1116 Liver Untargeted and Mitigation of Adenovirus Induced Hepatic Toxicity by Pretreatment with Trimeric Soluble Car Molecular Therapy �������� ��� ���� ���������������� �������� ���� ������®��������[.]
CANCER TARGETED GENE THERAPY III 1115 Treatment Resistant Tumour Eradication Using Adenovirus Mediated Oxygen Regulated GDEPT Rachel L Cowen,1 Kaye J Williams,1 Naomi Chadderton,1 Mohammed Jaffar,1 Brian A Telfer,1 Ian J Stratford.1 Pharmacy, University of Manchester, Manchester, United Kingdom The refractive nature of hypoxic tumour cells to radiation and chemotherapy makes them an important target for new anti-cancer approaches To this end we have developed a GDEPT strategy in which the prodrug, expression of the activating enzyme, and ultimately the delivery vehicle itself, are all regulated by oxygen We use bioreductive drugs as prodrugs as they are ‘switched on’ at low oxygen to cytotoxic free radical species As the selectivity of bioreductive drugs also depends upon the presence of elevated levels of cellular reductase enzymes we achieve this using adenoviral mediated reductase gene delivery Further, we are able to restrict reductase enzyme over-expression, therefore drug toxicity, to hypoxic cells by employing hypoxia responsive promoters within the therapeutic cassette Initially we used adenovirus to constitutively express the reductase enzyme human P450 reductase (P450R) within MDA 468 human breast tumour xenografts Untransduced tumours were nonresponsive (Treated/control [T/C] = 105%) to the clinically used bioreductive drug, mitomycin C (MMC) In contrast, MDA468 tumours transduced with the adenovirus (5 x 108 pfu) were sensitised to MMC treatment (T/C = 50.5%, p=0.008) However, MMC is poorly selective for hypoxic cells compared to the phase II/III bioreductive drug Tirapazamine (TPZ) and to our lead indolequinone drug 5-aziridinyl-3-hydroxymethyl-1methylindole-4,7-dione (prodrug 629) TPZ and 629 have also been shown to exhibit selectivity for activation by P450R Both drugs have been partnered with Adenovirus LDH-A P450R encoding for hypoxia induced P450R expression driven by a hypoxia responsive promoter derived from lactate dehydrogenase A Intra-tumoural (i.t.) injection with this virus results in focally high levels of reductase expression which co-localise with the hypoxic marker pimonidazole Again using the MDA468 model and the same dosing regime used with MMC we sensitise tumours to prodrug 629 by pre-i.t injection with Ad CMV P450R (T/C = 69%) However, using the hypoxia specific vector Ad LDH-A P450R this tumour response is significantly enhanced (T/C = 53%) Further, when this approach is combined with 10Gy radiation we are able to completely control the hypoxic tumour fraction and achieve 100% tumour regression (Figure 1) We are now evaluating this approach in a faster growing HT1080 (human fibrosarcoma) xenograft model Like the MDA468 model these tumours are refractive to prodrug 629 and TPZ alone and are not controlled by 10Gy radiation Also pre-transduction of these tumours with Ad LDH-A P450R does not sensitise these tumours to 629 or to TPZ However, when 10Gy radiation is included in the regimen the combined Ad LDH-A P450R/10Gy/629 treatment significantly enhances tumour response (T/C = 30.9%) and Ad LDHA P450R/10Gy/TPZ results in complete regression in 70% of treated tumours S430 1116 Liver Untargeted and Mitigation of Adenovirus Induced Hepatic Toxicity by Pretreatment with Trimeric Soluble Car Waleed O Arafat,1,2,3 Donald J Buchsbaum,3 Scott Chiz,3 Igor Dmitriev,1,2 David T Curiel.1,2 Division of Human Gene Therapy, Departments of Medicine, Pathology, and Surgery, University of Alabama at Birmingham, Birmingham, AL, United States; 2Gene Therapy Center, University of Alabama at Birmingham, Birmingham, AL, United States; 3Department of Radiation Oncology, University of Alabama at Birmingham, Birmingham, AL, United States Potential adenovirus (Ad) leakage after intratumoral (IT) injection is a major problem in human clinical trials Liver has been the main organ reported to be affected Ad infection is mediated by binding of the knob domain of the fiber protein to a host cell primary receptor, Coxsackie/Adenovirus receptor (CAR) The extracellular domain of CAR is sufficient for virus attachment and infection We hypothesized that high levels of soluble CAR in the blood stream could bind to systemically leaked virus and ablate its systemic toxic effects We have constructed a two recombinant Ad encoding a secretory form of monomeric and trimeric CAR ectodomain (Ads m CAR and Ad s T CAR