT’.sup.3177 (SEQ ID. NO:1)) as an immunogenic peptide to generate specific epitope monoclonal antibodies. The methods used for monoclonal antibody development were as previously described (Barascuk).
(15) Briefly, 4-6-week-old Balb/C mice were immunized subcutaneously with 200 μl emulsified antigen with 60 μg of the immunogenic peptide. Consecutive immunizations were performed at 2-week intervals in Freund's incomplete adjuvant, until stable sera titer levels were reached, and the mice were bled from the 2nd immunization on. At each bleeding, the serum titer was detected and the mouse with highest antiserum titer and the best native reactivity was selected for fusion. The selected mouse was rested for 1 month followed by intravenous boosting with 50 μg of immunogenic peptide in 100 μl 0.9% sodium chloride solution 3 days before isolation of the spleen for cell fusion.
(16) The fusion procedure has been described elsewhere (Gefter). Briefly, mouse spleen cells were fused with SP2/0 myeloma fusion partner cells. The fusion cells were raised in 96-well plates and incubated in the CO2-incubator. Here standard limited dilution was used to promote monoclonal growth. Cell lines specific to the selection peptide and without cross-reactivity to neither elongated peptide (KPGVISVMGTA (SEQ ID. NO:2), Chinese Peptide Company, China) nor truncated peptide (KPGVISVMG (SEQ ID. NO:3), American Peptide Company, USA) were selected and sub-cloned. At last the antibodies were purified using an IgG column.
(17) The antibodies generated were sequenced and the CDRs determined.
(18) The sequence of the chains are as follows (CDRs underlined and in bold):
(19) TABLE-US-00008 Heavy Chain Sequence (mouse IgG1 isotype) (SEQ ID. NO: 5) EVQLQQSGPVMVKPGTSVKTSCKASGYTFTDFNMNWVKQSHGKSLEWIGA INPHNGATSYNQKFSGKATLTVDKSSSTAYMELNSLTSDDSAVYYCARWG NGKNSWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFP EPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNV AHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTP KVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELP IMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQ MAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVY SKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK CDR-H1: (SEQ ID. NO: 6) DFNMN CDR-H2: (SEQ ID. NO: 7) AINPHNGATSYNQKFSG CDR-H3: (SEQ ID. NO: 8) WGNGKNS Light Chain Sequence (mouse Kappa isotype) (SEQ ID. NO: 9) DVVMTQTPLSLPVNLGDQASISCRSSQRIVHSNGITFLEWYLQKPGQSPK LLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGLYYCFQGSHVP LTFGAGTRLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDIN VKWKIDGSERQNGVLNSVVTDQDSKDSTYSMSSTLTLTKDEYERHNSYTC EATHKTSTSPIVKSFNRNEC CDR-L1: (SEQ ID. NO: 10) RSSQRIVHSNGITFLE CDR-L2: (SEQ ID. NO: 11) RVSNRFS CDR-L3: (SEQ ID. NO: 12) FQGSHVPLT
Pro-C6 Assay Protocol:
(20) ELISA-plates used for the assay development were Streptavidin-coated from Roche (cat.: 11940279). All ELISA plates were analyzed with the ELISA reader from Molecular Devices, SpectraMax M, (CA, USA). We labeled the selected monoclonal antibody with horseradish peroxidase (HRP) using the Lightning link HRP labeling kit according to the instructions of the manufacturer (Innovabioscience, Babraham, Cambridge, UK). A 96-well streptavidin plate was coated with biotinylated synthetic peptide biotin-KPGVISVMGT (SEQ ID. NO:4) (Chinese Peptide Company, China) dissolved in coating buffer (40 mM Na.sub.2HPO.sub.4, 7 mM KH.sub.2PO.sub.4, 137 mM NaCl, 2.7 mM KCl, 0.1% Tween 20, 1% BSA, pH 7.4) and incubated 30 minutes at 20° C. 20 μL of standard peptide or samples diluted in incubation buffer (40 mM Na.sub.2HPO.sub.4, 7 mM KH.sub.2PO.sub.4, 137 mM NaCl, 2.7 mM KCl, 0.1% Tween 20, 1% BSA, 5% Liquid II, pH 7.4) were added to appropriate wells, followed by 100 μL of HRP conjugated monoclonal antibody 10A3, and incubated 21 hour at 4° C. Finally, 100 μL tetramethylbenzinidine (TMB) (Kem-En-Tec cat. 438OH) was added and the plate was incubated 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). The TMB reaction was stopped by adding 100 μL of stopping solution (1% H.sub.2SO.sub.4) and measured at 450 nm with 650 nm as the reference.
(21) Pro-C6 Technical Evaluation:
(22) The lowest limit of detection (LLOD) was determined from 21 zero samples (i.e. buffer) and calculated as the mean+3× standard deviation. The intra-assay variation and inter-assay variations were determined by 12 independent runs of 8 QC samples with each run consisting of double determinations of the samples. Dilution recovery was determined in 4 serum samples and 4 heparin plasma samples and was calculated as a percentage of recovery of diluted samples from the 100% sample.
