AB0047 ACTIVATION OF TLR2 BY ADAMTS-5-MEDIATED DEGRADATION FRAGMENTS OF CARTILAGE EXPLANTS IS INHIBITED BY THE ANTI-ADAMTS-5 NANOBODY®, M6495
Authors: Neha Sharma, Christian Thudium, Morten Karsdal, Anne-Christine Bay-Jensen, Thorbjørn Gantzel, Martin Michaelis, Christoph Ladel, Daniela Werkmann, Sven Lindemann
Journal: Annals of the Rheumatic Diseases (2019)

<h3>Background:</h3> 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). <h3>Objectives:</h3> 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 <h3>Methods:</h3> 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 <sup>o</sup>C. 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. <h3>Results:</h3> Aggrecan degradation in cartilage was confirmed by increased release of AGNx1 (p&lt;0.0001) (Fig. 1a) and FFGV (p&lt;0.01 at 48 hours and p&lt;0.001 at 72 hours) (Fig. 1b) in the DS compared to control. M6495 inhibited release of ADAMTS-5-mediated AGNx1 (p&lt;0.0001, Fig. 1a) and FFGV (p&lt;0.05 at 48 hours and p&lt;0.05 at 72 hours, Fig. 1b). ADAMTS-5mediated DS showed TLR2 activation in the SEAP based reporter system when compared to control (p&lt;0.05) (Fig. 1c). Adding M6495 blocked the ADAMTS-5-mediated DS TLR2 activation (p&lt;0.01) (Fig. 1c). <h3>Conclusion:</h3> ADAMTS-5-mediated cartilage degradation leads to release of aggrecan fragments, which activate the TLR2 receptor <i>in vitro</i> 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. <h3>Disclosure of Interests:</h3> 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, Thorbjørn 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