Effect of expression of adenine phosphoribosyltransferase on the in vivo anti-tumor activity of prodrugs activated by E. coli purine nucleoside phosphorylase

The use of E. coli purine nucleoside phosphorylase (PNP) to activate prodrugs has demonstrated excellent activity in the treatment of various human tumor xenografts in mice. E. coli PNP cleaves purine nucleoside analogs to generate toxic adenine analogs, which are activated by adenine phosphoribosyl transferase (APRT) to metabolites that inhibit RNA and protein synthesis. We created tumor cell lines that encode both E. coli PNP and excess levels of human APRT, and have used these new cell models to test the hypothesis that treatment of otherwise refractory human tumors could be enhanced by overexpression of APRT. In vivo studies with 6-methylpurine-2'-deoxyriboside (MeP-dR), 2-F-2'-deoxyadenosine (F-dAdo) or 9-β-D-arabinofuranosyl-2-fluoroadenine 5'-monophosphate (F-araAMP) indicated that increased APRT in human tumor cells coexpressing E. coli PNP did not enhance either the activation or the anti-tumor activity of any of the three prodrugs. Interestingly, expression of excess APRT in bystander cells improved the activity of MeP-dR, but diminished the activity of F-araAMP. In vitro studies indicated that increasing the expression of APRT in the cells did not significantly increase the activation of MeP. These results provide insight into the mechanism of bystander killing of the E. coli PNP strategy, and suggest ways to enhance the approach that are independent of APRT.     

The use of Trichomonas vaginalis purine nucleoside phosphorylase to activate fludarabine in the treatment of solid tumors

Treatment with fludarabine phosphate (9-β-D-arabinofuranosyl-2-F-adenine 5′-phosphate, F-araAMP) leads to regressions and cures of human tumor xenografts that express Escherichia coli purine nucleoside phosphorylase (EcPNP). This occurs despite the fact that fludarabine (F-araA) is a relatively poor substrate for EcPNP, and is cleaved to liberate 2-fluoroadenine at a rate only 0.3% that of the natural E. coli PNP substrate, adenosine. In this study, we investigated a panel of naturally occurring PNPs to identify more efficient enzymes that may be suitable for metabolizing F-araA as part of experimental cancer therapy. We show that Trichomonas vaginalis PNP (TvPNP) cleaves F-araA with a catalytic efficiency 25-fold greater than the prototypic E. coli enzyme. Cellular extracts from human glioma cells (D54) transduced with lentivirus stably expressing TvPNP (D54/TvPNP) were found to cleave F-araA at a rate similar to extracts from D54 cells expressing EcPNP, although much less enzyme was expressed per cell in the TvPNP transduced condition. As a test of safety and efficacy using TvPNP, human head and neck squamous cell carcinoma (FaDu) xenografts expressing TvPNP were studied in nude mice and shown to exhibit robust tumor regressions, albeit with partial weight loss that resolved post-therapy. F-araAMP was also a very effective treatment for mice bearing D54/TvPNP xenografts in which approximately 10% of tumor cells expressed the enzyme, indicating pronounced ability to kill non-transduced tumor cells (high bystander activity). Moreover, F-araAMP demonstrated activity against D54 tumors injected with an E1, E3 deleted adenoviral vector encoding TvPNP. In that setting, despite higher F-araA cleavage activity using TvPNP, tumor responses were similar to those obtained with EcPNP, indicating factors other than F-Ade production may limit regressions of the D54 murine xenograft model. Our results establish that TvPNP is a favorable enzyme for activating F-araA, and support further studies in combination with F-araAMP for difficult-to-treat human cancers.     

Excellent in vivo bystander activity of fludarabine phosphate against human glioma xenografts that express the escherichia coli purine nucleoside phosphorylase gene

Escherichia coli purine nucleoside phosphorylase (PNP) expressed in tumors converts relatively nontoxic prodrugs into membrane-permeant cytotoxic compounds with high bystander activity. In the present study, we examined tumor regressions resulting from treatment with E. coli PNP and fludarabine phosphate (F-araAMP), a clinically approved compound used in the treatment of hematologic malignancies. We tested bystander killing with an adenoviral construct expressing E. coli PNP and then more formally examined thresholds for the bystander effect, using both MuLv and lentiviral vectoring. Because of the importance of understanding the mechanism of bystander action and the limits to this anticancer strategy, we also evaluated in vivo variables related to the expression of E. coli PNP (level of E. coli PNP activity in tumors, ectopic expression in liver, percentage of tumor cells transduced in situ, and accumulation of active metabolites in tumors). Our results indicate that F-araAMP confers excellent in vivo dose-dependent inhibition of bystander tumor cells, including strong responses in subcutaneous human glioma xenografts when 95 to 97.5% of the tumor mass is composed of bystander cells. These findings define levels of E. coli PNP expression necessary for antitumor activity with F-araAMP and demonstrate new potential for a clinically approved compound in solid tumor therapy.     