Example 2: PRO-C6 in Muscle Loss Studies
(23) Measurement of Pro-C3, C6M Assays in Berlin Bed Rest Study:
(24) The level of C-terminal of α3 chain is expected to reflect the level of newly formed mature type VI collagen. In order to investigate the synthesis of type VI collagen, we developed the Pro-C6 ELISA kit described above targeting the C-terminal of α3 chain. In addition, type VI collagen is also a substrate of MMPs (Veidal 2011). Previous studies showed that both MMP-2 and MMP-9 are relevant to muscle atrophy (Reznick 2003 and Giannelli 2005). Therefore, type VI collagen degradation fragments generated by MMP-2 and MMP-9 are of interest in such a process.
(25) In this study, we measured three biomarkers Pro-C6 (measuring the C-terminal α3(VI) chain) and C6M (measuring type VI collagen fragment degraded by MMP-2 and MMP-9)(Veidal 2011), and Pro-C3 (measuring the true synthesis of type III collagen) (Nielsen), which directly measure the turnover of type III and VI collagen in the Berlin bed rest study—using bed rest immobilization and remobilization as a human model of muscle atrophy and hypertrophy.
(26) The Berlin bed rest study has been described elsewhere (Rittweger 2006 and Belavy 2009). Briefly, 20 healthy young men were recruited and underwent a strict 8-week bed rest study. The 20 young men were then randomly divided into two groups. The resistive vibration exercise group (RVE) group was assigned to resistive vibration exercise 11 times per week. The resistive vibration exercises were performed by a vibration exercise apparatus at the end of the beds and pulling the subject towards the vibration plate with waist and shoulder straps and handles for the subjects to pull themselves towards the plate. The control group (CTRL) was not allowed to perform any exercise during the 8-week bed rest. Serum samples were obtained 2 days before the study (BDC-2), in the bed rest period (BR+) and in the following recovery period (R+). The serum samples were stored at −80° C. until further measurement. The muscle mass of both groups were assessed by MRI and DXA during the three periods.
(27) The protocols of Pro-C3 and C6M assays have been described elsewhere (Nielsen 2013 and Kuo 1997). The Pro-C3 assay measures levels of a pro-peptide fragment of collagen type III. The C6M assay measures MMP degradation fragments of mature collagen type VI. Briefly, in Pro-C3 assay, a 96-well streptavidin plate was coated with biotinylated synthetic peptide and incubated 30 minutes at 20° C. 20 μL of standard peptide or 1:2 diluted serum samples were added to appropriate wells, followed by 100 μL of HRP conjugated monoclonal antibody NB61N-62, and incubated 20 hour at 4° C. Finally, 100 μL TMB was added and the plate was incubated 15 minutes at 20° C. in the dark. The TMB reaction was stopped by adding 100 μL of stopping solution (1% H.sub.2SO.sub.4) and measured at 450 nm with 650 nm as the reference. In C6M assay, biotinylated synthetic peptide is coated to a 96-well streptavidin plate. 20 μL of standard peptide or 1:2 diluted serum samples are added, followed by 100 μL of HRP conjugated monoclonal antibody, and incubated 1 hour at 20° C. The plate was read after the development by TMB.
(28) Results:
(29) The chosen antibody 10A3 specifically recognized the last 10 amino acids of C-terminal COL6A3 3168′KPGVISVMGT′3177 (SEQ ID. NO:1), but did not recognize elongated peptide KPGVISVMGTA nor truncated peptide KPGVISVMG (
(30) The measurement range of Pro-C6 competitive ELISA was determined by LLOD and ULOD, providing the range from 0.15 ng/ml to 58.39 ng/ml. The inter- and intra-assay variability is 15.2% and 4.8%, respectively. The dilution recovery in human serum and heparin plasma were both within 100±20% (Table 1). The correlation between human serum and each of heparin plasma, citrate plasma and EDTA plasma was relatively high (
(31) TABLE-US-00009 TABLE 1 Table depicting dilution recovery. Dilution Heparin Dilution Serum samples recovery plasma samples recovery undiluted 100 undiluted 100 dilution 1:2 91 dilution 1:2 105 dilution 1:4 91 dilution 1:4 100 dilution 1:8 80 dilution 1:8 109
(32) Samples were diluted in serial 2-fold dilution steps concentration was measured in these serial dilutions. Dilution recovery was obtained by multiplying measured concentrations with the dilution factor and expressed as percent of the concentration of the undiluted (starting) sample. The table shows that the signal dilutes linearly and stays within +/−20% within and 8-fold dilution range.
(33) Biomarker Profiles in Berlin Bed Rest Study:
(34) The levels of the three biomarkers referred to above measured in the Berlin Bed rest study (BBR) are seen in
(35) As seen in
(36) When we compared individual biomarker levels of PRO-C3 with LBM and changes therein, we found that individual levels of PRO-C3 correlated significantly with LBM at baseline (R.sup.2=0.2869, R=0.536, ρ=0.0149). Furthermore, we found that the level of biomarker at its peak at BR47, correlated significantly with the amount of LBM lost during immobilization (R.sup.2=0.2056, R=0.453, ρ=0.0447).
