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The Anti-ADAMTS-5 Nanobody® M6495 Protects Cartilage Degradation Ex Vivo

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The Anti-ADAMTS-5 Nanobody® M6495 Protects Cartilage Degradation Ex Vivo International Journal of Molecular Sciences Article The Anti-ADAMTS-5 Nanobody® M6495 Protects Cartilage Degradation Ex Vivo Anne Sofie Siebuhr 1,* , Daniela Werkmann 2, Anne-C. Bay-Jensen 1, Christian S. Thudium 1, Morten Asser Karsdal 1, Benedikte Serruys 3, Christoph Ladel 2, Martin Michaelis 2 and Sven Lindemann 2 1 ImmunoScience, Nordic Bioscience Biomarkers and Research, 2730 Herlev, Denmark; [email protected] (A.-C.B.-J.); [email protected] (C.S.T.); [email protected] (M.A.K.) 2 Merck KGaA, 64293 Darmstadt, Germany; [email protected] (D.W.); [email protected] (C.L.); [email protected] (M.M.); [email protected] (S.L.) 3 Ablynx, A Sanofi Company, 9052 Ghent, Belgium; [email protected] * Correspondence: [email protected]; Tel.: +45-(44)-52-52-52 Received: 1 July 2020; Accepted: 2 August 2020; Published: 20 August 2020 Abstract: Osteoarthritis (OA) is associated with cartilage breakdown, brought about by ADAMTS-5 mediated aggrecan degradation followed by MMP-derived aggrecan and type II collagen degradation. We investigated a novel anti-ADAMTS-5 inhibiting Nanobody® (M6495) on cartilage turnover ex vivo. Bovine cartilage (BEX, n = 4), human osteoarthritic - (HEX, n = 8) and healthy—cartilage (hHEX, n = 1) explants and bovine synovium and cartilage were cultured up to 21 days in medium alone (w/o), with pro-inflammatory cytokines (oncostatin M (10 ng/mL) + TNFα (20 ng/mL) (O + T), IL-1α (10 ng/mL) or oncostatin M (50 ng/mL) + IL-1β (10 ng/mL)) with or without M6495 (1000 0.46 − nM). Cartilage turnover was assessed in conditioned medium by GAG (glycosaminoglycan) and biomarkers of ADAMTS-5 driven aggrecan degradation (huARGS and exAGNxI) and type II collagen degradation (C2M) and formation (PRO-C2). HuARGS, exAGNxI and GAG peaked within the first culture week in pro-inflammatory stimulated explants. C2M peaked from day 14 by O + T and day 21 in co-culture experiments. M6495 dose dependently decreased huARGS, exAGNxI and GAG after pro-inflammatory stimulation. In HEX C2M was dose-dependently reduced by M6495. M6495 showed no effect on PRO-C2. M6495 showed cartilage protective effects by dose-dependently inhibiting ADAMTS-5 mediated cartilage degradation and inhibiting overall cartilage deterioration in ex vivo cartilage cultures. Keywords: ADAMTS-5; aggrecan; cartilage; biomarkers 1. Introduction The extracellular matrix (ECM) of cartilage is well described and has been studied for decades. It is recognized that not only cartilage, but several joint tissues are important in osteoarthritis (OA) pathology. However, cartilage is still the main focus area in development of disease modifying treatments for OA (DMOADs). The ECM of cartilage consists mainly of proteoglycans and collagens and the chondrocyte is the only cell type responsible for the maintenance of cartilage. With the few ECM molecules and the single cell type present in cartilage, the search for DMOADs should be simple, but the continuous failure of DMOADs in phase 2 and 3 trials tells otherwise. ECM turnover is dependent on a delicate regulation of synthesis and degradation during development and aging. During aging or arthritic diseases, the proteolytic degradation of cartilage ECM is increased compared to the formation. The main ECM proteins in cartilage are aggrecan and type Int. J. Mol. Sci. 2020, 21, 5992; doi:10.3390/ijms21175992 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, x 2 of 14 Int. J. Mol. Sci. 2020, 21, 5992 2 of 14 as key enzymes for proteolytic depletion of aggrecan during the development of OA [1,2]. Both ADAMTS-4II collagen. ADAMTS-4and ADAMTS-5 (aggrecanase-1) cleave the and NITEGE373 ADAMTS-5↓374ARGS (aggrecanase-2) bond in havethe interglobular been identified domain as key (IGD)enzymes of foraggrecan. proteolytic MMP-13 depletion has ofbeen aggrecan recognized during as the the development key protease of for OA initial [1,2]. Bothcollagen ADAMTS-4 type II degradationand ADAMTS-5 [3]. cleavePrevious the NITEGE373studies in 374ARGSmice have bond indicated in the interglobularthat while MMP-mediated domain (IGD) of aggrecan.cartilage # destructionMMP-13 has leads been to recognized irreversible as deterioration the key protease of the for joint initial matrix, collagen the typeaggrecanase-mediated II degradation [3]. Previouscartilage deteriorationstudies in mice was have fully indicated reversible that upon while correction MMP-mediated of the catabolic cartilage environmen destructiont leads[4–6]. toOther irreversible in vivo studiesdeterioration have found of the jointthat aggrecan matrix, the loss aggrecanase-mediated was reversible as long cartilage as the progression deterioration was was not