EFFECT OF ISOTYPE ON INTERNALIZATION AND CYTOTOXICITY OF CD19-RICIN-AIMMUNOTOXINS

We analyzed the effect of isotype variation on effectiveness of B-cell CD19 immunotoxins (IT) by using class switch variants (CLB-B4-IG1 and CLB-B4-IgG2a) conjugated to ricin A. Notably, IgG1-IT appeared to be similar to 100-fold more potent than IgG2a-IT toward B-cell lines Daud i and KM3. Binding and internalization studies with I-125-labeled mono clonal antibodies (mAbs) revealed a higher cellular uptake of IgG1 com pared to IgG2a, despite similar binding affinities. Following removal of the Fc part, both mAbs internalized at the same rate as IgG2a, indi cating that the Fc part of IgG1 is involved in enhanced cellular uptak e. The involvement of Fc gamma RII (CD32) in this process was demonstr ated 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 i soform responsible for this phenomenon, internalization of IgG1 and Ig G2a in 11 B-cell lines and malignant B-cells of 8 patients was compare d with expression of Fc gamma RII subclasses. In addition to four cell lines (Daudi, KM3, Nalm6 and Raji), the malignant B-cells of two pati ents showed enhanced uptake of IgG1 relative to IgG2a. Only the Fc gam ma RIIa transcript was found in all B-cells. Furthermore, enhanced upt ake of IgG1 correlated with resetting of erythrocytes sensitized with anti-glycophorin A mAb of IgGI isotype rather than with Fc gamma RIIa membrane expression levels. These data support the idea that functiona l Fc gamma RIIa is involved in the enhanced IgG1 uptake observed in a subset of B-cells. Our study, therefore, points to an important role f or the Fc region of IT in the delivery of cytotoxic effects.