(37) The PRO-C6 biomarker changed over time during the course of immobilization (significant time effect, p<0.0001) in the form of an increase after approximately one week of immobilization, reaching a peak level approximately 30% higher than baseline during the last couple of weeks of immobilization (being significantly higher than baseline from BR19 to R28, peaking at BR47, ρ=0.0002). There were no between-group differences during the immobilization period (no significant treatment effects or treatment*time interactions).
(38) During re-mobilization, both time and time*treatment interaction effects manifested. This was in the form of an increase that peaked one week into remobilization (an 20% increase relative to the last day of immobilization, BR56, ρ=0.011), followed by a gradual return to baseline values. The interaction effect was not manifested in any post hoc tests, owing to high variation at the R7 time point.
(39) When we compared individual biomarker levels of PRO-C6 with LBM and changes therein, we found that the level of PRO-C6 was not related to LBM at all, but positively related to change in LBM during immobilization (R.sup.2=0.2794, R=0.529, ρ=0.0166) meaning that higher levels of PRO-C6 were associated with less loss of LBM. We also found that PRO-C6 was negatively related to the amount of LBM (re)gained during remobilization (R.sup.2=0.3365, R=0.580, ρ=0.0073), meaning that higher levels were associated with less (re)gain of LBM during remobilization.
(40) The C6M biomarker was essentially unaffected by immobilization (no time effect in the immobilization time period), but increased briefly 30-40% at the beginning of remobilization (a significant time effect at p<0.0001 during the immobilization period). There were no treatment effects during immobilization and although it may appear as if the increase in the C6M signal is bigger in the CTRL group than in the RVE group, this did not reach significance (the time*treatment interaction did not reach significance and thus no post hoc test was made). There were no differences between the groups.
(41) When we compared individual biomarker levels of PRO-C6 with LBM and changes therein, we found that the level of PRO-C6 was not related to LBM at all, but positively related to muscle loss during immobilization (R.sup.2=0.2794, R=0.529, ρ=0.0166) and negative related to the amount of muscle (re)gained during remobilization (R.sup.2=0.3365, R=0.580, ρ=0.0073).
(42) TABLE-US-00010 TABLE 2 Correlation matrix for biomarker vs. anthropometric variables. BioM (Biomarker), Lean Body mass (LBM), Leg Muscle Volume (LMV, from MRI), Loss is the absolute LBM change during immobilization, i.e. higher negative equals bigger loss; Gain is total LBM regain during remobilization. PRO-C3 PRO-C6 C6M R p R p R p BioM.sub.Baseline vs. 0.536 0.0149* 0.022 0.9270 0.595 0.0057* LBM.sub.Baseline BioM.sub.BR47 vs. 0.453 0.0447* 0.529 0.0166* 0.102 0.6684 leg LBM.sub.Loss BioM.sub.R3 vs. −0.171 0.4705 −0.580 0.0073* −0.269 0.2509 leg LBM.sub.Gain
(43) PRO-C6 is seen to be a biomarker of remodelling associated with changes in physical activity and changes in LBM. Low PRO-C6 at baseline is associated with a phenotype that is more prone to changes in LBM, both gain and loss.
Example 3: PRO-C6 in COPD
(44) Study Design:
(45) Patients presenting with a hospital admission deemed by a medical consultant to be a COPD exacerbation during 2011 and 2012 were recruited within 24 hours of admission. Blood samples were collected at time of exacerbation and at recovery: a 4 week follow-up visit performed a median of 30 (IQR 28-34) days after admission. At follow-up visit, the patients underwent standard post-bronchodilator spirometry, and performed a six minute walking distance (6MWD). Patient-reported measures included assessments of dyspnoea, using the Medical Research Council (MRC) dyspnoea scale, at follow-up visit, and smoking history.
(46) The inclusion criterion was a clinical diagnosis of acute COPD exacerbation at hospital admission made by a consultant physician. A physician diagnosis or radiological evidence of pneumonia was an exclusion criterion. The study comprised 69 patients with paired samples and with airflow obstruction (ratio of forced expiratory volume in one second (FEV1) to forced vital capacity (FVC) of <0.7) confirmed at follow-up visits.
(47) ECM remodelling biomarkers:
(48) Serum and heparin plasma samples were stored at −80° C. until analyzed. C3M, C4M, Pro-C3, P4NP 7S, ELM7, and EL-NE, were measured in serum, while C6M, Pro-C6, and VCANM were measured in heparin plasma. An overview of the assays used in this study to assess extracellular matrix remodelling appears in Table 3.
