An improved circularly permuted interleukin 4-toxin is highly cytotoxic to human renal cell carcinoma cells. Introduction of gamma c chain in RCC cells does not improve sensitivity

We have previously demonstrated that a chimeric protein composed of human IL-4 and Pseudomonas exotoxin, termed IL4-PE4E, is cytotoxic to primary cells derived from human renal cell carcinoma (RCC). To improve the cytotoxicity of IL4-toxins such as IL4-PE4E and IL4-PE38KDEL to IL-4 receptor (IL-4R) positive tumor cells, a circularly permuted chimeric toxin was prepared by fusing a truncated PE gene encoding PE38KDEL 3' to a circularly permuted IL-4 mutant gene encoding IL4 amino acids 38-129, the linker GGNGG, and IL4 amino acids 1-37. The resulting chimeric protein, termed IL4(38-37)-PE38KDEL, was tested on five RCC cell lines and its cytotoxicity was compared to that of the native IL4-toxins IL4-PE4E and IL4-PE38KDEL. IL4(38-37)-PE38KDEL was found to be 5 to 10 times more cytotoxic to all cell cultures tested compared to either native IL4-toxin. The cytotoxic activity of IL4(38-37)-PE38KDEL was competible by excess IL-4 and was confirmed by clonogenic assay. IL4(38-37)-PE38KDEL bound to IL-4R on RCC cells with 6- to 12-fold higher affinity than IL4-PE38KDEL or IL4-PE4E. RCC tumor cells were found to lack the common gamma chain (gamma c) of the IL-4R reported to be present on immune cells. The stable transfection of RCC cells with the gamma c chain gene did not significantly change their sensitivity to IL4(38-37)-PE38KDEL. Taken together, our results indicate that the CPIL4-toxin IL4(38-37)-PE38KDEL is highly cytotoxic to human RCC cells due to increased binding affinity to IL-4R while it is not cytotoxic or slightly cytotoxic to T and B cells, monocytic cell lines, and fresh resting or activated bone marrow-derived cells. The gamma c does not seem to increase the internalization rate and/or processing of IL4-toxins in RCC cells. CPIL4-toxin may be a useful agent for the treatment of human RCC.