Rational design of a chimeric toxin: an intramolecular location for the insertion of transforming growth factor alpha within Pseudomonas exotoxin as a targeting ligand.
To investigate the potential utility of Pseudomonas exotoxin (PE) in forming rationally designed chemotherapeutic agents, we inserted a cDNA encoding transforming growth factor alpha (TGF alpha) at several locations in a gene encoding a mutant full-length PE (PE4E) which does not bind to the PE receptor. After expression in Escherichia coli, we purified the chimeric toxins to near homogeneity and showed that they were specifically cytotoxic to human epidermoid, ovarian, colon, and hepatocellular carcinoma lines. Like the previously reported TGF alpha-PE40, one of the new molecules (TGF alpha-PE4E) contains the ligand at the amino terminus. Two additional chimeras (PE4E-TGF alpha and PE4E-TGF alpha-598-613) each contain TGF alpha inserted near the carboxyl terminus of PE. We show that preservation of the correct PE carboxyl-terminal amino acid sequence, REDLK, allows the toxins containing TGF alpha carboxyl inserts to retain significant cytotoxicity against target cells, since another molecule (PE4E-TGF alpha-ILK) containing a nonfunctional carboxyl-terminal sequence was over 100-fold less active. The chimeric toxins with TGF alpha had the same binding affinity for the EGF receptor whether the ligand occupied the amino or carboxyl position. Molecules with TGF alpha near the carboxyl position were consistently less active against target cells but also less toxic to mice than those with TGF alpha at the amino terminus, indicating both types of molecules might be therapeutically effective. Our results establish that a ligand can be placed near the carboxyl terminus of PE, within the portion of the toxin that translocates to the cytosol. The amino-terminal position in such molecules is then available for the placement of other targeting ligands.
Kreitman
R J
RJ
Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
Chaudhary
V K
VK
Siegall
C B
CB
FitzGerald
D J
DJ
Pastan
I
I
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002470:Cell Survival / Q000187:drug effects; D015195:Drug Design; D005098:Exotoxins / Q000737:chemistry / Q000633:toxicity*; D005260:Female; D006801:Humans; D000922:Immunotoxins / Q000737:chemistry / Q000633:toxicity*; D007928:Lethal Dose 50; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000633:toxicity; D016211:Transforming Growth Factor alpha / Q000737:chemistry / Q000633:toxicity*; D014407:Tumor Cells, Cultured
D005098:Exotoxins; D000922:Immunotoxins; D011993:Recombinant Fusion Proteins; D016211:Transforming Growth Factor alpha; C062005:transforming growth factor type alpha-Pseudomonas exotoxin A