E. coli NfsA: an alternative nitroreductase for prodrug activation gene therapy in combination with CB1954.

Prodrug activation gene therapy is a developing approach to cancer treatment, whereby prodrug-activating enzymes are expressed in tumour cells. After administration of a non-toxic prodrug, its conversion to cytotoxic metabolites directly kills tumour cells expressing the activating enzyme, whereas the local spread of activated metabolites can kill nearby cells lacking the enzyme (bystander cell killing). One promising combination that has entered clinical trials uses the nitroreductase NfsB from Escherichia coli to activate the prodrug, CB1954, to a potent bifunctional alkylating agent. NfsA, the major E. coli nitroreductase, has greater activity with nitrofuran antibiotics, but it has not been compared in the past with NfsB for the activation of CB1954. We show superior in vitro kinetics of CB1954 activation by NfsA using the NADPH cofactor, and show that the expression of NfsA in bacterial or human cells results in a 3.5- to 8-fold greater sensitivity to CB1954, relative to NfsB. Although NfsB reduces either the 2-NO(2) or 4-NO(2) positions of CB1954 in an equimolar ratio, we show that NfsA preferentially reduces the 2-NO(2) group, which leads to a greater bystander effect with cells expressing NfsA than with NfsB. NfsA is also more effective than NfsB for cell sensitisation to nitrofurans and to a selection of alternative, dinitrobenzamide mustard (DNBM) prodrugs.

Escherichia coli nitroreductase plus CB1954 enhances the effect of radiotherapy in vitro and in vivo.

Escherichia coli nitroreductase (NTR) converts the prodrug CB1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide) into a bifunctional alkylating agent that causes DNA crosslinks. In this study, the ability of NTR to enhance the combined effects of CB1954 and radiation has been tested in vitro and in vivo. Stably transduced ovarian cancer cells (SKOV3-NTR) that are sensitive to CB1954 (IC(50)=0.35 muM) demonstrated enhanced cytotoxicity when treated with CB1954 and single-fraction irradiation. The NTR-CB1954 system mediated a bystander effect in combination with radiation on transfer of conditioned medium from SKOV3-NTR, but not SKOV3, cells to SW480 target cells. The ability of CB1954 to enhance radiation-induced cytotoxicity in SKOV3-NTR (but not SKOV3) cells was also demonstrated by fluorescence-activated cell sorting (FACS) with dual staining for propidium iodide/fluorescein diacetate, 4',6-diamidino-2-phenylindole dichloride staining of apoptotic cells and measurement of double-stranded DNA breaks by FACS and confocal microscopy for gammaH2AX foci. Adenoviral delivery of NTR, under constitutive cytomegalovirus or tissue-specific CTP1 promoters, increased the in vitro cytotoxicity of CB1954 plus radiation in MTT and clonogenic assays. Finally, adenoviral delivery of NTR plus CB1954 enhanced the effect of fractionated radiotherapy (12 Gy in four fractions) in SW480 xenograft tumours in nude mice.

Combining gene and immunotherapy for prostate cancer.

The nitroreductase (NR)/CB1954 enzyme prodrug system has given promising results in pre-clinical studies and is currently being assessed in phase I and II clinical trials in prostate cancer. Enhanced cell killing by apparent immune-mediated mechanisms has been shown in pancreatic and colorectal cancer models, by co-expressing murine granulocyte macrophage colony-stimulating factor (GM-CSF) with NR in a single replication deficient adenoviral vector. This consists of the CMV immediate early promotor driving expression of NR, with an internal ribosome entry site (IRES) and the gene for murine GM-CSF (mGM-CSF). To examine if similar enhancement of tumour cell killing could be produced in prostate cancer, the TRAMP model was chosen. Results illustrate that the combination of suicide gene therapy using NR and CB1954, with cytokine stimulation with mGM-CSF gives an improved response compared with either modality alone. The mechanism of this improved response is however likely to be non-immune based as it lacks a memory effect.

