How does hsPro-C2 ECLIA work? A Novel, High Sensitivity Marker, hsPro-C2, Of Cartilage Formation, Was Developed And Tested In A Phase II Clinical Trial Of PTH Yunyun Luo1, Yi He1, Inger Byrjalsen1, Kim Henriksen1, Natasja Gudmann1, Ali Mobasheri2, Gitte Hanse1, Morten Karsdal1, Anne-C. Bay-Jensen1 1 Nordic Bioscience A/S, Herlev Hovedgade 207, DK-2730, Herlev, Denmark 2D-BOARD EU Consortium for Biomarker Discovery, University of Surrey, Surrey, United Kingdom arthritic disease METHODS BACKGROUND Presently, measurement of cartilage formation relies on magnetic resonance imaging (MRI), which is a sensitive measure, but which need long follow-up time. Thus, there is an unmet need, in disease-modifying osteoarthritis drug (DMOAD) development, for an objective and non-invasive marker of cartilage formation, which can provide early indication of drug efficacy. The objective was to enable assessment of type IIB collagen synthesis in serum from human subjects. Such tool may be applicable as a non-invasive biomarker of cartilage repair and growth in the development of cartilage anabolic drugs. No cartilage anabolic DMOAD have to date been approved thus for proof of concept, we used an osteoporosis (OP) trial testing teriparatide (human parathyroid hormone (PTH) 1-34). Teriparatide has been shown to have potential chondro-protective and chondro-regenerative effects on articular cartilage in vitro and in vivo. However, it remains unclear whether the pro-anabolic effect of teriparatide translate to human OA. A high sensitivity competitive electro-chemiluminescence immunoassay for detection of PIIBNP (hsPro-C2 ECLIA) was developed and the technical performance evaluated. From a randomized, double-blind placebo-controlled study with an open-label active comparator/positive control (teriparatide) in postmenopausal women with OP (clinicaltrials.gov: NCT01321723), the biomarker sub-study included 64 Caucasian postmenopausal women (age 45-80 years) with OP duration of at least 5 years. Thirty-two women were treated with teriparatide, and 32 with placebo. Biomarkers of bone formation (PINP; procollagen type I N-terminal propeptide) and cartilage formation (hsPro-C2) were analyzed retrospectively at baseline, week 4, 12 and 24. Correlation between PINP and hsPro-C2 at baseline and change at week 4 relative to baseline were investigated by Pearson’s correlation. Structures of PIIBNP Median percent change in serum hsPro-C2 level was higher in teriparatide treated group compared to placebo, although not statistically significant Figure 1. Schematic illustration of PIIBNP (Pro-C2). Type II procollagen is synthesized in two splice forms, type IIA and IIB, as the result of alternative splicing of exon 2 in N-propeptide region. The monoclonal antibody utilized in Pro-C2 only recognizes the N-terminus of PIIBNP after the removal of the signal peptide. How does hsPro-C2 ECLIA work? Figure 3. Response as median percent change from baseline (± interquartile range) in hsPro-C2 (A) and PINP (B) in women receiving teriparatide (●) or placebo (○). Teriparatide was administered subcutaneously with 20 mg/day. The marker of cartilage formation (hsPro-C2; procollagen type IIB N-terminal propeptides), and marker of bone formation (PINP; procollagen type I N-terminal propeptides) were measured with regular intervals throughout the study period. The responses in teriparatide treated and placebo groups are plotted as the median percent change of individual baselines. The % change of Pro-C2 values between teriparatide treated and placebo groups were compared with a two-way ANOVA test. Asterisks indicate the following: *P < 0.05, **P < 0.01, ****P < 0.0001. Elevated bone formation was significantly associated with the change of marker in cartilage formation at week 4 and 24 Table 2. Associations between biomarkers of bone formation and cartilage formation percent changes relative to baseline in teriparatide treated group. Pearson’s correlation coefficient (r) are indicated. PINP, amino terminal propeptide of type I procollagen; pct: percent change relative to baseline Figure 2. Basic principle of hsPro-C2 competition electrochemiluminescence immunoassay (ECLIA). 1) High binding carbon electrodes in the bottom of Streptavidin microplates allow for easy attachment of biotinylated PIIBNP antigen (10X greater binding capacity than polystyrene). 2) Electrochemiluminescent labels (SULFO-TAG) that are conjugated to detection antibodies allow for ultra-sensitive detection. 3) Electricity is applied to the plate electrodes by an MSD instrument leading to light emission by SULFO-TAG labels. 4) Light intensity is then measured to quantify analytes in the sample. (Adapted from MSD LLC.) CONCLUSION In spite of the small sample size of this study there was a clear trend toward increased cartilage formation in the PTH treated group over time. Furthermore, hsPro-C2 changes correlated with the changes in PINP, which is believed to be a pharmacodynamics biomarker of teriparatide treatment, indicating that hsPro-C2 reflect a possible chondro-anabolic effect of PTH. It is concluded that hsPro-C2 may be a promising and novel marker of cartilage formation to be used in DMOAD development.