A HIGHLY SENSITIVE NEO-EPITOPE BIOMARKER OF TYPE II COLLAGEN C- TERMINAL IS ASSOCIATED WITH CARTILAGE FORMATION

Background Altered extracellular matrix (ECM) remodeling is a common event in rheumatic diseases. Type II collagen is the most abundant ECM protein in the cartilage and provides tensile elasticity and strength to enable support to the joints. Chondrocalcin, also known as the C-terminal propeptide of type II collagen, is among the most highly synthesized polypeptides in cartilage. It is cleaved off by BMP-1/C-endopeptidase during maturation and plays a role in assembly of type II collagen and in calcification of cartilage matrix. When cleaved off, the chondrocalcin fragments are released into circulation, where they can be quantified in a blood sample. Objectives This study aimed at developing an immunoassay targeting a neo-epitope of chondrocalcin, named CALC2. Moreover, we explored its biomarker potential to evaluate type II collagen formation in an ex vivo human osteoarthritis (OA) cartilage explant model (HEX) with anabolic treatment, and in serum from healthy controls, patients with rheumatoid arthritis (RA) and patients with ankylosing spondylitis (AS). Methods A novel direct immunoassay targeting the neo-epitope of type II collagen C-terminal (PIICP) was developed and technically validated. The technical validation included inter- and intra-variation, linearity, spiking recovery, stability, and specificity. Specificity of the monoclonal antibody was tested using an elongated peptide, a truncated peptide, and a non-sense peptide to exclude possible cross-reactivity. CALC2 levels were measured in supernatant from HEX cultured for 35 days in serum free DMEM/F12 medium with IGF-1 (Insulin-like Growth Factor-1) (100 ng/mL), including a control group without (w/o) treatment. The supernatant was harvested 3 times weekly and replaced with new culture medium with IGF-1. Biomarker results were confirmed by western blot. Serum samples from 18 healthy donors (mean age 35.8 ± SD 3.8, 100% Caucasian), 19 patients with AS (mean age 35.8 ± SD 3.2, 100 % Caucasian) and 18 patients with RA (mean age 35.8 ± SD 3.4), 100% Caucasian) were also measured by CALC2. Linear regression models with pairwise comparisons were performed. Results A technically robust and specific assay was developed. The inter- and intra-assay variation of CALC2 was determined as 12% and 7% respectively. CALC2 showed a good dilution recovery, spiking recovery, and storage /freeze-thaw stability (All, 100%±20%). CALC2 showed specificity towards the targeted sequence and did not show any reactivity towards the truncated peptide, elongated peptide, or non-sense peptide. CAL2 neoepitope levels were significantly elevated after 14, 21 and 28 days of IGF-1 treatment compared to untreated (p<0.01, p<0.0001 p<0.001, respectively). The western blot confirmed the CALC2 results by the presence of a band of ~35 kDa in all explants, corresponding to the weight of chondrocalcin previously stablished (1). Furthermore, the bands were more pronounced at day 21 in the IGF-1 treated explant compared to the untreated explant. CALC2 also showed significantly lower levels in patients with RA compared to controls ( p =0.003; mean 0.32 ng/mL ± SD 0.16 vs 0.64 ng/mL ± SD 0.31). Conclusion Higher levels of CALC2 were detected in supernatants from explants after 14, 21 and 28 days of IGF-1 treatment compared to untreated. Lower levels of CALC2 were present in patients with RA compared to healthy controls. Overall, this suggests that CALC2 may have potential as biomarker for type II collagen formation. However, further preclinical and clinical studies are required to validate these findings. References [1]Poole R, Choi H. Association of an extracellular protein (chondrocalcin) with the calcification of cartilage in endochondral bone formation. J Cell Biol. 1984;98(1):54–65. Figure 1. CALC2 measurements in HEX model. Disclosure of Interests Helena Port: None declared, Christian Thudium Shareholder of: Shareholder at Nordic Bioscience A/S, Thorbjørn Gantzel: None declared, Anne-Christine Bay-Jensen Shareholder of: Shareholder of Nordic Bioscience A/S, Morten Karsdal Shareholder of: Shareholder at Nordic Bioscience A/S, Signe Holm Nielsen Shareholder of: Shareholder at Nordic Bioscience A/S