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

Expression of prodrug-activating enzymes that convert non-toxic substrates to cytotoxic derivatives is a promising strategy for cancer gene therapy. However, their catalytic activity with unnatural, prodrug substrates is often suboptimal. Efforts to improve these enzymes have been limited by the inability to select directly for increased prodrug activation. We have focussed on developing variants of Escherichia coli (E. coli) nitroreductase (NTR) with improved ability to activate the prodrug 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954), and describe here a novel, direct, positive selection for improved enzymes that exploits the alternative life cycles of bacteriophage lambda. In lambda lysogens of E. coli, the activation of the prodrug CB1954 by NTR triggers the SOS response to DNA damage, switching integrated lambda prophages into lytic cycle. This provides a direct, positive selection for phages encoding improved NTR variants, as, upon limiting exposure of lysogenized E. coli to CB1954, only those encoding the most active enzyme variants are triggered into lytic cycle, allowing their selective recovery. We exemplify the selection by isolating highly improved 'turbo-NTR' variants from a library of 6.8 x 10(5) clones, conferring up to 50-fold greater sensitivity to CB1954 than the wild type. Carcinoma cells infected with adenovirus expressing T41Q/N71S/F124T-NTR were sensitized to CB1954 concentrations 40- to 80-fold lower than required with WT-NTR.

Inhibition of NF-kappaB enhances the cytotoxicity of virus-directed enzyme prodrug therapy and oncolytic adenovirus cancer gene therapy.

Virus-directed enzyme prodrug therapy utilizing the bacterial enzyme nitroreductase delivered by a replication-defective adenovirus vector to activate the prodrug CB1954 is a promising strategy currently undergoing clinical trials in patients with a range of cancers. Similarly, selectively replicating oncolytic adenoviruses are entering clinical trials. An understanding of interactions between vector and target cell are critical to the development of these strategies. We demonstrate that adenovirus vectors activate cellular pathways that promote cell survival in an NF-kappaB-dependent manner, and consequently have a negative effect on the efficacy of cell killing induced by cancer gene therapy strategies. This provides a potential therapeutic target to enhance the cytotoxicity of these approaches.

Enhanced efficacy of Escherichia coli nitroreductase/CB1954 prodrug activation gene therapy using an E1B-55K-deleted oncolytic adenovirus vector.

Viruses that replicate selectively in cancer cells constitute an exciting new class of anticancer agent. The conditionally replicating adenovirus (CRAd) dl1520, which lacks the E1B-55K gene, has elicited significant clinical responses in humans when used in combination with chemotherapy. A convergent development has been to use replication-defective viruses to express prodrug-activating enzymes in cancer cells. This can sensitize the cancer to prodrug, but depends upon achieving sufficient level, distribution and specificity of enzyme expression within the tumour. In this study, we have expressed the prodrug-activating enzyme nitroreductase (NTR) in the context of an E1B-55K-deleted adenovirus, CRAd-NTR(PS1217H6). We show that CRAd-NTR(PS1217H6) retains oncolytic growth properties, and expresses substantially more NTR than a comparable, replication-defective adenovirus. The combination of viral oncolysis and NTR expression results in significantly greater sensitization of SW480 and WiDr colorectal cancer cells to the prodrug CB1954 in vitro. In vivo, CRAd-NTR(PS1217H6) was shown to replicate in subcutaneous SW480 tumour xenografts in immunodeficient mice, resulting in more NTR expression and greater sensitization to CB1954 than with replication-defective virus. Combination therapy of CRAd-NTR(PS1217H6) with CB1954 reduced tumour growth from 13.5- to 2.8-fold over 5 weeks, and extended median survival from 42 to 81 days, compared with no treatment.

In vivo gene therapy for colon cancer using adenovirus-mediated, transfer of the fusion gene cytosine deaminase and uracil phosphoribosyltransferase.

