Effect of isotype on internalization and cytotoxicity of XXXX -ricin A immunotoxins.

@entity1 @entity222 @entity1075
We analyzed the effect of isotype variation on effectiveness of B-cell @entity1075 immunotoxins (IT) by using class switch variants (CLB-B4-IgG1 and CLB-B4-IgG2a) conjugated to ricin A. Notably, IgG1-IT appeared to be approximately 100-fold more potent than IgG2a-IT toward B-cell lines Daudi and KM3. Binding and internalization studies with 125I-labeled monoclonal antibodies (mAbs) revealed a higher cellular uptake of IgG1 compared to IgG2a, despite similar binding affinities. Following removal of the Fc part, both mAbs internalized at the same rate as IgG2a, indicating that the Fc part of IgG1 is involved in enhanced cellular uptake. The involvement of Fc gamma RII ( @entity222 ) in this process was demonstrated by a decreased cytotoxicity of IgG1-IT (and not IgG2a-IT) in the presence of Fc gamma RII-blocking mAbs AT10 or IV.3. To identify the isoform responsible for this phenomenon, internalization of IgG1 and IgG2a in 11 B-cell lines and malignant B-cells of 8 @entity1 was compared with expression of Fc gamma RII subclasses. In addition to four cell lines (Daudi, KM3, @entity1 , and Raji), the malignant B-cells of two @entity1 showed enhanced uptake of IgG1 relative to IgG2a. Only the @entity222 transcript was found in all B-cells. Furthermore, enhanced uptake of IgG1 correlated with rosetting of erythrocytes sensitized with anti-glycophorin A mAb of IgG1 isotype rather than with @entity222 membrane expression levels. These data support the idea that functional @entity222 is involved in the enhanced IgG1 uptake observed in a subset of B-cells. Our study, therefore, points to an important role for the Fc region of IT in the delivery of cytotoxic effects.
