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Thesis Salvatore Santamaria 27.9.13 Screening, Isolation and Characterisation of Antibodies That Block Aggrecanase Activity of ADAMTS-5 Salvatore Santamaria A thesis submitted for the degree of Doctor of Philosophy to Imperial College London September 2013 Department of Matrix Biology The Kennedy Institute of Rheumatology Division Faculty of Medicine Imperial College London 1 Abstract A Disintegrin and Metalloproteinase with ThromboSpondin Motif (ADAMTS)-5 is considered to play a key role in degradation of aggrecan during the progression of osteoarthritis (OA). Thus, ADAMTS-5 inhibitors are emerging as potential disease-modifying OA drugs. The generation of small molecule inhibitors of metalloproteinases has not achieved successful outcomes primarily due to the lack of selectivity. On the other hand, monoclonal antibodies were proved potent and selective binders of many biologically relevant targets. We therefore aimed to isolate inhibitory antibodies against ADAMTS-5. A human naïve single-chain variable fragment library was used to select and isolate anti- ADAMTS-5 antibodies. Among the isolated antibodies, clones 2D3 and 2D5 inhibited ADAMTS-5 cleavage both of a peptide substrate (Ki app values of 2 nM and 18 nM, respectively) and bovine aggrecan (Ki app values of 30 nM and 300 nM, respectively). Antibody 2B9 did not inhibit ADAMTS-5 activity against the peptide substrate, but it inhibited the cleavage of aggrecan (Ki app: 300 nM). Surface plasmon resonance studies with a series of deletion mutants of ADAMTS-5 indicated that 2D3 and 2D5 bind to the catalytic/disintegrin domain (KD values of 3 nM and 55 nM, respectively), whereas 2B9 binds to the spacer domain of this enzyme (KD value of 6 nM), thus being an exosite inhibitor. Kinetic studies indicated that the anti-ADAMTS-5 antibodies described here do not act through a zinc-chelating mechanism of inhibition. Moreover, inhibition and co- immunoprecipitation data suggested that for some antibodies, such as 2D5, the binding site is different from that of TIMP-3. The efficacy of ADAMTS-5-blocking antibodies was investigated using model systems of aggrecan degradation (IL-1α/oncostatin M-stimulated porcine and human articular cartilage explants). The results indicated that antibodies 2D3 and 2B9 effectively inhibited aggrecanase activity in these systems. These antibodies may therefore be useful as chondroprotectant agents in OA. 2 TABLE OF CONTENTS Abstract……………………………………………………………………………………………...2 List of Figures……………………………………………………………………………………….7 List of Tables……………………………………………………………………………………….10 Declaration of Originality…………………………………………………………………………12 Copyright Declaration……………………………………………………………………………..13 Acknowledgements………………………………………………………………………………...14 Abbreviations………………………………………………………………………………………15 CHAPTER 1: INTRODUCTION 1.1. Protease-mediated degradation of articular cartilage…………………………...19 1.2. ADAMTSs………………………………………………………………………......21 1.2.1. The ADAMTS family…………………………………………………………….....21 1.2.2. Biological role of ADAMTSs…………………………………………………….....22 1.2.3. ADAMTS domain structure………………………………………………………....26 1.2.4. ADAMTS regulation………………………………………………………………..28 1.2.4.1. Regulation of expression………………………………………………….....28 1.2.4.2. Post-transcriptional regulation………………………………………………31 1.2.4.3. Post-translational regulation………………………………………………...31 1.2.4.4. Regulation by endogenous inhibitors and cofactors………….……………..33 1.2.4.5. ADAMTS in cancer…………………………………………………………33 1.3. Aggrecanases………………………………………………………………………37 1.3.1. Aggrecan cleavage sites…………………………………………………………......37 1.3.2. Physiological and pathological catabolism of aggrecan…………………………….40 1.3.3. Role of exosites in aggrecanase activity…………………………………………….43 1.3.4. Other substrates of aggrecanases……………………………………………………45 1.3.5. ADAMTS-5 (aggrecanase-2) as a target in OA…………………………………….48 1.4. Strategies to develop disease-modifying osteoarthritic drugs...............................53 1.4.1. Small molecule inhibitors of metalloproteinases...………………………………….53 1.4.2. Monoclonal antibodies (mAbs)……………………………………………………...54 1.5. Aim of the thesis……………………………..……………………………………..57 3 CHAPTER 2: MATERIALS AND METHODS 2.1. Reagents…………………………………………………………………………………….59 2.1.1. Tissue and cell culture reagents……………………………………………………..59 2.1.2. SDS-PAGE and Western blot reagents……….……………………………………..59 2.1.3. Protein chemistry reagents…………………………………………………………..60 2.1.3.1.1. Proteins and protein purification…………………………………….60 2.1.3.1.2. Biotinylation…………………………………………………………60 2.1.3.1.3. Quenched -Fluorescent (QF) peptide cleavage assay and aggrecan cleavage……………………..………………………………………61 2.1.4. Phage display and molecular biology reagents……………………………………...61 2.1.5. Primary screening…………………………………………………………………...61 2.1.6. scFv-Fc characterisation….