Monoclonal antibodies to two mouse bladder carcinoma antigens

Carcinomas of the urinary bladder of mice were induced by 3-methyl-cholanthrene or fanft [(4-5-nitro-2-furyl)-2-thioazolyl formamide]. On transplantation in vivo, tumor lines consisting of well-differentiated transitional-cell carcinomas were established. Some tumors were also explanted in vitro. A rat was immunized with a pool of carcinomas and normal bladder tissue and its spleen cells were hybridized with NS-l mouse myeloma cells. Supernatants of hybrid cells ("hybridomas") were screened for antibody binding to antigens present in bladder carcinomas but not in normal syngeneic urinary bladder, with cell extracts as targets. Hybridomas that appeared to have the required specificity were cloned, tested further against transitional-cell bladder carcinomas, an anaplastic bladder tumor, rhabdomyosarcomas, a mammary carcinoma, myelomas and lymphomas, and normal adult urinary bladder, kidney, lung, spleen, heart, brain, thymus, and whole embryo. Antibody formed by one hybridoma, 2H5, gave significant binding to membranes from five of seven transitional-cell carcinomas but not to membranes from many other tissues. A second hybridoma, IE6, formed antibody to an antigen present in bladder carcinomas and normal liver and, in smaller amounts, in several other normal and neoplastic tissues. Fluorescence microscopy established that both antigens were present at the cell surface of transitional-cell bladder carcinomas. Immunoprecipitation and SDS-polyacrylamide gel electrophoresis were used to identify the target antigens from 125I-labelled cell membrane proteins. The antibody formed by 2H5 was found to identify a protein with a molecular weight in the range of 140 kilodaltons, which was detected in transitional-cell bladder. The molecular nature of the antigen defined by hybridoma IE6 is not known.