In vivo antitumor activity of intratumoral fludarabine phosphate in refractory tumors expressing E. coli purine nucleoside phosphorylase

Purpose

Systemically administered fludarabine phosphate (F-araAMP) slows growth of human tumor xenografts that express Escherichia coli purine nucleoside phosphorylase (PNP). However, this treatment has been limited by the amount of F-araAMP that can be administered in vivo. The current study was designed to (1) determine whether efficacy of this overall strategy could be improved by intratumoral administration of F-araAMP, (2) test enhancement of the approach with external beam radiation, and (3) optimize recombinant adenovirus as a means to augment PNP delivery and bystander killing in vivo.

Methods

The effects of systemic or intratumoral F-araAMP in mice were investigated with human tumor xenografts (300?mg), in which 10?% of the cells expressed E. coli PNP from a lentiviral promoter. Tumors injected with an adenoviral vector expressing E. coli PNP (Ad/PNP; 2?×?10¹¹ viral particles, 2?times per day?×?3?days) and the impact of radiotherapy on tumors treated by this approach were also studied. Radiolabeled F-araAMP was used to monitor prodrug activation in vivo.

Results

Intratumoral administration of F-araAMP in human tumor xenografts expressing E. coli PNP resulted in complete regressions and/or prolonged tumor inhibition. External beam radiation significantly augmented this effect. Injection of large human tumor xenografts (human glioma, nonsmall cell lung cancer, or malignant prostate tumors) with Ad/PNP followed by intratumoral F-araAMP resulted in excellent antitumor activity superior to that observed following systemic administration of prodrug.

Conclusion

Activation of F-araAMP by E. coli PNP results in destruction of large tumor xenografts in vivo, augments radiotherapy, and promotes robust bystander killing. Our results indicate that intratumoral injection of F-araAMP leads to ablation of tumors in vivo with minimal toxicity.     

Targeting gene therapy for hepatocarcinoma cells with the E. coli purine nucleoside phosphorylase suicide gene system directed by a chimeric alpha-fetoprotein promoter

For hepatocarcinoma (HCC) gene therapy, the tumoricidal efficacy and selective expression of therapeutic gene remain two major challenges. The Escherichia coli (E. coli) purine nucleoside phosphorylase (PNP)/9-(2-deoxy-beta-dribofuranosyl)-6-methylpurine (MeP-dR) suicide gene system exhibits excellent anti-tumor effects, indicating this system directed by a HCC-specific promoter would offer a possibility of targeting gene therapy for HCC. To test this hypothesis, here, we prepared a plasmid (p[HRE]AF/PNP) containing the E. coli PNP/MeP-dR system and a chimeric human alpha-fetoprotein (AFP) promoter, [HRE]AF. We introduced this plasmid into AFP-positive and low-AFP-generating human HCC cells, and evaluated its therapeutic effects on both human HCC cell lines. In the presence of hypoxia, the E. coli PNP gene directed by the [HRE]AF promoter were HCC-specifically expressed in two human HCC cell lines and, moreover, the [HRE]AF-PNP/MeP-dR therapy would yield significant and selective cytotoxicity in both AFP-positive and low-AFP-generating HCC cells. Our findings suggest the [HRE]AF-PNP/MeP-dR therapy has worthy potentialities as an effective strategy for targeting therapy of AFP-positive, and especially AFP-negative or low-AFP-generating HCC.     

