Immunochemical and biochemical characterizations of two monoclonal antibody-reacting antigens associated with human bladder carcinoma.

Two murine monoclonal antibodies (Mabs), 3G2-C6 and C3, which react to surface components on human bladder carcinoma cells, were produced using cultured human bladder tumor cells as immunogens. The expression of these antigens is highly restricted to malignant cells, and as such these Mabs are potentially useful for cancer diagnosis and treatment. We report here the immunochemical characterizations and molecular size determinations of these two Mab-reacting bladder tumor-associated antigens. The 3G2-C6 antigen has a molecular weight of 92,000, while the C3 antigen is a macromolecule with a molecular weight of about 600,000, consisting of 4 subunits of identical size, as determined by high pressure liquid chromatography gel filtration and sodium dodecyl sulfate and gradient-gel electrophoresis. The apparent affinity constants for the binding of these two Mabs and the number of antigenic determinants per cell were determined in 4 human bladder tumor cell lines (MGH-U1 through MGH-U4) with different degrees of malignancy. The apparent affinity constants for 3G2-C6 ranged from 1.8 x 10(-10) to 1.7 x 10(-8) M, while those for C3 ranged from 1.9 x 10(-9) to 4.4 x 10(-8) M. The number of antigenic determinants per cell ranged from 3 x 10(5) to 2.9 x 10(3) for 3G2-C6 and from 2 x 10(6) to 1.2 x 10(5) for C3. This coincides with our earlier observation that bladder tumor cells of higher malignancy tend to express higher numbers of determinants of these antigens, particularly of 3G2-C6. Very low levels of both of the antigens were released from the cells; less than 10% of the antigens could be detected in 3-day spent culture medium. Radioactively labeled Mabs were used to assess the stability of the antibody bound to MGH-U1 cells. More than 70% of 3G2-C6 remained bound to the cell after 24 h, whereas more than 60% of C3 was lost from the cell and recovered in the culture medium as small fragments. This information may be useful for the clinical applications of these Mabs, including the improvement of in vitro detection of bladder cancer through identification of exfoliated tumor cells and determining the potential utilities of these Mabs in in vivo localization of in situ and metastatic bladder tumors.