…………………………………………………………62 2.2. Methods…………………………………………………………………………………….62 2.2.1. SDS-PAGE and Western Blots……………………………………………………..62 2.2.1.1. SDS-PAGE………………………………………………………………….62 2.2.1.2. Gel staining………………………………………………………………….62 2.2.1.2.1. Coomassie Brilliant Blue R-250 staining……………………………62 2.2.1.2.2. Silver staining………………………………………………………..63 2.2.1.3. Western blot analysis………………………………………………………..63 2.2.2. Antigen preparation and characterisation……………………………………….......64 2.2.2.1. Expression and purification of recombinant proteins..……………………...64 2.2.2.2. QF peptide cleavage assay.………………………………...………………..65 2.2.2.3. Aggrecan digestion assay……………………………………………………65 2.2.2.4. Biotinylation of ADAMTS-5 and ADAMTS-4……………………………..65 2.2.3. Phage Display Selections……………………………………………………………66 2.2.3.1. Solution-phase phage display selection………………...…………………...69 2.2.3.2. Solid-phase phage display selection (biopanning)……...…………………..71 2.2.3.3. Polyclonal Phage ELISA………………………………....…………………71 2.2.4. Primary Screening…………………………………………………………………...71 2.2.4.1. Subcloning selected populations into pSANG10-3F and pBIOCAM5 expression vectors …………………………………………..……………..71 2.2.4.2. Bacterial screening…………………………………………………………..72 2.2.4.2.1. Expression of scFv in E. coli………………………………………...72 2.2.4.2.2. scFv-ELISA……………………………………….…………………73 4 2.2.4.3. Mammalian screening……………………………………………………….73 2.2.4.3.1. High-throughput DNA Purification…………………………………73 2.2.4.3.2. Transient transfection of HEK293-F cells…………………………..74 2.2.4.3.3. scFv-Fc-ELISA……………………………………………………...74 2.2.5. Secondary screening………………………………………………………………...75 2.2.5.1. Sequence analysis…………………………………………………………...75 2.2.5.2. Determination of scFv-Fc concentration in cell supernatant by sandwich ELISA………………………………………………………………….75 2.2.5.3. Purification of scFv-Fv and scFv-Fc antibodies…………………………….75 2.2.6. scFv-Fc characterisation…………………………………………………………….76 2.2.6.1. Surface Plasmon Resonance (SPR)………………………………………….76 2.2.6.2. Investigation of inhibition mechanism………………………………………76 2.2.6.3. Dual inhibition studies………………………………………………………76 2.2.6.4. Solid-phase binding assay for ADAMTS-5 binding to heparin......................77 2.2.6.5. Immunoprecipitation of ADAMTS-5 with scFv-Fc antibodies......................77 2.2.7. Cartilage explant degradation studies……………………………………………….77 2.2.7.1. Cartilage explant culture…………………………………………………….77 2.2.7.2. Cartilage explant culture conditions for aggrecan degradation……………..77 2.2.7.3. Dimethylmethylene blue (DMMB) assay…………………………………...78 2.2.7.4. Analysis of fragments in the conditioned medium………………………….78 2.2.8. Phylogram…………………………………………………………………………...78 CHAPTER 3: PREPARATION OF ANTIGEN FOR PHAGE DISPLAY SELECTION 3.1. Introduction………………………………………………………………………………...81 3.2. ADAMTS-5 purification…………………………………………………………………..82 3.3. Characterisation of aggrecanase activity of ADAMTS5-2………...……………………84 3.4. ADAMTS-5 biotinylation………………………………………………………………….87 3.5. Discussion…………………………………………………………………………………..89 CHAPTER 4: PHAGE DISPLAY SELECTIONS AND SCREENING 4.1. Introduction……………………………………………………………………………….92 4.2. Phage display selections…………..………………………………………………………93 4.3. Primary screening………………………………………………………………………...98 4.3.1. Bacterial screening…………………………………………………………………99 5 4.3.2. Mammalian screening…………………………………………………………….103 4.3.3. Comparison between bacterial and mammalian screening……………………….108 4.3.4. Primary screening of anti-ADAMTS-5 antibodies..……………………………...109 4.4. Secondary screening……………………………………………………………………...115 4.4.1. Expression and purification of scFv-Fc lead antibodies……………………….......115 4.4.2. Investigation of the inhibitory potential of scFv-Fc lead antibodies on ADAMTS-5. ………………………………………………………….………………………….118 4.4.3. Recognition of ADAMTS-5 deletion mutants on Western Blot…...………………122 4.5.Discussion…………………………………………………………………………………127 CHAPTER 5: CHARACTERISATION OF scFv-Fc ANTIBODIES 5.1. Introduction……………………………………………………………………………….131 5.2. Inhibition of ADAMTS-5 cleavage of QF-peptide……………………………………...131 5.3. Inhibition of mouse ADAMTS-5………………………………………………………...133 5.4. Determination of dissociation constant (KD) and domain mapping by SPR………….136 5.5. Recognition of ADAMTS-5 on Western blot…………...………………………………141 5.6. Investigation of mechanism of inhibition of QF peptide cleavage…………………….143 5.7. Dual inhibition studies……………………...……………………………………………147 5.7.1. Effect of GM6001………………………………………………………………….147 5.7.2. Effect of heparin…………………………………………………………………...150 5.7.3. Effect of TIMP-3…………………………………………………………………..154 5.7.4. Effect of antibody combination……………………………………………………158 5.8. Effect of scFv-Fc antibodies on aggrecan breakdown in cartilage explants treated with IL-1 and OSM....................................................................................................................162
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