(49) TABLE-US-00011 TABLE 3 Assay overview. Intra- and Reference Detection inter-assay level range variation (ng/ml), Assay Target (ng/ml) (%) mean (SD) References C3M Collagen type III 5.52-177 3.4 and 9.8 15.3 (3.8) [28] degraded by MMPs C4M Collagen type IV 22.8-748 4.2 and 18.5 55.4 (17.8) [29] degraded by MMPs C6M Collagen type VI 4.88-420 8.0 and 11.0 8.85 (5.1) [30] degraded by MMPs ELM7 Elastin degraded by 1.16-36.6 8.1 and 9.1 2.23 (0.74) Preliminary MMP-7 data EL-NE Elastin degraded by 1.76-167 8.6 and 12.9 4.09 (2.24) Preliminary neutrophil elastase data VCANM Versican degraded by 0.78-7.13 3.0 and 7.6 1.20 (0.23) [31] MMPs Pro-C3 Collagen type III 5.32-96.4 6.5 and 12.4 12.3 (4.4) [32] propeptide (N-terminal) P4NP 7S Collagen type IV 7S 32.9-3460 9.4 and 14.2 263 (91.3) [33] domain (internal) Pro-C6 Collagen type VI 2.81-117 4.8 and 15.2 4.37 (0.69) Preliminary propeptide (C-terminal) data
(50) The reference level was provided by the manufacturer (Nordic Bioscience) and refers to the biomarker level of a healthy population in the relevant matrix, i.e. serum (C3M, C4M, Pro-C3, P4NP 7S, ELM7, EL-NE) or heparin plasma (C6M, Pro-C6, VCANM). SD, standard deviation; MMP, matrix metalloproteinase.
(51) Patient demographics and clinical characteristics are summarised in Table 4. Patients were mostly men (71%) and ex-smokers (55%). They were hospitalised for a median [IQR] of 3 [2-6] days, and follow-up visit was performed at 30 [28-34] days after admission.
(52) TABLE-US-00012 TABLE 4 Basic characteristics of the COPD population at follow-up visit 4 weeks after exacerbation onset. Variable Patients (n = 69) Age (years), median (IQR) 67 (61-75) Female sex, n (%) 20 (29) BMI (kg/m.sup.2) 25.7 (6.3) Current smokers, n (%) 31 (45) Smoking pack years (years) 52 (26) Length of hospitalisation (days), median (IQR) 3 (2-6) FEV.sub.1 (liters) 1.19 (0.50) FEV.sub.1 (% of predicted) 45.8 (16.1) FVC (liters) 2.55 (0.81) FVC (% of predicted) 77.5 (19.0) FEV.sub.1/FVC ratio 0.46 (0.11) 6MWD (meters) 166 (119) MRC dyspnoea score, median (IQR) 4 (3-4)
(53) Variables are listed as mean (standard deviation) unless otherwise stated. IQR, interquartile range; BMI, body mass index; FEV.sub.1, forced expiratory volume in one second; FVC, forced vital capacity; 6MWD, 6 minute walking distance; MRC, Medical Research Council.
(54) Circulating levels of protein fragments released at time of exacerbation and at a clinically stable disease period at 30-days follow-up are presented in Table 5.
(55) TABLE-US-00013 TABLE 5 Levels of circulating protein fragments at exacerbation and 30-days follow-up. Exacerbation (ng/ml) Follow-up (ng/ml) P value C3M 29.24 [26.32-32.49] 22.64 [20.78-24.67] <0.0001 C4M 95.96 [85.83-107.28] 73.30 [66.59-80.69] <0.0001 C6M 19.78 [16.82-23.27] 13.27 [11.56-15.23] <0.0001 ELM7 4.50 [3.91-5.17] 3.79 [3.37-4.27] <0.0001 EL-NE 7.79 [6.30-9.63] 5.23 [4.41-6.21] <0.0001 VCANM 1.69 [1.58-1.80] 1.87 [1.78-1.97] 0.0001 Pro-C3 12.10 [10.60-13.81] 12.79 [11.35-14.42] 0.2549 P4NP 7S 510.99 [440.91-592.21] 359.20 [312.28-413.17] <0.0001 Pro-C6 5.36 [4.81-5.99] 6.38 [5.71-7.14] <0.0001
(56) Results are presented as geometric mean [95% confidence interval] and corresponding P values comparing circulating levels of protein fragments at time of exacerbation and follow-up.
(57) Degradation fragments of collagen type III (C3M), collagen type IV (C4M), collagen type VI (C6M), and elastin (ELM7 and EL-NE) were significantly elevated at exacerbation compared to follow-up (all P<0.0001;
(58) The balance between degradation and formation of collagens was investigated by calculating the ratio between fragments of degradation and formation for collagen types III, IV, and VI (
(59) At follow-up, BMI was negatively associated with C3M (ρ=−0.271, P=0.029), Pro-C3 (ρ=−0.357, P=0.010), and Pro-C6 (ρ=−0.338, P=0.017). Age was negatively associated with C6M (ρ=−0.249, P=0.039) and Pro-C6 (ρ=−0.310, P=0.026). No associations were seen with smoking pack years, MRC score, length of hospitalisation, sputum production, or white blood cell counts. Pro-C3 levels were positively associated with FEV1% of predicted value (% pred) and FVC % pred, and these remained significant after correcting for age, gender, BMI, smoking pack years, and smoking status (Table 4). 6MWD was negatively associated with C3M, C4M, C6M, and P4NP 7S (Table 4). Following correction for age, gender, BMI, smoking pack years, and smoking status, associations with C3M and C6M remained significant, while C4M was borderline significant and P4NP 7S was non-significant (Table 6).
