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The type II collagen C-terminal pro-peptide is one of the most abundant polypeptides in cartilage. The purpose of this study was to develop a competitive chemiluminescence enzyme-linked immunosorbent assay, CALC2, targeting this pro-peptide as a marker of cartilage formation. Technical assay parameters were evaluated. CALC2 level was measured after in vitro cleavage of recombinant type II collagen with bone morphogenetic protein-1 (BMP-1) and treatment of ex vivo human osteoarthritis (OA) cartilage explant model (HEX) with insulin-like growth factor-1 (IGF-1). Serum CALC2 levels were assessed in 18 patients with rheumatoid arthritis (RA), 19 patients with ankylosing spondylitis (AS), and 18 age- and sex-matched controls in cohort 1 and 8 patients with OA and 14 age- and sex-matched controls in cohort 2. Type II collagen cleavage with BMP-1 increased the CALC2 level. IGF-1 treatment increased the CALC2 levels in HEX compared with the untreated explants (p < 0.05). Results were confirmed using Western blot analysis. CALC2 levels were decreased in the patients with RA and AS compared with the healthy controls (p = 0.01 and p = 0.02, respectively). These findings indicate that CALC2 may be a novel biomarker of type II collagen formation. However, further preclinical and clinical studies are required to validate these findings.

Osteoarthritis (OA) and rheumatoid arthritis (RA) are serious and painful diseases. Protease-activated receptor 2 (PAR2) is involved in the pathology of both OA and RA including roles in synovial hyperplasia, cartilage destruction, osteophyogenesis and pain. PAR2 is activated via cleavage of its N-terminus by serine proteases. In this study a competitive ELISA assay was developed targeting the 36-amino acid peptide that is cleaved and released after PAR2 activation. Technical assay parameters including antibody specificity, intra- and inter-assay variation (CV%), linearity, accuracy, analyte stability and interference were evaluated. PAR2 pro-fragment release was confirmed after in vitro cleavage of PAR2 recombinant protein and treatment of human synovial explants with matriptase. Serum levels of 22 healthy individuals, 23 OA patients and 15 RA patients as well as a subset of RA patients treated with tocilizumab were evaluated. The PAR2 antibody was specific for the neo-epitope and intra-inter assay CV% were 6.4% and 5.8% respectively. In vitro cleavage and matriptase treated explants showed increased PAR2 pro-fragment levels compared to controls. In serum, PAR2 pro-fragment levels were increased in RA patients and decreased in RA patients treated with tocilizumab. In conclusion, PAR2 may be a potential biomarker for monitoring RA disease and pharmacodynamics of treatment.

Sprifermin, recombinant human fibroblast growth factor 18 (rhFGF18), induces cartilage regeneration in knees of patients with osteoarthritis (OA). We hypothesized that a temporal multiphasic process of extracellular matrix (ECM) degradation and formation underlie this effect. We aimed to characterize the temporal ECM remodeling of human knee OA articular cartilage in response to sprifermin treatment. Articular cartilage explants from patients with knee OA (npatients = 14) were cultured for 70 days, with permanent exposure to sprifermin (900, 450, 225 ng/mL), FGF18 (450 ng/mL), insulin-like growth factor-1 (100 ng/mL, positive control) or vehicle (nreplicates/treatment/patient = 2). Metabolic activity (AlamarBlue) and biomarkers of type IIB collagen (PIIBNP) formation (Pro-C2 enzyme-linked immunosorbent assay [ELISA]) and aggrecanase-mediated aggrecan neo-epitope NITEGE (AGNx1 ELISA) were quantified once a week. At end of culture (day 70), gene expression (quantitative reverse transcription polymerase chain reaction) and proteoglycan content (Safranin O/Fast green staining) were quantified. The cartilage had continuously increased metabolic activity, when treated with sprifermin/FGF18 compared to vehicle. During days 7–28 PIIBNP was decreased and NITEGE was increased, and during days 35–70 PIIBNP was increased. At end of culture, the cartilage had sustained proteoglycan content and relative expression of ACAN

Summary Objective Links between pain and joint degradation are poorly understood. We investigated the role of activation of Toll-like receptors (TLR) by cartilage metabolites in initiating and maintaining the inflammatory loop in OA causing joint destruction. Methods Synovial membrane explants (SMEs) were prepared from OA patients’ synovial biopsies. SMEs were cultured for 10 days under following conditions: culture medium alone, OSM + TNFα, TLR2 agonist - Pam2CSK4, Pam3CSK4 or synthetic aggrecan 32-mer, TLR4 agonist - Lipid A. Release of pro-inflammatory and degradation biomarkers (acMMP3 and C3M) were measured by ELISA in conditioned media along with IL-6. Additionally, human cartilage was digested with ADAMTS-5, with or without the ADAMTS-5 inhibiting nanobody - M6495. Digested cartilage solution (DCS) and synthetic 32-mer were tested for TLR activation in SEAP based TLR reporter assay. Results Western blotting confirmed TLR2 and TLR4 in untreated OA synovial biopsies. TLR agonists showed an increase in release of biomarkers - acMMP3 and C3M in SME. Synthetic 32-mer showed no activation in the TLR reporter assay. ADAMTS-5 degraded cartilage fragments activated TLR2 in vitro. Adding M6495 – an anti-ADAMTS-5 inhibiting nanobody®, blocked ADAMTS-5-mediated DCS TLR2 activation. Conclusion TLR2 is expressed in synovium of OA patients and their activation by synthetic ligands causes increased tissue turnover. ADAMTS-5-mediated cartilage degradation leads to release of aggrecan fragments which activates the TLR2 receptor in vitro. M6495 suppressed cartilage degradation by ADAMTS-5, limiting the activation of TLR2. In conclusion, pain and joint destruction may be linked to generation of ADAMTS-5 cartilage metabolites.