Pharmacodynamics and Proposed Mechanism of Therapeutic Action and Host Toxicity of 9-β-D-Arabinofuranosyl-2-Fluoroadenine Monophosphate (F-araAMP) in P388 Murine Leukemia-Bearing Mice

The cellular metabolism of 9-β-D-arabinofiiranosyl-2-fluoroadenine 5'monophosphate (F-araAMP), a soluble nucleoside analog with proven antileukemic activity in animal and human tumors, has been studied in mice bearing P388 leukemia. Earlier studies showed markedly less in vivo accumulation of F-ara ATP the principal active metabolite, in gastrointestinal mucosa (GI) and bone marrow (BM) compared with P388 after F-araA or F-araAMP administration. To elucidate the mechanism of toxicity this work has examined the pharmacodynamics of F-araAMP anabolites, F-araATP and F-ATP, in P388 cells, BM and GI mucosa tissues after nontoxic (LD₁) and toxic (LD₅₀) doses of F-araAMP. F-araATP was the major triphosphate metabolite in acid-soluble extracts from P388 cells, BM, and GI mucosa tissues. F-araATP accumulated to approximately 1 mM in P388 cells after either LD₁or LD₅₀ treatment of F-araAMP and was eliminated with a t_1/2eiof approximately 5 h. The ratio of the area under the concentration-time curve (AUC 0 - ∞) of F-araATP was 1.01 after the LD₅₀over LD₁ doses of F-araAMP. BM and GI mucosa tissues accumulated 40-fold less F-araATP than the concentration in P388 cells. 2-Fluoro-ATP, a second toxic anabolite, accumulated in P388 cells to 156 ± 39 pM and 447 ± 79 pM after the two doses of the drug, respectively. The ratio of area under the curve (AUC) of F-ATP in P388 cells after the two doses of F-araAMP was 38.77, which approaches the ratio of % lethality (LD₅₀/LD; = 50). F-ATP was also quantitated in BM and GI mucosa reaching one-fifth to one-half the concentration ofF-araATP after the LD50 dose of F-araAMP. The AUC values of F-ATP (0 - 24 h) were 9.5-to 12.5-fold higher after the LD₅₀ than after the LD₁ dose of F-araAMP. These results suggest that there is a selective therapeutic action of F-araAMP against P388 and that the increased cellular concentration of F-ATP in both the tumor cells and the host tissues (BM and GI mucosa) could explain the mode of toxicity of F-araAMP.     

AF0.3启动子调控的PNP/MeP-dR系统对AFP阳性肝癌细胞的特异杀伤作用

背景与目的:甲胎蛋白(α-fetoprotein,AFP)启动子调控下的目的基因能在AFP阳性肝癌组织中特异性表达;嘌呤核苷磷酸化酶(Escherichiacolipurinenucleosidephosphorylase,PNP)/6-甲基嘌呤-2′-脱氧核糖核苷(6-methylpurine-2-deoxyriboside,MeP-dR)自杀基因系统具有强杀瘤效应。本研究旨在探讨AF0.3启动子调控下的PNP/MeP-dR系统对AFP阳性肝癌细胞的特异性杀伤作用。方法:构建甲胎蛋白启动子AF0.3调控下PNP基因表达载体pAF0.3/PNP,导入AFP阳性肝癌细胞HepG2和阴性的肝癌细胞株SMMC7721,利用G418筛选获得稳定转染PNP基因的HepG2/0.3-PNP及SMMC7721/0.3-PNP细胞。RT-PCR检测二者在细胞中的表达。台盼蓝拒染法检测细胞增殖效应,MTT法和流式细胞仪检测两株细胞对MeP-dR的敏感性及旁观者效应,高效液相色谱法(highperformanceliquidchromatography,HPLC)检测PNP基因产物活性。结果:不论有氧还是缺氧条件,HepG2/0.3-PNP对MeP-dR均较为敏感,SMMC7721/0.3-PNP则对MeP-dR完全不敏感。在任何一种条件下,HepG2/AF0.3-PNP在混合细胞中比例达25%后,就可致明显旁观者效应;而在同样条件下,SMMC7721/0.3-PNP不导致明显的旁观者效应。HPLC结果显示,pAF0.3/PNP在HepG2细胞中可以将MeP-dR转化为6-MP,但其在SMMC7721中则不具有转化活性。结论:AF0.3启动子调控下的PNP/MeP-dR系统对AFP阳性HepG2细胞有较好的杀伤作用。     

Lymphoma chemovirotherapy: CD20-targeted and convertase-armed measles virus can synergize with fludarabine

