MODIFIED CYTOKERATINS EXPRESSED ON THE SURFACE OF CARCINOMA-CELLS UNDERGO ENDOCYTOSIS UPON BINDING OF HUMAN MONOCLONAL-ANTIBODY AND ITS RECOMBINANT FAB FRAGMENT

Previously, we hare reported on successful imaging of colon, rectal, a nd 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 wa s cloned by phage display selection and the human Feb fragment mas exp ressed in bacteria, Analysis by confocal laser scanning microscopy dem onstrated that COU-1 bound in a uniform punctate pattern to the surfac e of viable carcinoma cells stained at 4 degrees C, and binding increa sed significantly when cells were cultured on fibronectin, laminin, or collagen IV, In the case of fibronectin, COU-1 staining was particula rly enhanced at intercellular junctions, When carcinoma cells were cul tured with COU-1 at 37 degrees C for 6 hr, the antibody was found in l arge perinuclear vesicles and the punctate surface staining was signif icantly 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 norma l cytokeratin 8 and 18, could differentiate between malignant and norm al colon epithelia, and between colon cancer metastasis in the liver a nd 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 cytoker atin at the surface of carcinoma cells may represent a ne-cv target fo r immunoconjugates and may explain the promising results of the phase I/II clinical study.