(60) TABLE-US-00014 TABLE 6 Associations between levels of circulating protein fragments and clinical parameters. FEV1 % pred FVC % pred 6MWD C3M 0.020 −0.182 −0.370** (−0.311*) C4M −0.002 −0.148 −0.313* (−0.252£) C6M −0.012 −0.224 −0.354** (−0.354**) ELM7 −0.041 −0.175 −0.125 EL-NE −0.016 −0.125 −0.189 VCANM 0.021 −0.084 −0.096 Pro-C3 0.391** (0.320*) 0.312* (0.305*) −0.009 P4NP 7S 0.042 −0.186 −0.278* (−0.230) Pro-C6 0.058 −0.013 −0.188
(61) Results are presented as Spearman correlation coefficients (p) for each marker. In brackets are given multivariate correlation coefficients for markers showing significant p. The multivariate linear regression analysis included age, gender, BMI, smoking pack years, and smoking status as additional explanatory variables. Significance levels: £P<0.07, *P<0.05, **P<0.01. FEV1, forced expiratory volume in one second; % pred, percentage of predicted value; FVC, forced vital capacity; 6MWD, 6 minutes walking distance.
(62) All assays employed a monoclonal antibody directed against either a protein fragment produced by MMP cleavage during degradation or formation or an internal protein sequence. An overview of the assays used in this study and their technical specifications is given in Table 3. All samples were measured within the quantification range of each assay and any sample with values below the lower limit of detection (LLOD) was assigned the value of LLOD.
(63) The above results demonstrate that ECM remodelling, assessed systemically by biomarkers of protein remodelling fragments, is accelerated during an exacerbation of COPD where disease activity is high.
Example 4: Pro-C6 in Diabetes Type II
(64) Treatment of diabetic patients with full agonists of peroxisome proliferator-activated receptor gamma (PPARγ) improves insulin sensitivity, but is associated with weight gain, heart failure, peripheral oedema, and bone loss. Endotrophin, the C-terminal fragment of the α3 chain of procollagen type VI (also called Pro-C6), is involved in both adipose tissue matrix remodeling and metabolic control. We established a serum assay for endotrophin to assess if this novel adipokine could identify type 2 diabetes (DM2) patients who respond optimally to PPARγ agonists, improving the risk to benefit ratio.
(65) Study Design
(66) The BALLETS (Birmingham and Lambeth Liver Evaluation
(67) Testing Strategies) study was a phase III, randomized, double-blind, parallel-group, placebo and active comparator-controlled clinical study to determine the efficacy and safety of six months' treatment of balaglitazone or pioglitazone in subjects with type 2 diabetes on stable insulin therapy. The baseline demographics, CONSORT diagram as well as efficacy and safety data have previously been published (Henriksen, 2011). In the current study we used the per protocol population of the BALLETS study, which consisted of 299 subjects spread evenly over four groups (placebo, two doses of balaglitazone, and one dose of piogliatazone) as previously described (Henriksen, 2011), all with baseline and up to six follow-up parameters related to blood sugar control and Pro-C6 determinations under therapy.
(68) Statistical Analysis
(69) The analysis included subjects from the per protocol population having a baseline measurement of serum Pro-C6. Subjects were grouped into one of three tertiles based on their baseline Pro-C6 value. Tertile 1 included subjects with baseline serum Pro-C6 of 6.2 ng/mL or below; tertile 2 had baseline serum Pro-C6 of 6.3 ng/mL to 7.7 ng/mL, and tertile 3 had baseline serum Pro-C6 of 7.8 ng/mL or above. Baseline characteristics between the three subgroups were compared by analysis of variance (ANOVA), and comparison of the proportion of genders in each tertile was compared by Fisher's exact test.
(70) Spearman's ranked correlation was conducted on baseline levels of serum Pro-C6, fasting serum glucose (FSG), blood HbA1c, body mass index (BMI), and the derived parameters of insulin resistance (HOMA-IR) and fatty liver index (FLI). The HOMA-IR was calculated according to the homeostatis model assessment including serum glucose and insulin (Feigh, 2011) and FLI was calculated (as described by Bedogni et al, 2006) using the equation:
(71)
(72) Changes from baseline in FSG, blood HbA1c, and serum Pro-C6 were studied as a function of time and treatment in each of the three tertiles. The least squares means (LS Means) and standard error were estimated from a mixed-effect repeated measure model with the change from baseline as the dependent variable; baseline level, visit (after 12 weeks on treatment) and end of treatment (after 26 weeks), and the baseline level vs visit and end of treatment vs visit interaction as fixed effects, and an unstructured covariance structure for subject.