Combination chemotherapy regimen incorporating CD20 antibodies are commonly used in the treatment of CD20-positive non-Hodgkin's lymphoma (NHL). Fludarabine phosphate (F-araAMP), cyclophosphamide, and CD20 antibodies (Rituximab) constitute the FCR regimen for treating selected NHL, including aggressive mantle cell lymphoma (MCL). As an alternative to the CD20 antibody, we generated a CD20-targeted measles virus (MV)-based vector. This vector was also armed with the prodrug convertase purine nucleoside phosphorylase (PNP) that locally converts the active metabolite of F-araAMP to a highly diffusible substance capable of efficiently killing bystander cells. We showed in infected cells that early prodrug administration controls vector spread, whereas late administration enhances cell killing. Control of spread by early prodrug administration was also shown in an animal model: F-araAMP protected genetically modified mice susceptible to MV infection from a potentially lethal intracerebral challenge. Enhanced oncolytic potency after extensive infection was shown in a Burkitt's lymphoma xenograft model (Raji cells): After systemic vector inoculation, prodrug administration enhanced the therapeutic effect synergistically. In a MCL xenograft model (Granta 519 cells), intratumoral (i.t.) vector administration alone had high oncolytic efficacy: All mice experienced complete but temporary tumor regression, and survival was two to four times longer than that of untreated mice. Cells from MCL patients were shown to be sensitive to infection. Thus, synergy of F-araAMP with a PNP-armed and CD20-targeted MV was shown in one lymphoma therapy model after systemic vector inoculation.     

Transcription-targeted gene therapy for androgen-independent prostate cancer

The Escherichia coli enzyme (purine nucleoside phosphorylase, PNP) gene is delivered directly into PC3 tumors by one injection of replication-deficient human type-5 adenovirus (Ad5). Expressed PNP converts the systemically administered prodrug, 6MPDR, to a toxic purine, 6MP, causing cell death. We sought to increase the specificity of recombinant Ad vectors by controlling PNP expression with the promoter region from the androgen-dependent, prostate-specific rat probasin (Pb) gene. To increase its activity, the promoter was combined with the SV40 enhancer (SVPb). Cell lines were transfected with plasmids containing both a reporter gene, under SVPb control, and a reference gene cassette to allow normalization of expression levels. Plasmids expressed approximately 20-fold more reporter in prostate cancer than in other cells, but surprisingly, the SVPb element was both androgen-independent and retained substantial prostate specificity. Killing by Ad5-SVPb-PNP vector of cell lines cultured with 6MPDR for 6 days was 5- to 10-fold greater in prostate cancer than in liver or lung cells. In vivo, a single intratumoral injection of Ad5-SVPb-PNP (4 x 10(8) pfu), followed by 6MPDR administration twice daily for 6 days, significantly suppressed the growth of human prostate tumors in nude mice and increased their survival compared to control animals. Thus, the androgen-independent, prostate-targeting Ad5 vector reduces human prostate cancer growth significantly in vitro and in vivo. This first example of an androgen-independent vector points the way toward treatment of emerging androgen-independent prostate cancer in conjunction with hormone ablation therapy at a time when the tumor burden is low.     

Armed and targeted measles virus for chemovirotherapy of pancreatic cancer

No curative therapy is currently available for locally advanced or metastatic pancreatic cancer. Therefore, new therapeutic approaches must be considered. Measles virus (MV) vaccine strains have shown promising oncolytic activity against a variety of tumor entities. For specific therapy of pancreatic cancer, we generated a fully retargeted MV that enters cells exclusively through the prostate stem cell antigen (PSCA). Besides a high-membrane frequency on prostate cancer cells, this antigen is expressed on pancreatic adenocarcinoma, but not on non-neoplastic tissue. PSCA expression levels differ within heterogeneous tumor bulks and between human pancreatic cell lines, and we could show specific infection of pancreatic adenocarcinoma cell lines with both high- and low-level PSCA expression. Furthermore, we generated a fully retargeted and armed MV-PNP-anti-PSCA to express the prodrug convertase purine nucleoside phosphorylase (PNP). PNP, which activates the prodrug fludarabine effectively, enhanced the oncolytic efficacy of the virus on infected and bystander cells. Beneficial therapeutic effects were shown in a pancreatic cancer xenograft model. Moreover, in the treatment of gemcitabine-resistant pancreatic adenocarcinoma cells, no cross-resistance to both MV oncolysis and activated prodrug was detected.     