fully too reversible advance [7–9].upon correctionIn a murine of the model catabolic inhibition environment of ADAMTS-5 [4–6]. Other preventedin vivo studies overall have cartilage found thatdegradation, aggrecan suggestingloss was reversible that ADAMTS-5 as long as may the progression be the primary was notaggrecanase too advance responsible [7–9]. In afor murine aggrecan model degradation inhibition inof ADAMTS-5mice [10]. Inhibiting prevented MMP-13, overallcartilage another degradation,protease upregulated suggesting during that ADAMTS-5 OA, reduced may collagen be the primarydegradation, aggrecanase but not responsibleaggrecan degradation for aggrecan [11]. degradation These results in mice indicate [10]. Inhibiting that inhibiting MMP-13, aggrecan another proteasedegradation upregulated by targeting during aggrecanases OA, reduced may collagen be mo degradation,re chondroprotective but not aggrecan than targeting degradation collagen [11]. Thesedegradation. results indicateIn addition, that inhibitingstudies have aggrecan demonstrat degradationed a superior by targeting effect aggrecanases of selective maymonoclonal be more antibodychondroprotective to ADAMTS-5 than targetingversus ADAMTS-4 collagen degradation.in human OA In cartilage addition, explants studies [12]. have Thus, demonstrated ADAMTS-5 a issuperior an attractive effect target of selective in the monoclonaldevelopment antibody of a therapeutic to ADAMTS-5 agent for versus OA [13]. ADAMTS-4 in human OA cartilageIn general, explants degradation [12]. Thus, ADAMTS-5 of the aggrecan is an attractive net results target in in thethe developmentgeneration and of a therapeuticrelease of glycosaminoglycanagent for OA [13]. (GAG) fragments [14]. Specific cleavage with ADAMTS-5 in aggrecan exposes the ARGSIn general, neo-epitope; degradation the of N-terminal the aggrecan neo- netepitope results in after the generationcleavage andof the release NITEGE373 of glycosaminoglycan↓374ARGSV (GAG)bond in fragments aggrecan [14[15]]. SpecificThe ARGS cleavage fragment with is ADAMTS-5 rapidly excreted in aggrecan from exposesthe ECM the in ARGS cartilage neo-epitope; explant culturethe N-terminal models neo-epitope and is also after present cleavage in serum, of the NITEGE373 urine and synovial374ARGSV fluid bond of inar aggrecanthritis patients [15] The [16,17]. ARGS # Quantificationfragment is rapidly of the excreted ARGS from fragment the ECM in synovial in cartilage fluid, explant instead culture of the models full length and is aggrecan, also present have in serum, been foundurine and to be synovial a more fluid sensitive of arthritis biomarker patients to [16 distinguish,17]. Quantification injured of and the diseased ARGS fragment subjects in from synovial normal, fluid, thaninstead other of the aggrecanase-derived full length aggrecan, biomarkers have been found [14,18]. to be a more sensitive biomarker to distinguish injured and diseasedIn this subjectsstudy we from investigated normal, than the other inhibitory aggrecanase-derived effect of the biomarkers Nanobody [14®, 18M6495,]. specifically inhibitingIn this ADAMTS-5, study we investigated in ex vivo thecartilage inhibitory models. effect M6495 of the Nanobodyis a bivalent® M6495,and bifunctional specifically Nanobody inhibiting ADAMTS-5,of 28.1 kilodalton in ex (kDa) vivo comprising cartilage models. two sequence-optimized M6495 is a bivalent variable and domains bifunctional derived Nanobody from heavy of chain-only28.1 kilodalton llama (kDa) antibodies, comprising i.e., one two N-terminal sequence-optimized ADAMTS-5-neutralizing variable domains building derived block from and one heavy C- terminalchain-only human llama serum antibodies, albumin i.e., (HSA)-binding one N-terminal block ADAMTS-5-neutralizingfor in vivo half-life extension building (HLE) (See block Figure and S1).one The C-terminal two building human blocks serum are albumin separated (HSA)-binding by a glycine-serine block linker for in (35GS). vivo half-life extension (HLE) (See Figure S1). The two building blocks are separated by a glycine-serine linker (35GS). 2. Results 2. Results 2.1. M6495 Specificity towards ADAMTS-5 2.1. M6495 Specificity towards ADAMTS-5 M6495 showed a high affinity for its target ADAMTS-5, with a KD of 3.65 pM (95% CI: 2.27 pM– 5.86 pM)M6495 (n showed= 3, CV a20%). high affinityAlso, M6495 for its targetdemonstrated ADAMTS-5, full with functionality a KD of 3.65 against pM (95% its CI:target, 2.27 pM–5.86as indicated pM) (byn =a concentration-dependent3, CV 20%). Also, M6495 and demonstrated complete inhibiti full functionalityon of the enzymatic against its activity target, of as ADAMTS-5 indicated by by a M6495concentration-dependent in an enzymatic activity and complete assay inhibition(unpublished of the data). enzymatic In contrast activity to of its ADAMTS-5 binding to
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