Development and characterization of anti-renal cell carcinoma x antichelate bispecific monoclonal antibodies for two-phase targeting of renal cell carcinoma.

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 monoclonal antibodies against the chelate 1,4,7-triazaheptane-N,N',N"-pentaacetic acid (DTPA) were produced and characterized. Competitive binding assays indicated that the anti-DTPA antibodies reacted with DTPA chelated with indium, yttrium, chromium, iron, or zinc. The affinity constants of the anti-DTPA antibodies for 111In-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 DTIn1, DTIn2, and DTIn4 share a N(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 hybridomas 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 chromatography 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 radionuclides.