Potentiation of 2'-deoxyguanosine cytotoxicity by a novel inhibitor of purine nucleoside phosphorylase, 8-amino-9-benzylguanine

We have synthesized and evaluated a series of 9-substituted analogues of 8-aminoguanine, a known inhibitor of human purine nucleoside phosphorylase (PNP) activity. The ability of these agents to inhibit PNP has been investigated. All compounds were found to act as competitive (with inosine) inhibitors of PNP, with Ki values ranging from 0.2 to 290 microM. The most potent of these analogues, 8-amino-9-benzylguanine; exhibited a Ki value that was 4-fold lower than that determined for the parent base, 8-aminoguanine. As a metabolically stable compound in human blood, 8-amino-9-benzylguanine was more effective than 8-aminoguanine at potentiating the toxicity of 2'-deoxyguanosine to MOLT-4 T-lymphoblasts in culture. 8-Amino-9-benzylguanine is the most potent base or nucleoside inhibitor of human PNP reported to date, and it is a promising lead compound in the development of more effective PNP inhibitors.     

Delivery of replication-competent retrovirus expressing Escherichia coli purine nucleoside phosphorylase increases the metabolism of the prodrug, fludarabine phosphate and suppresses the growth of bladder tumor xenografts

We have developed unique replication-competent retroviral (RCR) vectors based on murine leukemia virus that provide improved efficiency of viral delivery, allow for long-term transgene expression and demonstrate an intrinsic selectivity for transduction of rapidly dividing tumor cells. The purpose of this study was to evaluate the in vivo transduction efficiency and the therapeutic efficacy of the RCR vector mediated delivery of Escherichia coli purine nucleoside phosphorylase (PNP) in combination with fludarabine phosphate for bladder cancer. We constructed vectors containing green fluorescent protein (GFP) gene (ACE)-GFP) or PNP gene (ACE-PNP). KU-19-19 bladder tumors exhibited 28.3+/-16.1, 46.6+/-5.8 and 93.7+/-7.8% of GFP expression on 14, 18 and 26 days after intratumoral injection of ACE-GFP, respectively. GFP expression could not be observed in normal tissues surrounding the injected tumors. No detectable polymerase chain reaction products of GFP gene could be observed in any distant organs. Intratumoral injection of ACE-PNP, followed by systemically administered fludarabine phosphate, significantly inhibited the growth of pre-established KU-19-19 tumors. Our results indicate that RCR vectors are a potentially efficient gene delivery method and that the RCR vector mediated PNP gene transfer and fludarabine phosphate treatment might be a novel and potentially therapeutic modality for bladder cancer.     

Enzyme activities controlling adenosine levels in normal and neoplastic tissues

Adenosine is known to be associated with effects such as inhibition of immune response, coronary vasodilation, stimulation of angiogenesis, and inhibition of inflammatory reactions. Some authors suggest that adenosine may also have similar functions in tumor tissues. Tissue levels of adenosine are under close regulation by different enzymes acting at different levels. Adenosine is produced from AMP by the action of 5′-nucleotidase (5′-NT) and is converted back into AMP by adenosine kinase (AK) or into inosine by adenosine deaminase (ADA). Inosine is converted into purine catabolites by purine nucleoside phosphorylase (PNP), whereas AMP is converted into ADP and ATP by adenylate kinase (MK). The aim of this study was to analyze the activities of the above enzymes in fragments of neoplastic and apparently normal mucosa, obtained less than 5 cm and at least 10 cm from tumors, in 40 patients with colorectal cancer. The results showed much higher activities of ADA, AK, 5′-NT, and PNP in tumor tissue than in neighboring mucosa (p>0.01 for ADA, AK, and PNP; p>0.05 for 5′-NT), suggesting that the activities of purine metabolizing enzymes increase to cope with accelerated purine metabolism in cancerous tissue. The simultaneous increase in ADA and 5′-NT activities might be a physiological attempt by cancer cells to provide more substrate to accelerate salvage pathway activity.     

Experimental therapy of allogeneic solid tumors induced in athymic mice with suicide gene-transducing replication-competent foamy virus vectors