Virus-directed enzyme prodrug therapy (VDEPT) utilising cytosine deaminase (CD) converts 5-fluorocytosine (5-FC) into the chemotherapy agent, 5-fluorouracil (5-FU), and has entered into a clinical trial for metastatic colon cancer. To improve this system, a replication-deficient adenovirus, containing a bifunctional fusion gene, CD:uracil phosphoribosyltransferase (UPRT), was constructed (AdCDUPRT). UPRT enhances the conversion of 5-FU into its active metabolites, which inhibit DNA and RNA synthesis. In vitro, AdCDUPRT infection of colon cancer cells resulted in a marked increase in sensitisation to 5-FU, compared with AdCD-infected or uninfected cells. The corollary is a approximately 100-fold and approximately 10 000-fold increase in sensitisation to 5-FC in AdCDUPRT-infected cells, compared to AdCD-infected and uninfected cells, respectively. There was a strong bystander effect in vitro, 70% of tumour cells were killed by 5-FC when only 10% of cells expressed CDUPRT. In vivo, athymic mice with colon cancer xenografts treated with intratumoral AdCDUPRT and intraperitoneal 5-FC, significantly reduced tumour growth rates compared with untreated controls (P = 0.02), whereas AdCD/5-FC treated mice did not. At higher AdCDUPRT virus doses, 5-FC and 5-FU were equally effective at delaying tumour growth compared with controls. In summary, VDEPT for colon cancer utilising AdCDUPRT is more effective than AdCD and the bifunctional CDUPRT gene enables the use of either 5-FC or 5-FU as prodrugs.

The structure of Escherichia coli nitroreductase complexed with nicotinic acid: three crystal forms at 1.7 A, 1.8 A and 2.4 A resolution.

Escherichia coli nitroreductase is a flavoprotein that reduces a variety of quinone and nitroaromatic substrates. Its ability to convert relatively non-toxic prodrugs such as CB1954 (5-[aziridin-1-yl]-2,4-dinitrobenzamide) into highly cytotoxic derivatives has led to interest in its potential for cancer gene therapy. We have determined the structure of the enzyme bound to a substrate analogue, nicotinic acid, from three crystal forms at resolutions of 1.7 A, 1.8 A and 2.4 A, representing ten non-crystallographically related monomers. The enzyme is dimeric, and has a large hydrophobic core; each half of the molecule consists of a five-stranded beta-sheet surrounded by alpha-helices. Helices F and F protrude from the core region of each monomer. There is an extensive dimer interface, and the 15 C-terminal residues extend around the opposing monomer, contributing the fifth beta-strand. The active sites lie on opposite sides of the molecule, in solvent-exposed clefts at the dimer interface. The FMN forms hydrogen bonds to one monomer and hydrophobic contacts to both; its si face is buried. The nicotinic acid stacks between the re face of the FMN and Phe124 in helix F, with only one hydrogen bond to the protein. If the nicotinamide ring of the coenzyme NAD(P)H were in the same position as that of the nicotinic acid ligand, its C4 atom would be optimally positioned for direct hydride transfer to flavin N5. Comparison of the structure with unliganded flavin reductase and NTR suggests reduced mobility of helices E and F upon ligand binding. Analysis of the structure explains the broad substrate specificity of the enzyme, and provides the basis for rational design of novel prodrugs and for site-directed mutagenesis for improved enzyme activity.

Gene therapy for colorectal cancer: therapeutic potential.

Colorectal cancer is a leading cause of cancer mortality in Western countries. Gene therapy has been proposed as a potential novel treatment modality for colorectal cancer, but it is still in an early stage of development. The preclinical data have been promising and numerous clinical trials are underway. This brief review aims to summarise the current status of clinical trials of different gene therapy strategies, including immune stimulation, mutant gene correction, prodrug activation and oncolytic virus therapy, for patients with colorectal cancer. Data from phase I trials have proven the safety of the reagents but have not yet demonstrated significant therapeutic benefit. In order to achieve this and extend the scope of the treatment, continuing efforts should be made to improve the antitumour potency, efficiency of gene delivery and accuracy of gene targeting.

