title: Development of an in vivo antibody-mediated killing (IVAK) model, a flow cytometric method to rapidly evaluate therapeutic antibodies.
abstract: The efficacy and mechanism of action of therapeutic antibodies that target cancer cells have typically been evaluated using in vitro assays and long-term in vivo tumor models. To allow for a more efficient assessment of the function of candidate therapeutic antibodies, we have developed a flow cytometric-based method that rapidly and directly quantifies antibody-mediated killing in a short term in vivo assay. Target cells that express human CD52, including huCD52(+) splenocytes from huCD52 transgenic mice and Ramos cells, a CD52(+) human B cell lymphoma line, and CD52(-) reference cells were differentially labeled by using two fluorescent dyes to distinguish target and reference cell populations. Labeled cells were injected into mice with or without Campath-1H (Alemtuzumab) and then recovered for flow cytometric analysis 5 h later. We found that huCD52(+) transgenic splenocytes and Ramos cells were selectively depleted in Campath-treated animals but not in animals treated with a negative control antibody. Furthermore, it is likely that the cells were depleted in vivo by a complement-dependent mechanism since target cell depletion was significantly reversed after complement inactivation using cobra venom factor. This report demonstrates the feasibility and utility of a powerful method for the rapid evaluation in vivo of therapeutic antibody candidates for cancer.
authors.affiliation: Genzyme Corporation, P.O. Box 9322, Framingham, MA 01701, USA.
authors.forename: Cheryl A
authors.initials: CA
authors.lastname: Guyre
authors.forename: Danilo
authors.initials: D
authors.lastname: Gomes
authors.forename: Karen A
authors.initials: KA
authors.lastname: Smith
authors.forename: Johanne M
authors.initials: JM
authors.lastname: Kaplan
authors.forename: Michael A
authors.initials: MA
authors.lastname: Perricone
mesh_terms: D000074323:Alemtuzumab; D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000494:pharmacology*; D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D061067:Antibodies, Monoclonal, Humanized; D000912:Antibodies, Neoplasm / Q000494:pharmacology*; D000920:Antibody-Dependent Cell Cytotoxicity; D015703:Antigens, CD / Q000276:immunology; D000951:Antigens, Neoplasm / Q000276:immunology; D000074301:CD52 Antigen; D045744:Cell Line, Tumor; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D006023:Glycoproteins / Q000276:immunology; D006801:Humans; D007167:Immunotherapy / Q000379:methods; D016393:Lymphoma, B-Cell / Q000276:immunology* / Q000628:therapy; D051379:Mice; D008810:Mice, Inbred C57BL; D016513:Mice, SCID; D008822:Mice, Transgenic; D013047:Specific Pathogen-Free Organisms
chemical_list: D000906:Antibodies; D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D000912:Antibodies, Neoplasm; D015703:Antigens, CD; D000951:Antigens, Neoplasm; D000074301:CD52 Antigen; C000613862:CD52 protein, human; D005456:Fluorescent Dyes; D006023:Glycoproteins; D000074323:Alemtuzumab