A replication competent foamy virus derived retroviral vector expressing suicide genes has been constructed and characterized in vitro. Here we used vectors expressing the purine nucleoside phosphorylase (FOV-7/pnp), the nitroreductase (FOV-7/ntr), or the thymidine kinase (FOV-7/tk) suicide gene in an in vivo athymic (nude) mice/human glioblastoma tumor model. Gliomas were induced by subcutanous injection of U87 tumor cells. The virus vector was injected when the tumor became visible. Mice with vector virus-injected tumors were treated with the respective prodrug. The treatment resulted in significant inhibition of tumor growth. Surprisingly, in mice with vector virus-injected tumors without prodrug treatment a similar suppression of tumor growth was observed. In 65% (pnp vector), 75% (ntr vector) and 37% (tk vector) of these mice the tumors stopped growing or vanished and the animals remained tumor free for the 25 weeks of the experiment, whereas all mice of the control groups had to be killed because of the tumor growth. In control experiments, the suppression of tumor growth could also be observed when wild-type foamy virus was injected instead of the suicide gene-transducing vectors. Similar results were obtained using the nude mice/G59 human glioblastoma tumor model. In conclusion, the experiments demonstrate an oncolytic activity of foamy virus replication in a nude-mice glioblastoma xenograft tumor model. The analysis of vector virus DNA by PCR revealed that the vector persisted in different organs of the animals irrespective of the use of a prodrug or the elimination of a tumor.     

Efficient electrogene therapy for pancreatic adenocarcinoma treatment using the bacterial purine nucleoside phosphorylase suicide gene with fludarabine

The aim of this study was to demonstrate the potential of electrogene therapy with the bacterial purine nucleoside phosphorylase gene (ePNP), on pancreatic carcinoma (PC) large tumors. The in vivo electroporation (EP) conditions and efficacy were investigated on both subcutaneous xenografts of human PC cells in immunocompromised mice and orthotopic intrapancreatic grafts of rat PC cells in syngenic rats. After intratumoral injection of naked plasmid DNA, EP was performed using a two-needle array with 25-msec pulses and either a 300 V/cm field strength for subcutaneous or a 500 V/cm field strength for orthotopic PC, parameters providing the best electrotransfer as reflected by the measurements of both luciferase activity and ePNP mRNA. As expected, tumors developed sensitivity to prodrug treatment (6-methylpurine deoxyribose or fludarabine phosphate). We observed both significant inhibition of tumor growth and extended survival of treated mice. In fact, after prodrug treatment, PC growth in the subcutaneous model was delayed by 50-70% for ePNP-expressing tumors. In an orthotopic pancreatic tumor model, the animal survival was significantly prolonged after ePNP electrogene transfer followed by fludarabine treatment, with one animal out of 10 being tumor-free 6 months thereafter. The current study demonstrates for the first time on PC the in vivo feasibility of electrogene transfer and its therapeutic efficiency using the suicide gene/prodrug system, ePNP/fludarabine. These findings suggest that electrogene therapy strategy must be considered for pancreatic cancer treatment, particularly at advanced stages of the disease.     

Suicide gene/prodrug therapy using salmonella-mediated delivery of Escherichia coli purine nucleoside phosphorylase gene and 6-methoxypurine 2'-deoxyriboside in murine mammary carcinoma 4T1 model

Attenuated salmonella have been reported to be capable of both selectively growing in tumors and expressing exogenous genes for tumor-targeted therapy. As 6-methoxypurine 2'-deoxyriboside (MoPdR) is similar to 6-methylpurine 2'-deoxyriboside in structure, we aimed to evaluate the antitumoral effect of the Escherichia coli purine nucleoside phosphorylase (ePNP) gene, using an attenuated salmonella-mediated delivery system, in combination with MoPdR. A novel mutant serovar Typhimurium (SC36) was used to carry the pEGFP-C1-ePNP vector that contains an enhanced green fluorescent protein and an ePNP gene under the control of the cytomegalovirus promoter. The function of the ePNP expression vector was confirmed in vitro using the enzymic conversion of MoPdR into methoxypurine. We also observed a high bystander effect induced by the ePNP/MoPdR system with a very low proportion (1%) of ePNP-positive cells and 5 µg/mL MoPdR, although the growth of parental cells was affected appreciably by MoPdR. The killing effect and increased apoptosis induced by SC36 carrying the ePNP expression vector (SC/ePNP) were detected by cytotoxicity assay and propidium iodide staining flow cytometry analysis, in combination with MoPdR. SC/ePNP was given orally to mice bearing mammary carcinomas, and its antitumor effect was evaluated. SC/ePNP plus MoPdR significantly inhibited tumor growth by approximately 86.6–88.7% and prolonged the survival of tumor-hosting mice. Our data support the view that MoPdR combined with the ePNP gene could be used in gene-directed enzyme prodrug therapy. Attenuated salmonella could be a promising strategy to improve ePNP/MoPdR bystander killing due to its preferential accumulation and anticancer activity in tumors. ( Cancer Sci 2008; 99: 1172–1179)     

