title: Targeting of human squamous carcinomas by SPA470-doxorubicin immunoconjugates.
abstract: In a quest to identify a favorable target for head and neck cancers and squamous cell carcinoma, we sought to determine if Hsp47/CBP2 could be used as a target and whether the expression of this target was influenced by hypoxia. Moreover, we determined if doxorubicin (DOX) immunoconjugates directed against Hsp47/CBP2 that linked monoclonal antibodies (MAbs) to the 13-keto position of the drug possessed high cytotoxic drug activity and antibody-directed killing of antigen bearing tumor target cells. Experiments were performed using established cell lines of human oral squamous carcinoma cells (SCCs) (SCC-4, -9, -15 and -25) obtained from American Type Culture Collection (ATCC) (Manassas, VA). In addition, the UMB2 cell line is a spontaneous mutant of SCC-9 that does not express Hsp47/CBP2 was also used. Synthesis of the immunoconjugates was accomplished by thiolating the MAbs with 2 IT and reacting the MAbs with the DOX-hydrazone. The binding of MAb-DOX conjugates to SCC cells was determined by indirect immunofluorescence and analyzed using a Becton Dickinson FACS scan with Cell Quest software. Comparison of the cytotoxicity of DOX, MAb-DOX conjugates and MAb+DOX were determined using a limited dilution assay and colony survival assays during normoxia and hypoxia. These studies revealed that SCC cells treated with the SPA470-DOX conjugate for 2 h retained the original binding activity for targeted SCC cells and was significantly more potent that unconjugated DOX, DOX-hydrazone or equivalent MAb protein+DOX. Also, SPA47-DOX produced equal to and at lower concentrations greater cell killing than equivalent dose of free DOX. During hypoxia cells treated with SPA470-DOX demonstrated a small increase in colony survival and a diminishment in cytotoxicity. SPA470-DOX conjugates target SCC cells that express Hsp47/CBP2. The demonstration that SPA470-DOX is effective during hypoxia or conditions that mimic hypoxia presumes the further utility of SPA470-DOX in treating head and neck cancers.
authors.affiliation: Department of Dignostic Sciences and pathology University of Maryland, Baltimore, MD 21201, USA.
authors.forename: C
authors.initials: C
authors.lastname: Hebert
authors.forename: K
authors.initials: K
authors.lastname: Norris
authors.forename: J J
authors.initials: JJ
authors.lastname: Sauk
mesh_terms: D000911:Antibodies, Monoclonal / Q000008:administration & dosage / Q000737:chemistry / Q000494:pharmacology*; D000970:Antineoplastic Agents / Q000008:administration & dosage / Q000737:chemistry / Q000494:pharmacology*; D002294:Carcinoma, Squamous Cell / Q000473:pathology; D015687:Cell Hypoxia; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004317:Doxorubicin / Q000008:administration & dosage / Q000737:chemistry / Q000494:pharmacology*; D019084:Fluorescent Antibody Technique, Indirect; D050958:HSP47 Heat-Shock Proteins; D006258:Head and Neck Neoplasms / Q000473:pathology; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D018796:Immunoconjugates / Q000008:administration & dosage / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
chemical_list: D000911:Antibodies, Monoclonal; D000970:Antineoplastic Agents; D050958:HSP47 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D018796:Immunoconjugates; C494660:SERPINH1 protein, human; D004317:Doxorubicin