title: Antibody-mediated "universal" osteoclast targeting platform using calcitonin as a model drug.
abstract: OBJECTIVE
  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.
authors.affiliation: Faculty of Pharmacy & Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta T6G2N8, Canada.
authors.forename: Madhuri
authors.initials: M
authors.lastname: Newa
authors.forename: Krishna Hari
authors.initials: KH
authors.lastname: Bhandari
authors.forename: Lili
authors.initials: L
authors.lastname: Tang
authors.forename: Rohit
authors.initials: R
authors.lastname: Kalvapalle
authors.forename: Mavanur
authors.initials: M
authors.lastname: Suresh
authors.forename: Michael R
authors.initials: MR
authors.lastname: Doschak
mesh_terms: D000135:Acid Phosphatase / Q000276:immunology; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D015153:Blotting, Western; D002116:Calcitonin / Q000008:administration & dosage* / Q000494:pharmacology; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D016503:Drug Delivery Systems; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D018796:Immunoconjugates / Q000008:administration & dosage* / Q000494:pharmacology; D007150:Immunohistochemistry; D007527:Isoenzymes / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D008903:Minerals / Q000378:metabolism; D010010:Osteoclasts / Q000166:cytology / Q000276:immunology*; D053245:RANK Ligand / Q000276:immunology*; D018003:Receptors, Calcitonin / Q000378:metabolism; D011994:Recombinant Proteins / Q000276:immunology; D000071681:Tartrate-Resistant Acid Phosphatase
chemical_list: D000911:Antibodies, Monoclonal; D018796:Immunoconjugates; D007527:Isoenzymes; D008903:Minerals; D053245:RANK Ligand; D018003:Receptors, Calcitonin; D011994:Recombinant Proteins; C028815:salmon calcitonin; D002116:Calcitonin; D000135:Acid Phosphatase; C000605511:Acp5 protein, mouse; D000071681:Tartrate-Resistant Acid Phosphatase