Development and Characterization of Anti-Renal Cell Carcinoma x Antichelate Bispecific Monoclonal Antibodies for Two-Phase Targeting of Renal Cell Carcinoma1

To test a two-step approach for radioimmunotargeting of renal cell cancer, quadroma cells secreting antichelate X anti-renal cell carcinoma bispecific antibodies were obtained by somatic cell fusion. Five monoclo­ nal antibodies against the chelate l,4,7-triazaheptane-A^N',/V"-pentaacetic acid (DTPA) were produced and characterized. Competitive binding assays indicated that the anti-DTPA antibodies reacted with DTPA che­ lated with indium, yttrium, chromium, iron, or zinc. The affinity constants of the anti-DTPA antibodies for m In-DTPA ranged from 0.19 to 0.23 nM_1, Using different chelates, a remarkable chelate specificity of the anti-DTPA antibodies was demonstrated. The chelates recognized by the antibodies DTInl, DTIn2, and DTIn4 share a A^N'^-diacetic acid group, whereas the chelates recognized by DTIn3 share a N *-acetic acid group, suggesting the presence of different essential structures within the DTPA molecule that determine the reactivity of the antibodies. Five anti-DTPA antibody-producing hvbridomas were used for somatic cell fusion with hybridoma G250 directed against renal cell carcinoma, resulting in three bispecific antibody-producing quadroma cell lines. The bispecific monoclonal antibodies were purified from ascites fluid using protein A affinity chromatography followed by hydroxylapatite chroma­ tography and/or cation exchange chromatography. Of the total IgG amount present in the ascites fluid, 10-15% represented the bispecific antibodies« These bispecific antibodies will allow testing and optimization of a two-step approach for radioimmunotargeting of chelated radionu­ clides.