Monoclonal antibodies against cell surface antigens present on human urinary bladder cancer cells

After hybridomas were prepared by cell fusion between the mouse myeloma cell line P3x63Ag8.653 and the spleen cells of a BALB/c mouse hyperimmune to the human bladder cancer KU-1 cell line, they were cloned. Monoclonal antibodies (HBA4, HBE3, HBE10, HBF2, HBG9, and HBH8) from the hybridoma clones were selected for their serologic reactivity to cell surface antigens of KU-1 cells and for their unresponsiveness to 2 human lymphoma cell lines. They were cross-reactive with a characteristic portion of human epithelial tumor cell lines and with surgically resected bladder cancer tissues. With regard to reactivity to normal human cells and tissues, HBG9 alone was reactive to epidermis and both HBF2 and HBH8 were reactive to erythrocytes, but none of the other 3 monoclonal antibodies was reactive to any normal cells or tissues. The biochemical analysis of the antigens defined at least three antigenic systems. One system was a glycopeptide complex recognized by HBA4, HBE3, and HBG9, and it consisted of molecules having molecular weights of 78,000 (major) and 40,000 and 130,000, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the 125I-radiolabeled extracts of KU-1 cells. Treatment of KU-1 cells with tunicamycin or neuraminidase lowered the reactivity to these antibodies, suggesting that the antigenic determinants reside in sialoglycoproteins. Antigenic determinants against HBG9 and the other two antibodies (HBA4 and HBE3) appeared to be different, as judged by the serologic reactivities. The other two antigenic systems detected by HBE10, HBF2, and HBH8 were defined as neutral glycolipids, according to heat stability, solubility in organic solvents, and characters in lipid fractionation. When examined by thin-layer chromatography in chloroform-methanol-0.25% KCl (30:15:4), a lipid component recognized by HBE10 migrated to a single zone [retardation factor (Rf), 0.33]. The other two antibodies (HBF2 and HBH8) did detect two lipid components (Rf, 0.23 and 0.33). Although the lipid component detected by HBE10 showed the same migration distance as one of the components detected by HBH8 (or HBF2), the antigenic determinants defined by these antibodies appeared to be different, as judged by their serologic reactivities.