caa a22 4500 445313994 CHVBK 20180317142639.0 cr unu---uuuuu 170323e20110501xx s 000 0 eng 10.1007/s11095-011-0376-y doi (NATIONALLICENCE)springer-10.1007/s11095-011-0376-y Antibody-Mediated "Universal” Osteoclast Targeting Platform using Calcitonin as a Model Drug [Elektronische Daten] [Madhuri Newa, Krishna Bhandari, Lili Tang, Rohit Kalvapalle, Mavanur Suresh, Michael Doschak] ABSTRACT: Purpose: To generate and characterize a specific monoclonal antibody (mAb) against recombinant human RANK receptor and to develop an antiresorptive strategy using this mAb as an osteoclast-targeting platform that selectively targets osteoclast cells whilst delivering an attached (i.e. chemically conjugated) active drug cargo. Methods: Using hybridoma technology, we generated a specific monoclonal antibody (mAb) against recombinant human RANK receptor and characterized by SDS PAGE, ELISA, Western Blot and immunocytochemistry, then synthesized osteoclast-targeting bioconjugates of salmon calcitonin (sCT) using this antibody by generating thiol groups on mAb using 2-Iminothiolane and subsequently reacting them with sCT-PEG-MAL synthesised from sCT and NHS-PEG-MAL. To test the efficacy of the conjugate in vitro, osteoclasts were generated from precursor RAW 264.7 cells by dosing with the cytokines macrophage-colony-stimulating factor (M-CSF), and RANK Ligand (RANKL) and TRAP activity assay, Resorption Pit Assay, TRAP staining were performed. Cytotoxicity of the mAb-sCT conjugate was also evaluated in RAW 264.7 cells; sCT bioactivity and CTR binding potential were evaluated by in vitro intracellular cAMP stimulation assay in human T47D breast cancer cells. Results: Generation of antibody against human RANK receptor was confirmed by SDS PAGE, ELISA and Western Blot. Immunocytochemistry confirmed the osteoclast targeting potential of the antibody. Successful conjugation of the antibody with sCT was confirmed by SDS PAGE and ELISA.Multinucleated osteoclast formation was confirmed by staining for tartrate-resistant acid phosphatase (TRAP). Conjugate functionality was confirmed by TRAP activity and Resorption Pit assay, showing the inhibitory effect on osteoclast differentiation. cAMP assay confirmed the retention of calcitonin bioactivity after conjugation. Conclusions: Our strategy offers the potential for a "universal” osteoclast-targeting platform—one that targets the RANK receptor on osteoclast cells by simply altering the conjugated cargo in order to affect the specific regulation of osteoclast cells. The Author(s), 2011 drug delivery system nationallicence monoclonal antibody nationallicence osteoclast nationallicence osteoporosis nationallicence post-traumatic osteoarthritis nationallicence RANK receptor nationallicence salmon calcitonin nationallicence Newa Madhuri Faculty of Pharmacy & Pharmaceutical Sciences, University of Alberta, T6G 2N8, Edmonton, Alberta, Canada aut Bhandari Krishna Faculty of Pharmacy & Pharmaceutical Sciences, University of Alberta, T6G 2N8, Edmonton, Alberta, Canada aut Tang Lili Faculty of Pharmacy & Pharmaceutical Sciences, University of Alberta, T6G 2N8, Edmonton, Alberta, Canada aut Kalvapalle Rohit Faculty of Pharmacy & Pharmaceutical Sciences, University of Alberta, T6G 2N8, Edmonton, Alberta, Canada aut Suresh Mavanur Faculty of Pharmacy & Pharmaceutical Sciences, University of Alberta, T6G 2N8, Edmonton, Alberta, Canada aut Doschak Michael Faculty of Pharmacy & Pharmaceutical Sciences, University of Alberta, T6G 2N8, Edmonton, Alberta, Canada aut Pharmaceutical Research Springer US; http://www.springer-ny.com 28/5(2011-05-01), 1131-1143 0724-8741 28:5<1131 2011 28 11095 https://doi.org/10.1007/s11095-011-0376-y text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s11095-011-0376-y text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Newa Madhuri Faculty of Pharmacy & Pharmaceutical Sciences, University of Alberta, T6G 2N8, Edmonton, Alberta, Canada aut NATIONALLICENCE 700 1- Bhandari Krishna Faculty of Pharmacy & Pharmaceutical Sciences, University of Alberta, T6G 2N8, Edmonton, Alberta, Canada aut NATIONALLICENCE 700 1- Tang Lili Faculty of Pharmacy & Pharmaceutical Sciences, University of Alberta, T6G 2N8, Edmonton, Alberta, Canada aut NATIONALLICENCE 700 1- Kalvapalle Rohit Faculty of Pharmacy & Pharmaceutical Sciences, University of Alberta, T6G 2N8, Edmonton, Alberta, Canada aut NATIONALLICENCE 700 1- Suresh Mavanur Faculty of Pharmacy & Pharmaceutical Sciences, University of Alberta, T6G 2N8, Edmonton, Alberta, Canada aut NATIONALLICENCE 700 1- Doschak Michael Faculty of Pharmacy & Pharmaceutical Sciences, University of Alberta, T6G 2N8, Edmonton, Alberta, Canada aut NATIONALLICENCE 773 0- Pharmaceutical Research Springer US; http://www.springer-ny.com 28/5(2011-05-01), 1131-1143 0724-8741 28:5<1131 2011 28 11095 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer