We reported previously that circularly permuted interleukin-4 (IL4), composed of amino acids 38–129 of IL4 connected by a linker peptide GGNGG to amino acids 1–37, is preferable to native IL4 for fusing to the amino terminus of truncated Pseudomonas exotoxin (PE) to make a recombinant toxin, because the new ligand-toxin junction results in improved IL4 receptor (IL4R)-binding (R. J. Kreitman et al., Proc. Natl. Acad. Sci. USA, 91: 6889–6893, 1994). We now report that the improved binding of circularly permuted IL4-toxin is associated with improved antitumor activity in tumor-bearing mice. For in vivo testing, we made an improved circularly permuted IL4-toxin, termed IL4(38–37)-PE38KDEL. It contains an N38D mutation at the amino terminus, allowing improved expression and large-scale production in Escherichia coli. It also contains the truncated toxin PE38KDEL, which is composed of amino acids 253–364 and 381–608 of PE, followed by KDEL. To evaluate antitumor activity, nude mice carrying s.c. tumors composed of IL4R-bearing human A431 epidermoid carcinoma cells were injected with recombinant toxins i.v. every other day for three doses. IL4(38–37)-PE38KDEL induced complete remissions in 80% of mice receiving 50 µg/kg × 3 and 100% of mice receiving 100 µg/kg × 3, while only 70% of mice receiving 200 µg/kg × 3 of the native IL4-toxin IL4-PE38KDEL obtained complete remission. Disease-free survival after obtaining complete remissions was higher in mice treated with IL4(38–37)-PE38KDEL 50 µg/Kg QOD × 3 than with IL4-PE38KDEL 200 µg/Kg QOD × 3 (P < 0.03). IL4(38–37)-PE38KDEL and IL4-PE38KDEL exhibited similar toxicity and pharmacokinetics in the mice, indicating that the improved antitumor activity of the circularly permuted IL4-toxin was due to its improved binding to the IL4R on the target cells.

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