title: Modified cytokeratins expressed on the surface of carcinoma cells undergo endocytosis upon binding of human monoclonal antibody and its recombinant Fab fragment.
abstract: Previously, we have reported on successful imaging of colon, rectal, and pancreatic carcinomas in patients by using a radiolabeled all-human monoclonal antibody, COU-1, directed against modified cytokeratin. To further develop this antibody for use as an immunoconjugate, COU-1 was cloned by phage display selection and the human Fab fragment was expressed in bacteria. Analysis by confocal laser scanning microscopy demonstrated that COU-1 bound in a uniform punctate pattern to the surface of viable carcinoma cells stained at 4 degrees C, and binding increased significantly when cells were cultured on fibronectin, laminin, or collagen IV. In the case of fibronectin, COU-1 staining was particularly enhanced at intercellular junctions. When carcinoma cells were cultured with COU-1 at 37 degrees C for 6 hr, the antibody was found in large perinuclear vesicles and the punctate surface staining was significantly reduced. Similar results were obtained using intact IgM COU-1 and the recombinant Fab fragment. Immunohistological studies indicated that COU-1, in contrast to murine monoclonal antibodies against normal cytokeratin 8 and 18, could differentiate between malignant and normal colon epithelia, and between colon cancer metastasis in the liver and surrounding normal hepatocytes. Within biopsies of malignant tissue, COU-1 exhibited membrane-associated staining of proliferating cells, while resting cells had a filamentous pattern. Thus, modified cytokeratin at the surface of carcinoma cells may represent a new target for immunoconjugates and may explain the promising results of the phase I/II clinical study.
authors.affiliation: Department of Clinical Immunology, Copenhagen University Hospital, Rigshospitalet, 2200 Copenhagen, Denmark. hditzel@scripps.edu
authors.forename: H J
authors.initials: HJ
authors.lastname: Ditzel
authors.forename: U
authors.initials: U
authors.lastname: Garrigues
authors.forename: C B
authors.initials: CB
authors.lastname: Andersen
authors.forename: M K
authors.initials: MK
authors.lastname: Larsen
authors.forename: H J
authors.initials: HJ
authors.lastname: Garrigues
authors.forename: A
authors.initials: A
authors.lastname: Svejgaard
authors.forename: I
authors.initials: I
authors.lastname: Hellström
authors.forename: K E
authors.initials: KE
authors.lastname: Hellström
authors.forename: J C
authors.initials: JC
authors.lastname: Jensenius
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology*; D061067:Antibodies, Monoclonal, Humanized; D001435:Bacteriophages / Q000235:genetics; D001666:Binding Sites, Antibody; D002460:Cell Line; D003106:Colon / Q000276:immunology / Q000378:metabolism; D003110:Colonic Neoplasms / Q000276:immunology / Q000378:metabolism*; D004705:Endocytosis; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000276:immunology*; D007413:Intestinal Mucosa / Q000276:immunology / Q000378:metabolism; D007633:Keratins / Q000276:immunology / Q000378:metabolism*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; C107416:COU-1 antibody; D007140:Immunoglobulin Fab Fragments; D011994:Recombinant Proteins; D007633:Keratins