Tumour-specific therapeutic adenovirus vectors: repression of transgene expression in healthy cells by endogenous p53.

Approximately 50% of human tumours lack functional p53 suppressor protein. A promoter that is repressed by p53 in healthy cells could thus provide tumour-specific gene expression for a huge subset of tumours. In this report we describe a double recombinant adenovirus vector, 'Ad.p53R', encoding a therapeutic gene that is indirectly repressed by endogenous wild-type p53. Ad.p53R contains two independent expression cassettes; (1) the E. coli nitroreductase gene (NTR) driven by the human hsp70 promoter containing LacI binding sites (hsp70lacO-NTR) and (2) a p53-inducible lac repressor gene (tkGC3-lacI). In p53 null cells (Hep3B), Ad.p53R directed the same level of NTR expression as Ad.p53NR which lacks the tkGC3-lacI cassette. Moreover, injection of SW480 xenografts (mutated p53) with Ad.p53R resulted in a clear inhibition of growth in response to the prodrug CB1954. In cells retaining wt p53 (HepG2 and primary human endothelial cells), Ad.p53R expressed significantly less NTR (approximately 70%) than Ad.p53NR. Ad.p53R administered by i.v. injection also produced significantly less NTR than Ad.p53NR in normal tissues in vivo. Finally, adenovirus infection per se of cultured HepG2 cells at low MOI induced p53 stabilisation suggesting that adenovirus-mediated gene delivery may contribute to p53-based selectivity.

Combined adenovirus-mediated nitroreductase gene delivery and CB1954 treatment: a well-tolerated therapy for established solid tumors.

Gene-directed enzyme prodrug therapy (GDEPT) is a refinement of cancer chemotherapy that generates a potent cell-killing drug specifically in tumor cells by enzymatic activation of an inert prodrug. We describe in vivo studies that evaluate the efficacy and safety of intratumoral (i.t.) injection of an adenovirus vector (CTL102) expressing Escherichia coli nitroreductase (NTR) combined with systemic prodrug (CB1954) treatment. A single i.t. injection of CTL102 (7.5 x 10(9) to -2 x 10(10) particles) followed by CB1954 treatment produced clear anti-tumor effects in subcutaneous (s.c.) xenograft models of four cancers that are likely candidates for GDEPT (i.e., primary liver, head and neck, colorectal and prostate). Virus dose-response studies (s.c. liver model) revealed a steep increase and subsequent rapid plateauing of both NTR gene delivery and anti-tumor efficacy. Evidence of minor virus spread (toxicity) was observed in a s.c. head and neck xenograft model. This was eliminated by passive immunization with neutralizing anti-Ad5 antibodies prior to virus injection without reducing the magnitude of the anti-tumor effect. Preexisting anti-Ad5 neutralizing antibodies may therefore be an advantage rather than an issue in the clinical use of this new therapy.

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.

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.

Sensitisation of human carcinoma cells to the prodrug CB1954 by adenovirus vector-mediated expression of E. coli nitroreductase.

