Optimized clostridium-directed enzyme prodrug therapy improves the antitumor activity of the novel DNA cross-linking agent PR-104.
We have previously shown that spores of the nonpathogenic clostridial strain C. sporogenes genetically engineered to express the E. coli-derived cytosine deaminase gene are effective in converting systemically injected nontoxic 5-fluorocytosine into the toxic anticancer drug 5-fluorouracil, thereby producing tumor-specific antitumor activity. To improve the expression of E. coli-derived genes with this system, we first replaced the original fdP promoter in the vector with one of two powerful endogenous clostridial promoters: that of the thiolase gene (thlP) and that for the clostridial transcription factor abrB310 (abrBP). These substitutions improved protein expression levels of the prodrug-activating genes by 2- to 3-fold in comparison with fdP-driven expression. However, despite these strong promoters, we found much higher expression of the nitroreductase (NTR) protein in the E. coli host compared with the clostridial host, which we hypothesized could be the result of different codon use between the two organisms. To test this, we constructed new expression vectors with an artificially synthesized NTR gene using optimized clostridial codons (sNTR). Results from both enzymatic assays and Western blots of cell extracts from clostridial transformants harboring plasmid constructs of thlP-sNTR and abrBP-sNTR showed that the expression and activity of the NTR gene product was increased by approximately 20-fold compared with the original construct. In vivo studies with i.v. administered sNTR-expressing C. sporogenes spores in SiHa tumor-bearing mice showed significantly improved antitumor efficacy when combined with either 5-aziridinyl-2,4-dinitrobenzamide (CB1954) or the novel dinitrobenzamide mustard prodrug, PR-104.
Liu
Shie-Chau
SC
Department of Radiation Oncology, Stanford University School of Medicine, Stanford, California, USA.
Ahn
G-One
GO
Kioi
Mitomu
M
Dorie
Mary-Jo
MJ
Patterson
Adam V
AV
Brown
J Martin
JM
D000818:Animals; D018906:Antineoplastic Agents, Alkylating / Q000378:metabolism / Q000627:therapeutic use; D001483:Base Sequence; D002277:Carcinoma / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D003013:Clostridium / Q000235:genetics / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000378:metabolism / Q000627:therapeutic use*; D004273:DNA, Neoplasm / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005260:Female; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D008969:Molecular Sequence Data; D009588:Nitrogen Mustard Compounds / Q000378:metabolism / Q000627:therapeutic use*; D011355:Prodrugs / Q000378:metabolism* / Q000627:therapeutic use*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000378:metabolism / Q000627:therapeutic use; D013171:Spores, Bacterial / Q000235:genetics / Q000378:metabolism; D002583:Uterine Cervical Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D023041:Xenograft Model Antitumor Assays
D018906:Antineoplastic Agents, Alkylating; D003432:Cross-Linking Reagents; D004273:DNA, Neoplasm; D009588:Nitrogen Mustard Compounds; C522381:PR-104; D011355:Prodrugs; D011994:Recombinant Proteins