respectively) We validated our viruses for secretion of CAR by western blot and ELISA To test our hypothesis, we intravenously injected AdsCAR or a control virus (AdGFP) at a dose of 5X1010 PFU into mice harboring subcutaneous nodules of DU145 prostate cancer cells One day later, replicative Ad encoding luciferase (AdLuc) was injected IT or IV at a dose of 5X1010 PFU or 5X107 respectively Immunohistochemistry (IH) of liver sections for s CAR was performed DNA and RNA from various organs was collected and analyzed for the presence of Adenovirus E1 gene and transgene Luc by quantitative PCR and RT-PCR Total protein from various organs was also extracted for luciferase analysis Liver toxicity was determined by morphological analysis of liver sections with H&E stain Our two viruses produced a robust quantity of s CAR which was detected pericellularly by IH of liver in AdsCAR pretreated animals only Adenovirus E1 gene was detected at a lower copy no./ng of DNA in the liver of animals pretreated with AdsCAR than those pretreated with the control virus A similar ratio was observed in cardiac, kidney and lung tissue However, spleen showed a higher copy no of E1 in animals treated with AdsCAR that AdGFP Luc assay showed two-fold reduction of Luc protein detected in animals pretreated with smCAR than non-treated animal Most importantly, the liver of animals pretreated with trimeric CAR showed three-fold reduction in Luc expression than the msCAR and five-fold more reduction than the control virus Additionally, liver toxicity as seen by H&E staining in ,form of inflammatory reaction hepatocyte necrosis and lymphocyte Molecular Therapy Vol 7, No 5, May 2003, Part of Parts Copyright ® The American Society of Gene Therapy CANCER TARGETED GENE THERAPY III infiltration, was significantly lower in animals pretreated with AdsCAR than in animals pretreated with control virus High levels of systemically administered stCAR provide a mean to ablate adenovirus-mediated toxicity in vivo 1117 Incorporation of Polypeptide Ligands into the Fiber Knob Protein of Ad5 To Allow Targeting to Receptor Molecules That Are Upregulated in Human Pancreatic Cancer Michael A van Geer,1 Piter J Bosma,1 Kitty N de Groot,1 Koert F Kuhlmann,2 Dirk J Gouma,2 John G Wesseling.1 Lab of Experimental Hepatology, AMC Liver Center, University of Amsterdam, Amsterdam, Netherlands; 2Dept of Surgery, Academic Medical Center, University of Amsterdam, Amsterdam, Netherlands Pancreatic cancer is the 5th leading cause of cancer death in the Western world with no effective treatment Adenoviral (Ad) vectors have been studied for gene delivery into human pancreatic cancer, but they revealed a limited transduction efficiency, due to the low amount of coxsackie- and adenovirus receptor (CAR) molecules present on these tumor cells Insertion of the RGD-4C (Arg-GlyAsp-Cys) peptide into the HI-loop of the fiber knob of Ad5 has revealed expanded tropism by which this motif targets to the αv integrins present on pancreatic tumor cells Since RGD-targeting is not truly tumor-specific, i.e normal cells also express αv integrins, we sought for other peptides to be inserted into the the fiber knob Insertion in the HI-loop has the advantage that it should not affect fiber trimerization and leaves the peptides exposed on the outside of the virion, while maintaining part of the native virus tropism Therefore, we selected polypeptides that would serve as targeting ligands to receptors that are elevated expressed on pancreatic tumor cells The choice of the receptors was based on the binding of antibodies to the receptors on pancreatic carcinoma cells in vitro as revealed by flow cytometry studies It was also based on peptides that were already identified by the phage display approach in other tumor cells, combined with the identification of genes that are differentially expressed in pancreatic cancer We have selected five pepides from genes encoding receptors that are expressed on pancreatic tumor cells: 1) a 15 a.a Thomsen-Friedenreich antigenspecific peptide P-30 that binds to the T antigen, 2) two ephrin peptides (12 a.a each) that bind to the EphA2 receptor, 3) a peptide (12 a.a.) that binds to the Vascular Endothelial Growth Factor (VEGF)-receptor KDR (kinase domain receptor) and 4) a neurotensin-peptide (14 a.a.) that binds to the neurotensin receptor NTR3 The peptide sequences were assembled with oligonucleotides, annealed in pairs, and cloned into the unique BstB1 and/or Cla1 site that were introduced into the HI-loop of the fiber gene of vector plasmid pAdHM15, containing the whole Ad5 genome, with the CMV-eGFP-poly(A) cassette cloned into the deleted E1 region The fiber modified vector plasmids were digested with Pac1 and transfected into 293 cells The viral constructs which induced cytopathic effect on 293 cells were grown to viral stocks and were checked for incorporation of the peptide sequences into the HIloop by PCR Subsequently, human pancreatic tumor cell lines were infected with these fiber modified AdGFP viruses and their transduction efficiency was compared with wild type AdGFP and with AdGFP-RGD-4C using fluorescence microscopy and FACs analysis A number of the fiber modified AdGFP viruses revealed an enhanced gene transfer in human pancreatic carcinoma cells as compared to wt AdGFP We conclude that Ad vectors with polypeptide ligands cloned into the HI-loop of the fiber knob and targeted to receptors that are highly expressed on pancreatic tumor cells will be useful for gene therapy of pancreatic cancer Molecular Therapy Vol 7, No 5, May 2003, Part of Parts Copyright ® The American Society of Gene Therapy 1118 Development of a Genetically-Modified Adenovirus Vector That Improves the Efficiency and Specificity of Gene Transfer for Peritoneal Cancers Selva Murugesan,1 Masaki Akiyama,2 Peter Roelvink,1 David Einfeld,1 C Richter King.1 GV; 2GVF The development of genetically modified adenovirus vectors capable of targeting tumors located in the peritoneal cavity or elsewhere in the body is an important goal in cancer gene therapy Achieving this goal requires that the native adenovirus coat protein/ receptor interactions are removed and replaced with new tumorselective ligand/receptor interactions Towards this goal we have created a vector, AdL**RGD, that is ablated for native receptor binding and contains the avb3/5-specific peptide motif, RGD4C, in the HI loop of fiber The AdL**RGD vector increased the transduction of avb3/5 integrin-positive, but not avb3/5 integrinnegative cells, compared to a similar vector without the RGD motif (AdL**) In addition, AdL**RGD significantly increased gene transfer to CAR-deficient cells compared to the native tropism, AdL vector The AdL**RGD vector was next compared to a panel of control and tropism-modified vectors to determine whether it could reduce the transduction of non-target organs in vivo AdL**RGD, together with AdL (unmodified tropism), AdL+ (ablated for integrin binding), AdL* (ablated for CAR binding), or AdL** (ablated for CAR plus integrin binding) were administered intraperitoneally to mice followed by analysis of transgene expression in the liver, kidney and spleen The results of this analysis showed that transduction of the liver, kidney, and spleen following intraperitoneal administration is strongly CAR-dependent and weakly integrin dependent In addition, the AdL**RGD vector displayed significant reductions of 85-, 139-, and 18-fold in the liver, kidney, and spleen, respectively compared to AdL Based upon the above results using luciferase-containing vectors, a similar panel of vectors was constructed that expressed the human tumor necrosis factor (hTNF) gene under control of a CMV-enhanced promoter The individual vectors were native tropism (AdhTNF), CAR and integrin ablated (AdTNF**), or CAR and integrin ablated plus the RGD-4C motif in the fiber (AdTNF**RGD) Similar to the luciferase-expressing vector panel, both the AdTNF** and AdTNF**RGD vectors, demonstrated 237-fold and 134-fold reductions in transgene expression, respectively, compared to AdTNF as measured by serum TNF levels following intraperitoneal administration In addition, these observed reductions in serum TNF levels correlated with decreased toxicity as measured by body weight loss These results suggest that receptor-based targeting of adenovirus vectors to tumor-selective receptors has the potential to significantly improve both the efficiency and specificity of gene transfer for the treatment of cancers in the peritoneal cavity Stockholders in GenVec, Inc S431 ... pretreated with AdsCAR than in animals pretreated with control virus High levels of systemically administered stCAR provide a mean to ablate adenovirus- mediated toxicity in vivo 1117 Incorporation of. .. of coxsackie- and adenovirus receptor (CAR) molecules present on these tumor cells Insertion of the RGD-4C (Arg-GlyAsp-Cys) peptide into the HI-loop of the fiber knob of Ad5 has revealed expanded... Wesseling.1 Lab of Experimental Hepatology, AMC Liver Center, University of Amsterdam, Amsterdam, Netherlands; 2Dept of Surgery, Academic Medical Center, University of Amsterdam, Amsterdam, Netherlands