A NEO-EPITOPE FRAGMENT OF CARTILAGE DEGRADATION GENERATED FROM TYPE II COLLAGEN PROCESSING: A NOVEL SERUM BIOMARKER TO ACCESS TYPE II COLLAGEN DEGRADATION IN JOINT DEGENERATIVE DISEASES

Background: Altered extracellular matrix (ECM) remodelling is an important part of the pathology seen in joint degenerative diseases. Type II collagen is the most abundant ECM protein in the cartilage and provides the tissue with essential tensile strength in order to withstand high compressive loading. During cartilage erosion, type II collagen is cleaved by matrix metallopeptidases (MMPs) which generates new protein fragments called neo-epitopes. These fragments are released into circulation and may potentially serve as biomarkers by indicating the degree of cartilage destruction. Objectives: The aim of this study is to develop a highly specific immunoassay targeting a neo-epitope fragment of type II collagen cleaved, named T2CM. Moreover, we investigated the assays potential to evaluate type II collagen degradation in an ex vivo bovine full-depth cartilage explants model (BEX) with catabolic treatment and in healthy controls and osteoarthritis (OA) patients. Methods: A monoclonal antibody was raised in mouse against the C-terminus from protease cleavage site of type II collagen and a direct competitive ELISA was developed and technically validated. The assay specificity was evaluated for the standard peptide excluding cross-reactivity with elongated and truncated peptides, and a non-sense coating peptide. Human OA cartilage was cleaved with MMP-1, -2, -9 and -13 and measured with the T2CM-assay to investigate which MMPs generated the neo-epitope. T2CM levels were measured in supernatant from BEX explants cultured for 21 days in serum free DMEM/F12 medium with six different doses of OSM+TNF-α (O+T) treatment (20/10, 20/20, 20/40, 10/10, 10/20, 10/40 ng/mL) including a control group without (w/o) treatment. The supernatant was harvested 3 times weekly and replaced with new culture medium with O+T treatment. Biomarker results were confirmed by western blot, where T2CM was measured in supernatant from explants with O+T treatment 20/20 ng/mL and 20/40 ng/mL harvested on day 14 and day 21. To confirm the preclinical data, serum samples from 23 healthy controls (age range from 44-59 years with mean 51.4 ± SD 5.1, gender distribution was 56% female and 44% male, and 100% Caucasian) and 23 OA patients (age range from 41-77 years with mean 57.7 ± SD 13.7, gender distribution was 61% female and 39% male, and 100% Caucasian) were measured by T2CM. Results: A technically robust and T2CM-specific assay was developed. The assay linearity and spike-recovery were accepted with percentage of 99.69% and 93.15%. The assay showed no cross-reaction with the elongated, truncated or non-sense coating peptide. In addition, it was demonstrated that the T2CM neo-epitope was derived from MMP-1 and MMP-13 cleavage of type II collagen. O+T treatment induced the T2CM release in BEX compared to the untreated ( Figure 1 -2). Moreover, the western blot confirmed the T2CM results by the presence of two T2CM bands on day 21 from O+T treated explant compared to day 14 where no bands appeared. T2CM showed to be significantly elevated in patients with OA compared to controls ( p =0.036; mean 3.262 ng/mL ± SD 1.065 vs 2.698 ng/mL ± SD 1.118). Conclusion: The newly developed assay was specific for the T2CM neo-epitope and was determined to be generated by MMP-1 and MMP-13. Additionally, the assay detected elevated levels of T2CM in supernatant from explants treated with O+T after 19 days of treatment compared to untreated. This was further confirmed in human OA patients, where the level of T2CM was elevated compared to healthy controls. This suggests that T2CM may have potential as biomarker for type II collagen degradation. Future preclinical and clinical studies are needed to validate these findings. Figure 1-2. T2CM measurements in BEX model. OSM + TNF-a (O+T) ng/mL. Disclosure of Interests: Solveig Skovlund Groen Employee of: Nordic Bioscience, Dovile Sinkeviciute Grant/research support from: Industrial PhD Student, Employee of: Industrial PhD Student, Christian Thudium Employee of: Employee at Nordic Bioscience A/S., Patrik Önnerfjord: None declared, Morten Karsdal Shareholder of: Nordic Bioscience A/S., Employee of: Full time employee at Nordic Bioscience A/S., Anne-Christine Bay-Jensen Shareholder of: Nordic Bioscience A/S, Employee of: Full time employee at Nordic Bioscience A/S., Signe Holm Nielsen Employee of: Full time employee at Nordic Bioscience Citation: Ann Rheum Dis, volume 79, supplement 1, year 2020, page 236Session: OA, aetiology, pathology and animal models (Poster Presentations)