Analysis of a murine anti-ganglioside GD2 monoclonal antibody expressing both IgG2a and IgG3 isotypes: monoclonality, apoptosis triggering, and activation of cellular cytotoxicity on human melanoma cells.

In this study we have documented a hybridoma secreting an unusual MAb, which expresses both IgG3 and IgG2a subclasses with a lambda-light chain. How this dual expression of isotypes was exactly brought about is not clear. To resolve this problem, it will have to wait the complete sequence analysis the heavy chain gene of MAb 9C4. Although the expression of IgG2a was about 50% that of IgG3, antibody titration studies showed the major binding affinity of MAb 9C4 to GD3-positive cells being mostly contributed by the IgG3 rather than IgG2a part of the antibody. This antibody could induce apoptosis in melanoma cells in 10-15% of cells in vitro, but the generality of this phenomenon is yet to be confirmed by the use of different cell targets and different anti-GD2 MAbs other than 9C4. Aside from the demonstrated indirect killing mechanisms of many anti-GD2 MAbs through CDC and ADCC, MAb 9C4 induction of apoptosis represents an alternative mechanism of tumor cell killing, by which direct killing of anti-GD2 antibody takes its effect. This apoptotic effect was demonstrated for the first time with an anti-ganglioside monoclonal antibody. From the therapeutic point of view, the cytolytic activity of MAb 9C4-targeted ADCC/LAK killing against GD2-positive tumor cells to be more effective than that of LAK alone and a possibility for dendritic cells to effectively acquire antigen through pulsing with MAb-induced apoptotic cells are both of great clinical importance. Further studies are warranted aiming at elucidating the molecular basis of bi-isotypic specificity and aberrant isotype switching, molecular pathway of anti-GD2 antibody-induced apoptosis, and ways to improve clinical utility of this unusual hybridoma/MAb 9C4.