title: Increased antitumor activity of a circularly permuted interleukin 4-toxin in mice with interleukin 4 receptor-bearing human carcinoma.
abstract: 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 micrograms/kg x 3 and 100% of mice receiving 100 micrograms/kg x 3, while only 70% of mice receiving 200 micrograms/kg x 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 micrograms/Kg QOD x 3 than with IL4-PE38KDEL 200 micrograms/Kg QOD x 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.
authors.affiliation: Laboratory of Molecular Biology, National Cancer Institute, NIH, Bethesda, Maryland 20892, USA.
authors.forename: R J
authors.initials: RJ
authors.lastname: Kreitman
authors.forename: R K
authors.initials: RK
authors.lastname: Puri
authors.forename: I
authors.initials: I
authors.lastname: Pastan
mesh_terms: D036002:ADP Ribose Transferases; D000818:Animals; D001427:Bacterial Toxins / Q000737:chemistry / Q000493:pharmacokinetics / Q000627:therapeutic use*; D002277:Carcinoma / Q000188:drug therapy* / Q000378:metabolism; D002478:Cells, Cultured; D004354:Drug Screening Assays, Antitumor; D004926:Escherichia coli / Q000187:drug effects; D005098:Exotoxins / Q000737:chemistry / Q000493:pharmacokinetics / Q000627:therapeutic use*; D005260:Female; D006801:Humans; D000922:Immunotoxins / Q000737:chemistry / Q000493:pharmacokinetics / Q000627:therapeutic use*; D015847:Interleukin-4 / Q000737:chemistry / Q000493:pharmacokinetics / Q000627:therapeutic use*; D051379:Mice; D008819:Mice, Nude; D010446:Peptide Fragments / Q000737:chemistry / Q000493:pharmacokinetics / Q000627:therapeutic use; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000493:pharmacokinetics / Q000627:therapeutic use*; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
chemical_list: D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D005098:Exotoxins; D000922:Immunotoxins; D015847:Interleukin-4; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A