(73) For each subject the mean change from baseline was calculated as area under the curve by the trapezoidal method, and the LS means and standard error were estimated from an analysis of covariance model (ANCOVA) with the mean change as the dependent variable, the baseline level as the covariate, and treatments as fixed effects. Each tertile within each of the active treatment groups was compared with the placebo group with the level of significance adjusted for multiple comparisons by the Dunnett method. Assessment of whether mean change from baseline was different from 0 was based on the standard error of the LS means.
(74) All statistical calculations were performed using the SAS software package. This study is registered with ClinicalTrials.gov identifier NCT00515632.
(75) Results
(76) Serum Endotrophin is Correlated to Metabolic Parameters.
(77) Efficacy of treatment as assessed by metabolic parameters and safety data in the BALLET trial have been published previously (Henriksen, 2011). The baseline correlations of endotrophin to parameters associated with the metabolic syndrome are presented in Table 7.
(78) TABLE-US-00015 TABLE 7 Demographics and baseline characteristics in subgroups of baseline PRO-C6 Endotrophin Endotrophin Endotrophin (2.4-6.2 ng/mL) (6.3-7.7 ng/mL) (7.8-16 ng/mL) n = 96 n = 101 n = 100 p-value Treatment Bala 10 mg: n = 27 Bala 10 mg: n = 21 Bala 10 mg: n = 25 — Bala 20 mg: n = 22 Bala 20 mg: n = 21 Bala 20 mg: n = 25 Pio 45 mg: n = 24 Pio 45 mg: n = 29 Pio 45 mg: n = 31 Placebo n = 23 Placebo n = 30 Placebo n = 19 Age (yrs) 57.6 (8.1) 60.6 (8.3) 63.4 (80) <0.0001 Gender Female: 21 (22%) Female: 32 (32%) Female: 43 (43%) p = 0.007 Male: 75 (78%) Male: 69 (68%) Male: 57 (57%) BMI (kg/m.sup.2) 32.0 (3.9) 33.6 (4.7) 34.9 (6.3) 0.0005 Waist 110 (10) 114 (12) 117 (14) 0.001 circumference (cm) Hip 109 (8) 111 (10) 115 (12) 0.0002 circumference (cm) DXA total body 30.8 (8.4) 33.86 (8.9) 36.1 (9.8) 0.0006 fat mass (kg) DXA trunk fat 18.3 (5.2) 20.0 (5.0) 21.7 (5.6) 0.0001 mass (kg) Blood HbA1C 8.7 (1.4) 8.4 (1.3) 8.8 (1.5) ns (%) Serum Glucose 9.4 (3.3) 9.2 (3.2) 9.8 (3.4) Ns (mmol/L) Serum AST 28 (12) 32 (13) 32 (12) Ns (U/L) Serum ALT 31 (15) 34 (19) 33 (17) Ns (U/L) Serum GGT 45 (38) 55 (56) 54 (47) Ns (U/L) Serum ALP 163 (49) 172 (46) 187 (56) 0.004 (U/L) Serum Bilirubin 9 (3.3) 9 (5.1) 9 (3.7) Ns (μmol/L) Serum 1.52 (0.94) 1.85 (1.16) 2 05 (1.07) 0.002 Triglycerides (mmol/L) Serum 4.34 (0.96) 4.28 (0.85) 4.45 (1.04) Ns Cholesterol (mmol/L)) Serum HDL 1.31 (0.35) 1.23 (0.29) 1.25 (0.27) Ns Chol (mmol/L) Serum LDL 2.61 (0.90) 2.54 (0.76) 2.61 (0.97) Ns Chol (mmol/L)
(79) Endotrophin levels were significantly correlated to HOMA-IR, FLI, triglycerides, and BMI, but not to FSG and HbA1c, supporting that endotrophin is indeed an adipokine, related to adipocyte function, fat mass, and some aspects of insulin sensitivity. Endotrophin levels were not correlated to cholesterol levels or liver enzymes.
(80) At the end of the six month treatment period, in the placebo group, the correlations between endotrophin and these metabolic parameters were maintained (Table 8, 9A). However, in those treated with either PPARγ agonist, the correlation between HOMA-IR and endotrophin was eliminated, while the correlation between endotrophin and BMI or FLI persisted and even showed a trend towards becoming stronger (Table 9B-9C).
