Characterization of the antigen (CAK1) recognized by monoclonal antibody K1 present on ovarian cancers and normal mesothelium.
K1 is a monoclonal antibody that reacts with a cell surface antigen (CAK1) found in human mesothelia and nonmucinous ovarian tumors. In this article, the characteristics of the CAK1 antigen have been examined in detail. Using immunofluorescence microscopy, we have found that the CAK1 signal is removed from the cell surface by treatment with proteases or by phosphatidylinositol-phospholipase C, but not by neuraminidase and beta-galactosidase. The phosphatidylinositol-phospholipase C-released material was found to contain the CAK1 antigen which was detected by a competition radioimmunoassay. The phosphatidylinositol-phospholipase C-released CAK1 antigen was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting and found to be approximately 40 kDa protein. The CAK1-K1 antibody complex remains on the cell surface and is poorly internalized, as shown by an acid wash immunofluorescence internalization assay. An immunotoxin composed of K1 and Lys-PE40, a mutant form of Pseudomonas exotoxin lacking the cell binding domain, was not cytotoxic, supporting the conclusion that the CAK1-K1 antibody complex is not internalized. However, an immunotoxin composed of K1 and native Pseudomonas exotoxin was selectively cytotoxic to cells expressing the CAK1 antigen. This cytotoxicity is due to the fact that domain I of Pseudomonas exotoxin promotes internalization of antigens which are not internalized or bound to antibody alone. Our results suggest that CAK1 is a polypeptide that is expressed on mesothelial cells and many ovarian cancers, and that K1 may be useful as a targeting agent for the immunotherapy of human ovarian cancer.
Chang
K
K
Laboratory of Molecular Biology, National Cancer Institute, NIH, Bethesda, Maryland 20892.
Pai
L H
LH
Batra
J K
JK
Pastan
I
I
Willingham
M C
MC
D000911:Antibodies, Monoclonal / Q000627:therapeutic use; D000936:Antigen-Antibody Complex / Q000378:metabolism; D015295:Antigens, Tumor-Associated, Carbohydrate / Q000032:analysis* / Q000378:metabolism; D004848:Epithelium / Q000276:immunology; D005260:Female; D006801:Humans; D000922:Immunotoxins / Q000627:therapeutic use; D000090204:Mesothelin; D010051:Ovarian Neoplasms / Q000276:immunology* / Q000378:metabolism; D010740:Phospholipases / Q000494:pharmacology; D011863:Radioimmunoassay; D014407:Tumor Cells, Cultured / Q000276:immunology
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D015295:Antigens, Tumor-Associated, Carbohydrate; D000922:Immunotoxins; C000715527:MSLN protein, human; D010740:Phospholipases; D000090204:Mesothelin