Establishment of anti-human neuroblastoma-selective isotype-switch variants

Isotype-switch variants of monoclonal antibodies occur spontaneously at low frequency in hybridoma cultures. We have established gamma 2b variants from the gamma 1/kappa antibody-secreting murine hybridoma CE7 and gamma 2a-secreting variants from a selected CE7 gamma 2b clone. The CE7 antibodies selectively recognize a cell surface glycoprotein of 185 kDa expressed on all human sympatho-adrenomedullary cells. The switch-variants were obtained by a stepwise cloning strategy and selected by an isotype-specific solid-phase sandwich ELISA. The frequency of the switch variants was 1-2 x 10(-5) for both isotypes. Using ELISA inhibition technique it was demonstrated that the selected variants were able to bind to the same epitope of neuroblastoma as the original CE7 gamma 1. Southern blot analysis showed that the functionally rearranged VH and VL genes of CE7 gamma 1, CE7 gamma 2a and CE7 gamma 2b antibodies were identical. The N terminal FR1 amino acid sequence of the L chains was identical in all three isotypes and the H chains were blocked for Edman degradation. Regarding the possible applications of CE7 antibodies the different isotypes were assayed for their cytolytic activity as measured by complement-mediated 51Cr release of IMR-32 neuroblastoma cells.