(81) TABLE-US-00016 TABLE 8 Spearman correlation coefficient (Rho) at baseline Endo- Serum- Baseline- HOMA- trophin glucose HbA1c IR FLI BMI PRO-C6 1 0.07 0.06 0.16** 0.32*** 0.24*** Serum- — 1 0.47*** 0.27*** 0.20*** 0.17** glucose Baseline- — — 1 0.15** 0.17** 0.10 HbA1c HOMA- — — — 1 0.42*** 0.33*** IR FLI — — — — 1 0.86*** BMI — — — — — 1
(82) TABLE-US-00017 TABLE 9A Spearman correlation coefficient at week 26 - Placebo group Endo- Serum- HOMA- trophin Glucose HbA1c IR FLI BMI PRO- 1 0.05 −0.07 0.28* 0.34** 0.26* C6 Serum- — 1 0.24* 0.23* 0.18 0.26* Glucose HbA1c — — 1 0.12 0.16 0.11 HOMA- — — — 1 0.35** 0.23 IR FLI — — — — 1 0.87*** BMI — — — — — 1
(83) TABLE-US-00018 TABLE 9B Spearman correlation coefficient at week 26 - Pioglitazone 45 mg Endo- Serum- HOMA- trophin Glucose HbA1c IR FLI BMI PRO- 1 −0.21 −0.31** 0.02 0.39*** 0.31** C6 Serum- — 1 0.48*** 0.02 −0.11 −0.14 Glucose HbA1c — — 1 −0.06 −0.13 −0.12 HOMA- — — — 1 0.30** 0.25* IR FLI — — — — 1 0.84*** BMI — — — — — 1
Endotrophin Identifies Responders to Glitazone Therapy
(84) Body weight and BMI were higher in the upper tertiles in all four treatment groups than in the lower tertile (Table 1). No differences were seen in glucose homeostasis between treatment groups.
(85) Absolute levels of FSG and HbA1c decreased in all three treatment arms as compared to placebo, but only in the two upper tertiles of endotrophin as compared to the baseline set as zero during the study (
(86) When assessing the mean absolute change over time from baseline to end of treatment (week 26) in FSG (
(87) The effect on serum endotrophin as a function of treatment and time (to study midpoint and end of treatment), expressed as percent change relative to baseline, is shown in
(88) Adverse Events
(89) Lower leg edema, when measured as volume increase due to water displacement, was correlated with baseline serum endotrophin tertiles. Glitazone therapy led to increased lower leg volume in the lower and middle tertile, while there were no differences between treatment and placebo groups in the upper tertile (
(90) TABLE-US-00019 TABLE 10 Adverse event profile in subgroups of baseline endotrophin in each treatment group N (%) E Balaglitazone Balaglitazone Pioglitazone Placebo 10 mg 20 mg 45 mg Tertile 1: AEs # Subjects n = 23 n = 27 n = 22 n = 24 All AEs 16 (70%) 30 20 (74%) 38 17 (77%) 33 19 (79%) 45 Serious AEs 0 (0%) 0 1 (4%) 1 1 (5%) 1 1 (4%) 1 Tertile 2: AEs # Subjects n = 30 n = 21 n = 21 n = 29 All AEs 23 (77%) 51 17 (81%) 35 15 (71%) 36 17 (59%).sup. Serious AEs 3 (10%) 3 1 (5%) 1 0 (0%) 0 2 (6%) 3 Tertile 3: AEs # Subjects n = 19 n = 25 n = 25 n = 31 All AEs 14 (74%) 41 20 (60%) 55 20 (60%) 43 25 (81%).sup. Serious AEs 0 (0%) 0 1 (4%) 1 6 (24%) 6 4 (13%) 6 Tertile 1: Severe AEs Heart failure 0 (0%) 0 0 (0%) 0 0 (0%) 0 0 (0%) 0 Cardiac ischaemia 1 (4%) 1 0 (0%) 0 2 (9%) 2 0 (0%) 0 Peripheral oedema 0 (0%) 0 2 (7%) 2 2 (9%) 2 5 (21%) 5 Total severe AEs 1 (4%) 1 2 (7%) 2 4 (18%) 4 5 (21%) 5 Tertile 2: Severe AEs Heart failure 0 (0%) 0 0 (0%) 0 0 (0%) 0 0 (0%) 0 Cardiac ischaemia 1 (3%) 1 1 (5%) 1 0 (0%) 0 1 (3%) 1 Peripheral oedema 1 (3%) 1 3 (14%) 3 2 (10%) 2 5 (17%) 5 Total severe AEs 2 (7%) 2 4 (19%) 4 2 (10%) 2 5 (17%) 6 Tertile 3: Severe AEs Heart failure 0 (0%) 0 0 (0%) 0 0 (0%) 0 1 (3%) 1 Cardiac ischaemia 1 (5%) 1 0 (0%) 0 1 (4%) 1 3 (10%) 4 Peripheral oedema 1 (5%) 2 1 (8%) 1 1 (4%) 1 2 (6%) 2 Total severe AEs 2 (11%) 3 1 (8%) 1 2 (8%) 2 6 (19%) 7
Discussion
(91) Serum endotrophin (Pro-C6) was predictive of a response to the insulin sensitizers, pioglitazone and balaglitazone, in patients with type 2 diabetes. Thus, patients with Pro-C6 serum levels in the two upper tertiles were 4 times more likely to have a treatment response when compared to patients in the lower tertile. As the glitazones are associated with safety concerns such as non-fatal heart failure and bone fractures, identifying the optimal responders who will gain the most treatment benefit with the fewest AEs is crucial for the continued use of these drugs, which still are considered highly effective insulin sensitizers. In direct agreement, patients in the upper tertiles of baseline Pro-C6 who responded with a decrease of FPG and HbA1c tertile developed no increase in lower leg oedema, one of the major AEs with glitazone treatment. These efficacy and safety data combined are highly relevant for an improved benefit to side effect prediction for patients treated with glitazones; this should also apply when their repurposing for other indications, especially the treatment of non-alcoholic steatohepatitis (NASH) is considered.
