A simple technique for the rapid enrichment of class and subclass hybridoma switch variants. A 1000-fold enrichment in half the time, for half the cost.

Switching parental hybrids in vitro to downstream switch variant clones producing more desirable monoclonal antibodies (MoAbs) requires either labor intensive and time consuming subcloning techniques, or fluorescence activated sorting of the desired clones. We tested the hypothesis that enrichment of downstream switch variant clones might be achieved by selective lysis of upstream hybridoma cells followed by expansion of the enriched downstream clone. Using a parental hybridoma with surface and secretory IgM, we attempted to enrich downstream switch variant clones producing class (IgG) and subclass (IgG1 or IgG2a) MoAbs. Enrichment of downstream IgG, IgG1 and IgG2a MoAb-producing switch variants was achieved by single or repeated antibody-dependent, complement-mediated lysis of the upstream IgM-bearing parental hybridoma cells followed by limited subcloning. Two exposures of parental hybridoma cells to lysis followed by plating at 100 cells/well enriched the frequency of switch variants up to 1235-fold, enabling the development of IgG1 or IgG2a-producing subclones exhibiting high yield antibody production. Using this protocol, production time and costs were reduced by > 50% when compared to the standard technique. This novel technique for the rapid isolation and expansion of switch variant clones should be ideal for most laboratories, particularly those without access to cell sorting capabilities.

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.

Lineage and stage specificity of isotype switching in humans.

The lineage and stage specificity of human isotype switch recombination was investigated by examining the IgH gene configuration in 61 hemopoietic malignancies representing different stages of B and T cell development. An unexpectedly high frequency (20%) of IgM-producing B cell leukemias and lymphomas had undergone CH gene rearrangements and deletions consistent with attempted switch recombination. These CH gene alterations were found on productive, non-productive, and 14q+ chromosomes. These data support the concept of a non-specific (common) switch recombinase activity that is often ineffective. No evidence of such switch recombination was found in 25 mu- or mu+ pre-B cell leukemias with the single exception of a mu- pre-B leukemia in which subsets of the cells were producing gamma- or alpha-H chains. The switch recombinase activity gamma- or alpha-H chains. The switch recombinase activity may be restricted to the B cell lineage, inasmuch as CH gene deletions were not observed in T lineage malignancies.