Expression of Escherichia coli B nitroreductase in established human tumor xenografts in mice results in potent antitumoral and bystander effects upon systemic administration of the prodrug CB1954

Abstract

Expression of the Escherichia coli enzyme nitroreductase (NTR) in mammalian cells enables them to activate the prodrug 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954), leading to interstrand DNA cross-linking and apoptosis in both proliferating and quiescent cells. In the work reported here, we used human hepatocellular carcinoma and squamous carcinoma cell lines constitutively expressing NTR to demonstrate that the ntr/CB1954 system results in potent, long-lasting antitumoral effects in mice. We also demonstrate that this enzyme/prodrug combination results in antitumoral effects in vivo when only a minority of tumor cells express the enzyme, using either cells constitutively expressing NTR or ntr gene delivery in situ. Cancer Gene Therapy (2000) 7, 721-731

Repeated cycles of Clostridium-directed enzyme prodrug therapy result in sustained antitumour effects in vivo

“…As we observe such a striking antitumour response, it must mean that not only the hypoxic but also aerobic cells are being killed as a result of an additional bystander effect. Such a phenomenon has indeed been described for NTR in combination with CB1954 (Djeha et al, 2000;Wilson et al, 2002). Therefore, the use of a prodrug activating enzyme not only is a further safeguard to the system (compared to expression of a direct cytotoxic protein which might produce host toxicity if it comes in circulation), but also provides considerable potential for treatment amplification.…”

“…Tumour colonisation was allowed f or 5 days before initiation of prodrug and sham treatment, while selection antibiotics (erythromycin at 60 mg l À1 ) were added to the drinking water. CB1954 prodrug (15 mg kg À1 ) was prepared as previously described (Djeha et al, 2000) and administrated intraperitoneally (i.p.) for 5 consecutive days.…”

Mechanism of cell death induced by the novel enzyme-prodrug combination, nitroreductase/CB1954, and identification of synergism with 5-fluorouracil

“…Similarly, in vivo in a human hepatoma murine xenograft model, a significant antitumour effect and improved survival were observed even when only 5% of cells expressed NR, confirming a significant bystander effect (Djeha et al, 2000). Interestingly, studies of other enzyme -prodrug combinations also suggest a 'distant bystander effect' thought to be a systemic immunemediated response to local tumour cell killing (Link et al, 1997).…”

Direct positive selection for improved nitroreductase variants using SOS triggering of bacteriophage lambda lytic cycle

“…4,5,8,21 Such an effect in bacteria could potentially reduce the efficiency of phage selection from a mixed population, as induction of the lytic cycle would be less dependent upon the NTR expressed from the resident prophage in that cell. To determine whether phage encoding active NTRs could be enriched from a mixed population, we derived two mixed populations of lysogens, using 90% empty vector and 10% phages encoding either WT or F124N-NTR.…”

“…1 This NTR was later shown to convert the prodrug CB1954 to a potent DNA-crosslinking agent, 2 leading to the investigation of this combination for cancer gene therapy. [3][4][5][6][7][8] Expression of NTR from a replication-defective adenovirus can increase the sensitivity of carcinoma cells to CB1954 by 41000-fold 7,9 and this combination has entered clinical trials. [10][11][12][13] The cell-cycle independence of CB1954-mediated toxicity may have advantages over the S-phase-dependent toxicity of ganciclovir (GCV) or 5-fluorocytosine (5FC) to cells expressing herpes simplex virus thymidine kinase (HSV-TK) or cytosine deaminase (CD), respectively.…”