Background: Patients with joint diseases such as osteoarthritis (OA) are believed to have an abundance of endogenous Toll like receptor (TLR) ligands in their joints, which might be responsible for activating TLRs that may ultimately initiate a self-perpetuating inflammatory loop in the disease. TLR ligands may be generated from the breakdown of articular cartilage, which in turn arises from the activity of two key enzymes: A disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS-5) and Matrix metalloproteinases (MMPs). Objectives: In this study, we investigate the effect of M6495, a novel anti ADAMTS-5 inhibiting nanobody® on TLR2 activation by ADAMTS-5 derived cartilage cleaved fragments Methods: Human cartilage biopsies were retrieved from OA patients undergoing total knee replacement. The tissue cartilage was snap frozen in liquid nitrogen to eliminate underlying metabolic activity and then digested with recombinant human ADAMTS-5 (4 μg/ml) for 24, 48 and 72 hours respectively at 37 oC. The undigested remaining cartilage was discarded after each timeframe and the digested cartilage solution (DS) was used for further testing. Tissue cleavage was assessed by measuring the release of aggrecan degradation biomarkers: Aggrecanase mediated aggrecan degradation (AGNx1) and MMP mediated aggrecan degradation (FFGV). DS was tested for TLR activation in a secreted embryonic alkaline phosphatase (SEAP) reporter gene based HEK hTLR2 (human Toll like receptors) cell line. Cartilage tissue in buffer alone was used as control at each time point. Results: Aggrecan degradation in cartilage was confirmed by increased release of AGNx1 (p Conclusion: ADAMTS-5-mediated cartilage degradation leads to release of aggrecan fragments, which activate the TLR2 receptor in vitro in a specialised reporter system. Anti-ADAMTS-5 inhibiting nanobody®, M6495, showed a suppression in release of degradation biomarkers leading to limited activation of TLR2. The data suggest a potential chondro-protective effect by M6495. Disclosure of Interests: Neha Sharma: None declared, Christian Thudium Employee of: I am a full time employee of Nordic Bioscience, Morten Karsdal Shareholder of: I own shares of Nordic Bioscience, Employee of: I am a full-time employee in Nordic Bioscience, Anne-Christine Bay-Jensen Shareholder of: I own shares of Nordic Bioscience, Employee of: I am a full-time employee in Nordic Bioscience, Thorbjorn Gantzel: None declared, Martin Michaelis Employee of: Employee of Merck KGaA, Darmstadt, Germany, Christoph Ladel Employee of: Employee of Merck KGaA, Darmstadt, Germany, Daniela Werkmann Employee of: Employee of Merck KGaA, Darmstadt, Germany, Sven Lindemann Employee of: Employed by Merck KGaA

Objective Rheumatoid arthritis (RA) is a chronic and degenerative autoimmune joint disease that leads to disability, reduced quality of life, and increased mortality. Although several synthetic and biologic disease-modifying antirheumatic drugs are available, there is still a medical need for novel drugs that control disease progression. As only 10% of experimental drug candidates for treatment of RA that enter phase I trials are eventually registered by the Food and Drug Administration, there is an immediate need for translational tools to facilitate early decision-making in drug development. In this study, we aimed to determine if the inability of fostamatinib (a small molecule inhibitor of Syk) to demonstrate sufficient efficacy in phase III of a previous clinical study could have been predicted earlier in the development process. Methods Biomarkers of bone, cartilage, and interstitial matrix turnover (C-telopeptide of type I collagen [CTX-I], matrix metalloproteinase-derived types I, II, and III collagen neoepitopes [C1M, C2M, and C3M]) were measured in 450 serum samples from the Oral Syk Inhibition in Rheumatoid Arthritis 1 study (OSKIRA-1, a phase III clinical study of the efficacy of fostamatinib in RA) at baseline and follow-up. Additionally, the same biomarkers were subsequently measured in conditioned media from osteoclast, cartilage, and synovial membrane cultured with the active metabolite of fostamatinib, R406, to assess the level of suppression induced by the drug. Results In OSKIRA-1 serum samples and osteoclast and cartilage cultures, fostamatinib suppressed the levels of CTX-I and C2M. In OSKIRA-1 serum samples and synovial membrane cultures, fostamatinib did not mediate any clinical or preclinical effect on either C1M or C3M, which have previously been associated with disease response and efficacy. Conclusion These data demonstrate that translational biomarkers are a potential tool for early assessment and decision-making in drug development for RA treatment.