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.

Figure 1

Deduced amino acid sequence of the variable heavy and light chain of HumAb COU-1 compared with the closest known germ-line sequences. FR, framework region; CDR, complementarity-determining region.

Figure 2

Binding of HumAb IgM COU-1 (0.5 μg/ml), recombinant Fab fragment of COU-1 (10 μg/ml), and normal human IgM (10 μg/ml) to a panel of solid-phase antigens tested by ELISA.

Figure 3

COU-1 localization, internalization, and influence of fibronectin on surface cytokeratin expression. Colon cancer cells [H3619 (a), colo137 (b)] were fixed, permeabilized, and stained with COU-1 (a) or Fab COU-1 (b) and FITC-labeled goat anti-human κ-chain antibody. Note intense fibrillar staining of the intermediate filament (arrowhead). In addition, vesicles (arrows) throughout the cytoplasm were routinely observed. Live H3619 cells incubated with COU-1 at 4°C gave dispersed punctate staining of the upper cell surface (c). When the H3619 cells were grown on fibronectin-coated slides, the level of punctate staining at the cell surface increased and COU-1 was enriched at intercellular junctions (arrows, d). When the colon cancer cells [H3619 (e, f), colo137 (g, h)] were incubated with COU-1 at 37°C, the dispersed punctate surface staining disappeared and COU-1 (e, f, h) or Fab COU-1 (g) localized in large cytoplasmic vesicles adjacent to the nucleus. (×1000.)

Figure 4

Comparison of the tissue distribution of COU-1 and murine anti-cytokeratin 8 antibody in ethanol-fixed tissues. Tumor cells within tissue sections bound COU-1, while surrounding normal cells were not stained (a–d). Fibrillar staining characteristic of intermediate filaments (a, arrow), membrane staining of single proliferating cells (b, arrow; note metaphase, arrowhead), and enrichment of COU-1 at intercellular junctions (arrows in c and d). In adjacent normal colon epithelia, weak staining was found only in a few cells of some biopsies (arrow, d). (e and f) Comparison of staining with COU-1 (e) and murine anti-cytokeratin 8 (M20) (f) on serial sections of malignant (m) and adjacent normal colon epithelium (n). (g and h) Adjacent sections of a colon cancer metastasis in the liver (m) and surrounding normal hepatocytes (h) incubated with COU-1 (g) and with M20 (h). (a and b, ×600; c and d, ×500; and e–h, ×200.)