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.
Puri
R K
RK
Laboratory of Molecular Tumor Biology, Food and Drug Administration, National Institutes of Health, Bethesda, Maryland 20892, USA.
Leland
P
P
Obiri
N I
NI
Husain
S R
SR
Mule
J
J
Pastan
I
I
Kreitman
R J
RJ
D036002:ADP Ribose Transferases; D001427:Bacterial Toxins / Q000235:genetics / Q000276:immunology / Q000633:toxicity*; D001853:Bone Marrow / Q000378:metabolism; D001854:Bone Marrow Cells; D002292:Carcinoma, Renal Cell / Q000235:genetics / Q000276:immunology*; D003114:Colony-Forming Units Assay; D005098:Exotoxins / Q000235:genetics* / Q000633:toxicity*; D005434:Flow Cytometry; D006801:Humans; D015847:Interleukin-4 / Q000235:genetics* / Q000276:immunology / Q000633:toxicity*; D007680:Kidney Neoplasms / Q000235:genetics / Q000276:immunology*; D011485:Protein Binding / Q000276:immunology; D011550:Pseudomonas aeruginosa / Q000276:immunology; D015375:Receptors, Interleukin-2 / Q000096:biosynthesis; D011993:Recombinant Fusion Proteins / Q000276:immunology / Q000633:toxicity*; D014162:Transfection / Q000276:immunology; D014407:Tumor Cells, Cultured; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D005098:Exotoxins; D015847:Interleukin-4; D015375:Receptors, Interleukin-2; D011993:Recombinant Fusion Proteins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A