The enzyme nitroreductase from E. coli can reduce the weak, monofunctional alkylating agent 5-(aziridin-1-yl)-2, 4-dinitrobenzamide (CB1954) to a potent cytotoxic species that generates interstrand crosslinks in DNA. Nitroreductase therefore has potential as a "suicide enzyme" for cancer gene therapy, as cells that express nitroreductase become selectively sensitive to the prodrug CB1954. We have incorporated a nitroreductase expression cassette into a replication-defective adenovirus vector (Ad-CMV-ntr), which allowed efficient gene transfer to SK-OV-3 or IGROV-1 ovarian carcinoma cells. Nitroreductase levels increased in line with multiplicity of infection, and this was reflected in increasing sensitisation of the cells to CB1954, reaching an optimum (approx. 2, 000-fold sensitisation) with 25-50 p.f.u. per cell. Similar Ad-CMV-ntr-dependent sensitisation to CB1954 was seen in 3 of 6 low-passage primary ovarian tumour lines. Cells grown at low-serum concentration to inhibit proliferation remained equally susceptible to the Ad-CMV-ntr-dependent cytotoxicity of CB1954, indicating a distinct advantage over retroviral gene delivery and other popular enzyme-prodrug systems for human tumours with a low rate of cell proliferation. Additionally, cisplatin-resistant cells were sensitised towards CB1954 by Ad-CMV-ntr as efficiently as the parental cells, indicating that the system could be effective in patients with cisplatin-resistant tumours. In a murine xenograft model for disseminated peritoneal carcinomatosis with ascites, treatment of nude mice bearing intraperitoneal SUIT2 tumours with Ad-CMV-ntr and CB1954 almost doubled the median survival from 14 to 26 days (p < 0.0001).

Prostate-specific antigen promoter/enhancer driven gene therapy for prostate cancer: construction and testing of a tissue-specific adenovirus vector.

A range of luciferase reporter vectors was constructed, incorporating 5'-flanking sequences from the prostate-specific antigen (PSA), human glandular kallikrein 2 (hKLK2), and cytomegalovirus (CMV) promoters for expression control. Tissue specificity was evaluated in the PSA-positive line LNCaP and PSA-negative cells from different tissues of origin (CoLo320, DG75, EJ, A2780, and Jurkat). The minimal 628-bp PSA and hKLK2 promoters showed only low-level expression in either PSA-positive or PSA-negative cells and showed no increase with the addition of androgen. Tandem duplication of the PSA promoter slightly increased expression in PSA-positive LNCaP cells. The addition of CMV enhancer sequences upstream of a single PSA or hKLK2 promoter substantially but nonspecifically increased luciferase expression in all cell lines tested. However, placing a 1455-bp PSA enhancer sequence upstream of either the PSA or hKLK2 promoters increased expression 20-fold in the PSA-positive cell line LNCaP but not in the PSA-negative lines. Tandem duplication of the PSA enhancer increased expression to approximately 50-fold higher than either promoter alone while retaining tissue-specific control. The level of expression was reduced by the addition of a third copy of the PSA enhancer. Expression from all enhancer constructs was increased 100-fold above basal levels when induced with the androgen dihydrotestosterone, with the PSA-based constructs consistently exhibiting roughly twice the level of expression of the hKLK2-based constructs at all androgen concentrations. Adenovirus vectors were produced in which either enhanced green fluorescent protein or nitroreductase could be expressed from the optimized PSA double enhancer-promoter construct and evaluated in LNCaP cells and the bladder-derived line EJ. Control vectors with the CMV promoter gave good levels of expression in both cell lines, whereas the PSA constructs only produced detectable levels of protein in the LNCaP cells as assessed by fluorescence of enhanced green fluorescent protein or by Western blotting of nitroreductase. LNCaP but not EJ cells were selectively sensitized to the prodrug CB1954 following infection with Ad-PSA(EEP)-NR. The PSA-based nitroreductase virus produced comparable amounts of nitroreductase and sensitization to CB1954 approaching that of the CMV-driven virus. Plasmid and adenovirus constructs combining PSA enhancer and promoter sequences demonstrate selective expression of linked genes in PSA-positive cells. The expression is induced by androgen and gives therapeutically relevant levels of effector proteins.

Gene therapy strategies for colon cancer.

Colorectal cancer is the second most common cause of cancer mortality in Western countries. Gene therapy represents a novel approach to the treatment of colorectal cancer, and this review addresses the current strategies and ongoing clinical trials, including gene correction, immunomodulatory approaches and virus-directed enzyme-prodrug systems. Although the pre-clinical results for these strategies have been encouraging, clinical trials have not yet reflected these data. However, gene therapy for colorectal cancer is still in the early stages of development, and its potential, particularly in combination with conventional cancer therapies, warrants further investigation.

