title: A novel pretargeting method for measuring antibody internalization in tumor cells.
abstract: A novel pretargeting method has been developed to quantitate antibody cellular internalization. In this study, the antibody was conjugated with a phosphorodiamidate morpholino oligomer (MORF) specific for the complementary MORF (cMORF) as an effector. Half the tumor cells were incubated with the MORF-antibody (pretargeting group) and the other half with the same MORF-antibody at the same concentration but radiolabeled (direct targeting group). After incubation, the same dosage of radiolabeled cMORF was added to the wells of the pretargeting group. The radioactivity of the direct targeting cells represented the sum of both internalized and cell-surface-bound antibodies, whereas the radioactivity of the pretargeting cells resulted only from the surface-bound antibodies, as the radiolabeled cMORF does not penetrate the cell surface. Therefore, the difference in radioactivity accumulation between pretargeting and direct targeting provides the internalized fraction. In this example, the internalization of a MORF conjugated anti-prostate-specific membrane antigen antibody, 3C6, in LNCaP cells was examined, and the average cell-surface residence time was determined as 2 hours. This method of measuring antibody internalization is directly applicable to pretargeting applications but can be a universal alternative to the conventional acid-wash method, with the advantage of leaving the cell membrane undamaged.
authors.affiliation: Division of Nuclear Medicine, Department of Radiology, University of Massachusetts Medical School, Worcester, MA 01655-0243, USA. guozheng.liu@umassmed.edu
authors.forename: Guozheng
authors.initials: G
authors.lastname: Liu
authors.forename: Shuping
authors.initials: S
authors.lastname: Dou
authors.forename: Dongguang
authors.initials: D
authors.lastname: Yin
authors.forename: Shayne
authors.initials: S
authors.lastname: Squires
authors.forename: Xinrong
authors.initials: X
authors.lastname: Liu
authors.forename: Yi
authors.initials: Y
authors.lastname: Wang
authors.forename: Mary
authors.initials: M
authors.lastname: Rusckowski
authors.forename: Donald J
authors.initials: DJ
authors.lastname: Hnatowich
mesh_terms: D000143:Acids; D000818:Animals; D000906:Antibodies / Q000008:administration & dosage / Q000032:analysis* / Q000276:immunology / Q000378:metabolism*; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D016503:Drug Delivery Systems / Q000379:methods*; D005434:Flow Cytometry; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D009369:Neoplasms / Q000276:immunology* / Q000378:metabolism* / Q000473:pathology; D023041:Xenograft Model Antitumor Assays
chemical_list: D000143:Acids; D000906:Antibodies