Phase I dose-escalating trial of Escherichia coli purine nucleoside phosphorylase and fludarabine gene therapy for advanced solid tumors

BackgroundThe use of Escherichia coli purine nucleoside phosphorylase (PNP) to activate fludarabine has demonstrated safety and antitumor activity during preclinical analysis and has been approved for clinical investigation.Patients and methodsA first-in-human phase I clinical trial (NCT 01310179; IND 14271) was initiated to evaluate safety and efficacy of an intratumoral injection of adenoviral vector expressing E. coli PNP in combination with intravenous fludarabine for the treatment of solid tumors. The study was designed with escalating doses of fludarabine in the first three cohorts (15, 45, and 75 mg/m²) and escalating virus in the fourth (10¹¹–10¹² viral particles, VP).ResultsAll 12 study subjects completed therapy without dose-limiting toxicity. Tumor size change from baseline to final measurement demonstrated a dose-dependent response, with 5 of 6 patients in cohorts 3 and 4 achieving significant tumor regression compared with 0 responsive subjects in cohorts 1 and 2. The overall adverse event rate was not dose-dependent. Most common adverse events included pain at the viral injection site (92%), drainage/itching/burning (50%), fatigue (50%), and fever/chills/influenza-like symptoms (42%). Analysis of serum confirmed the lack of systemic exposure to fluoroadenine. Antibody response to adenovirus was detected in two patients, suggesting that neutralizing immune response is not a barrier to efficacy.ConclusionsThis first-in-human clinical trial found that localized generation of fluoroadenine within tumor tissues using E. coli PNP and fludarabine is safe and effective. The pronounced effect on tumor volume after a single treatment cycle suggests that phase II studies are warranted.ClinicalTrials.gov IdentifierNCT01310179.     

Suicide gene therapy using E. coli beta-galactosidase

PURPOSE: Suicide gene therapy offers the potential to increase the selective toxicity of antitumor agents by intratumoral expression of exogenous enzymes that convert nontoxic prodrugs to toxic products. The use of herpes simplex virus thymidine kinase with ganciclovir, and E. coli cytosine deaminase with 5-fluorocytosine are well-known examples of this approach. The purpose of this study was to investigate a novel suicide gene therapy using E. coli beta-galactosidase (beta-gal) as the prodrug-activating enzyme. Advantages of this approach include: (1) the ability to use prodrugs that are cleaved by beta-gal to agents that are known to possess activity against human solid tumors, and (2) the extensive experience gained with targeting beta-gal to specific tumors in experimental animals and in humans. METHODS: Two different structural types of anthracycline prodrugs, N-[4"-(beta- D-galactopyranosyl)-3"-nitrobenzyloxycarbonyl]daunomycin (Daun02) and N-[(4" R,S)-4"-ethoxy-4"-(1"'- O-beta- D-galactopyranosyl)butyl]daunorubicin (gal-DNC4) were investigated. The prodrugs were evaluated as substrates for beta-gal. Cytotoxicity studies of Daun02 were conducted against a murine tumor (Panc02), two human breast tumors (MCF-7 and T47D), and three human prostate tumors (PC3, DU145 and LNCAP) that had been transduced to express beta-gal. Antitumor studies of Daun02 were conducted against mouse tumor Panc02 xenografts implanted subcutaneously. RESULTS: Daun02 was a good substrate for beta-gal. By comparison, gal-DCN4 was a poor substrate. Except for PC3, the beta-gal-transduced tumors showed 3- to 60-fold increased sensitivity to Daun02 compared with mock-transduced control cells. Daunomycin was formed from Daun02 in tissue culture medium containing beta-gal-transduced cell lines but was not observed in the medium from mock-transduced controls. In vivo therapeutic studies of Daun02 against the Panc02 tumor in athymic mice showed no significant inhibition of tumor growth. Pharmacokinetic studies showed limited distribution of the prodrug beyond the vascular space. CONCLUSIONS: E. coli beta-gal may be useful as a prodrug-activating enzyme in suicide gene therapy and has the potential to increase the selective toxicity of conventional antitumor agents. Although this approach worked well against tumor cells in vitro, it was not effective against a xenograft model in vivo, apparently because of poor drug-tissue distribution.     