Production of retroviral vectors for gene therapy with the human packaging cell line FLYRD18.

The development of gene therapy is hampered by the difficulty of producing large stocks of retroviral vectors at high titer. This study aimed to improve culture conditions and to intensify the production of retroviruses by FLYRD18, a packaging cell line derived from the HT1080 human fibrosarcoma line. Batch virus production proved to be feasible in unsupplemented basal medium and provided significantly higher titers and productivities than medium supplemented with 10% serum. For longer-term production, however, AIM-V complete serum-free medium and basal medium supplemented with 2% serum gave superior results. Serum supplementation should nevertheless be optimized to take into account the presence of inhibitors of viral production. In monolayer cultures with 0.2 mL/cm(2), the cell concentration was increased up to 2 x 10(6) cells/mL without loss of cell productivity. A semicontinuous production process, which enables the collection of larger amounts of viruses from the same culture, has also been successfully used. Suspension culture processes were prevented by the anchorage dependency of the FLYRD18 cell line. Microcarrier cultures were able to produce viruses but will require further investigation and optimization for their performance to become competitive with monolayer cultures. In the course of this study, more than a 10-fold increase of titer has been achieved.

Improved titers of retroviral vectors from the human FLYRD18 packaging cell line in serum- and protein-free medium.

The influence of serum on the production of retroviral vectors by the HT1080 human fibrosarcoma-derived packaging cell line FLYRD18 was investigated. A fourfold increase in virus titer was observed under serum-free conditions, as compared with medium supplemented with 10% fetal calf serum. A similar improvement was also seen for bulk transduction efficiency. Serum had a negative and dose-dependent effect on titer without affecting cell growth, virus stability, or infectivity. In contrast to virus from NIH 3T3-derived packaging cells [Hanenberg, H., et al. (1996). Nature Med. 2, 876-882], the FLYRD18-derived virus did not adhere to fibronectin or serum proteins adsorbed at the surface of culture flasks. Electron microscopy supports the conclusion that the effect of serum is at the level of virus production by the cells. Addition of soybean trypsin inhibitor had an inhibitory effect on virus production, while pretreatment of serum with trypsin was found to enhance the retroviral titer. These results suggest that protease inhibitors present in serum may be responsible for the inhibition of virus production. The exact mechanism remains, however, to be determined. As compared with medium supplemented with 10% serum, the combination of increased virus titer and absence of exogenous protein under serum-free conditions resulted in a 300-fold increase in the virus:total protein ratio in the supernatants harvested from the FLYRD18 packaging line. This improvement enhances prospects for further concentration and purification of the virus.

Virus-directed enzyme prodrug therapy using CB1954.

The virus-directed enzyme prodrug therapy (VDEPT) anti-cancer 'gene therapy' strategy relies on the use of viral vectors for the efficient delivery to tumour cells of a 'suicide gene' encoding an enzyme which converts a non-toxic prodrug to a cytotoxic agent. The prodrug 5-(aziridin-1-yl)-2,4 dinitrobenzamide, CB1954, has been proposed for use in enzyme-prodrug gene therapy systems with the Escherichia coli enzyme nitroreductase (Ntr). Ntr converts CB1954 to 2- and 4-hydroxylamino derivatives, whereupon the non-enzymatic reaction of the 4-hydroxylamino derivative with cellular thio- esters generates a potent cytotoxic bifunctional alkylating agent capable of cross-linking DNA. Ntr delivery has been achieved in vitro using retroviral and adenoviral vectors and confirmed by immunocytochemical demonstration of Ntr expression. The Ntr-expressing cells have been shown to be sensitized to CB1954 by up to 2000-fold. The Ntr-CB1954 system shows effective bystander killing in mixed populations of Ntr-expressing and non-expressing cells treated with CB1954. The efficacy of this enzyme-prodrug approach in model systems compared with other VDEPT approaches demonstrates the feasibility and future promise of this gene therapy strategy.