(92) Endotrophin mediated metabolic dysfunction in obesity is likely induced via induction of a pro-inflammatory state and fibrosis in adipose tissue coupled with a reduction of energy expenditure. Accordingly, its suppression improved insulin sensitivity and attenuated adipose tissue inflammation (Sun, 2014), which correlates well with our findings that elevated serum endotrophin levels are indicative of a response to PPARγ agonists. Furthermore, mRNA levels of the endotrophin precursor, procollagen α3(VI), are upregulated in obese adipose tissue, again paralleling adipose tissue inflammation and fibrosis, supporting an important role of procollagen type VI as a modulator of adipocytes and adipose tissue in general (Dankel, 2014). The ECM and especially procollagen type VI and endotrophin may be of particular relevance in fatty liver disease and its severe expression, NASH, a metabolic-fibrotic disorder of the liver that shows at least a partial overlap with type 2 diabetes. Accordingly, we expect that this novel biomarker will also assist in the diagnosis and management of NASH patients, where insulin sensitizers may be beneficial for subpopulations, both for the treatment of insulin resistance and liver fibrosis. Here, the ECM, in particular collagens/collagen type VI, and their functional role in transition of fatty liver to overt fibrotic NASH needs to be further investigated. In agreement, in the current study, we observed a strong correlation to serum triglycerides and the FLI index that correlates with NASH inflammatory activity and predicts more severe liver fibrosis (Bedogni, 2006). In support of a role for type VI collagen in NASH-related fibrosis, prior studies demonstrated its prominent expression in areas of active scar formation (Burt, 1990; Griffiths, 1992) and elevated serum levels of the collagen VI core structure (which lacks the endotrophin domain) have been shown to be associated with advanced liver fibrosis in rodents (Veidal, 2011) and patients (Lebensztejn, 2006; Stickel, 2001), and with elevated portal pressure (Leeming, 2013). The expression of procollagen α3(VI) is regulated by PPARγ which is in direct alignment with our findings. In fact, procollagen α3(VI) mRNA is suppressed by PPARγ, as demonstrated by an increase in its mRNA in adipocyte cultures treated with a siRNA against PPARγ and by a decrease in its transcripts in subcutaneous adipose tissue of patients with type 2 diabetes treated with the PPARγ agonist pioglitazone, especially in patients with high baseline tissue levels of procollagen α3(VI) mRNA. These data may in part explain the change in correlations, from baseline to the end of treatment, between endotrophin/Pro-C6 serum levels and HbA1c or HOMA-IR, in particular the lack of a correlation between endotrophin and the metabolic parameters following glitazone treatment. Thus the expression of the endotrophin precursor (as measured by procollagen α3(VI) mRNA) in peripheral adipose tissue was not dependent on BMI or total fat mass in severely obese, insulin-resistant patients. In another clinical study tissue endotrophin levels in obese subjects correlated with chronic inflammation and systemic insulin resistance (Park, 2013). Further proof of the direct link between procollagen VI, adipose tissue fibrosis and impaired glucose sensitivity is provided by a study in ob/ob mice (that lack a functional leptin gene) in the absence of collagen VI in white adipose tissue. These mice had a significantly improved insulin sensitivity in the absence of adipose tissue fibrosis and inflammation (Khan, 2009). On a first view these data appear to contradict the strong correlation between serum endotrophin and BMI, FLI, and HOMA-IR, as found in our study. However, the presence of procollagen VI is only a necessary but not a sufficient precondition for the proteolytic generation of the adipokine endotrophin. Therefore, it will be of interest to identify the endotrophin generating protease and to characterize its upstream regulation. In addition, leptin induced the expression of type VI procollagen, which further supports a link between leptin resistance, metabolic dysfunction, and endotrophin.
(93) As discussed before, the ECM has until now mostly been considered a passive scaffold. Type VI collagen has mostly been recognized through mutations in the genes COL6A1, COL6A2, and COL6A3 that encode its three constituent chains, which cause muscle disorders such as Bethlem myopathy, Ullrich congenital muscular dystrophy, limb-girdle muscular dystrophy, and autosomal recessive myosclerosis. (Lampe, 2005; Bonaldo, 1998; Bushby, 2014). This provides an interesting link to metabolic dysfunction since muscle represents an important regulator of insulin resistance. Therefore, all available evidence strongly suggests that collagen type VI is more than a passive ECM component, but an important mediator of adipose (and liver) metabolic dysfunction related to insulin resistance, type 2 diabetes, and NASH.
(94) In conclusion, circulating endotrophin which prominently derives from adipocytes and adipose tissue is elevated in relation to insulin resistance and predictive of the response to insulin sensitizers. This permits identification and monitoring of patients who will respond optimally to an insulin sensitizer, which improves the benefit to risk ratio of PPARγ agonists in the treatment of type 2 diabetes and likely NASH.
(95) 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.
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