Characterization of an engineered human purine nucleoside phosphorylase fused to an anti-her2/neu single chain Fv for use in ADEPT

Background

Antibody Directed Enzyme Prodrug Therapy (ADEPT) can be used to generate cytotoxic agents at the tumor site. To date non-human enzymes have mainly been utilized in ADEPT. However, these non-human enzymes are immunogenic limiting the number of times that ADEPT can be administered. To overcome the problem of immunogenicity, a fully human enzyme, capable of converting a non-toxic prodrug to cytotoxic drug was developed and joined to a human tumor specific scFv yielding a fully human targeting agent.

Methods

A double mutant of human purine nucleoside phosphorylase (hDM) was developed which unlike the human enzyme can cleave adenosine-based prodrugs. For tumor-specific targeting, hDM was fused to the human anti-HER2/neu single chain Fv (scFv), C6 MH3B1. Enzymatic activity of hDM with its natural substrates and prodrugs was determined using spectrophotomeric approaches. A cell proliferation assay was used to assess the cytotoxicity generated following conversion of prodrug to drug as a result of enzymatic activity of hDM. Affinity of the targeting scFv, C6 MH3B1 fused to hDM to Her2/neu was confirmed using affinity chromatography, surface plasmon resonance, and flow-cytometry.

Results

In vitro hDM-C6 MH3B1 binds specifically to HER2/neu expressing tumor cells and localizes hDM to tumor cells, where the enzymatic activity of hDM-C6 MH3B1, but not the wild type enzyme, results in phosphorolysis of the prodrug, 2-fluoro-2''-deoxyadenosine to the cytotoxic drug 2-fluoroadenine (F-Ade) causing inhibition of tumor cell proliferation. Significantly, the toxic small drug diffuses through the cell membrane of HER2/neu expressing cells as well as cells that lack the expression of HER2/neu, causing a bystander effect. F-Ade is toxic to cells irrespective of their growth rate; therefore, both the slowly dividing tumor cells and the non-dividing neighboring stromal cells that support tumor growth should be killed. Analysis of potential novel MHCII binding peptides resulting from fusion of hDM to C6 MH3B1 and the two mutations in hDM, and of the structure of hDM compared to the wild-type enzyme suggests that hDM-C6 MH3B1 should exhibit minimal immunogenicity in humans.

Conclusion

hDM-C6 MH3B1 constitutes a novel human based protein that addresses some of the limitations of ADEPT that currently preclude its successful use in the clinic.     

Catalytic activity of an in vivo tumor targeted anti-CEA scFv::carboxypeptidase G2 fusion protein

Antibody-directed enzyme prodrug therapy (ADEPT) targets an enzyme selectively to a tumor where it converts a relatively non-toxic prodrug to a potent cytotoxic drug. Previous clinical work using antibody-enzyme chemical conjugates has been limited by the moderate efficiency of tumor targeting of these molecules. To address this a recombinant fusion protein composed of MFE-23, an anti-carcinoembryonic antigen (CEA) single chain Fv (scFv) antibody, fused to the amino-terminus of the enzyme carboxypeptidase G2 (CPG2) has been constructed to achieve ADEPT in CEA-producing tumors. MFE-23::CPG2 fusion protein was overexpressed in Escherichia coli and purified using CEA affinity chromatography. Efficacy of MFE-23::CPG2 delivery to tumors in vivo was assessed by measuring catalytic activity after intravenous injection of purified MFE-23::CPG2 into nude mice bearing CEA-positive LS174T human colon adenocarcinoma xenografts. Recombinant MFE-23::CPG2 cleared rapidly from circulation and catalytic activity in extracted tissues showed tumor to plasma ratios of 1.5:1 (6 hr), 10:1 (24 hr), 19:1 (48 hr) and 12:1 (72 hr). (125)I-MFE-23::CPG2 was retained in kidney, liver and spleen but MFE-23::CPG2 catalytic activity was not, resulting in excellent tumor to normal tissue enzyme ratios 48 hr after injection. These were 371:1 (tumor to liver), 450:1 (tumor to lung), 562:1 (tumor to kidney), 1,477:1 (tumor to colon) and 1,618:1 (tumor to spleen). Favorable tumor : normal tissue ratios occurred at early time points when there was still 21% (24 hr) and 9.5% (48 hr) of the injected activity present per gram of tumor tissue. The high tumor concentrations and selective tumor retention of active enzyme delivered by MFE-23::CPG2 establish that this recombinant fusion protein has potential to give improved clinical efficiency for ADEPT.