439 Effect of Ablating CAR and Integrin Binding on the Biodistribution and Cellular Localization of Adenovirus Vectors Following Intravenous or Intraperitoneal Delivery Molecular Therapy �������� ���[.]
CANCER TARGETED GENE THERAPY II conditionally replicating vectors with either Ad.5 or chimeric, nonCAR interacting, short shafted Ad.5/35 fibers It is important to state that Ad.5/35 vectors were ∼3 fold less efficient in transducing our target cell population, HeLa cervical carcinoma cells, than Ad.5 However, human Ad.5 efficiently transduces a variety of mouse tissues, most importantly the liver In contrast, every mouse tissue we analyzed was resistant to transduction with Ad.5/35, including hepatocytes and Kupffer cells We established a two-dimensional in vitromodel of liver metastases by growing nests of HeLa cells surrounded by mouse HepSV40 hepatocytes, which are resistant to infection by Ad.5/35 When HeLa cells were grown alone, Ad.5/35 vectors induced less reporter gene expression (ΔE1) and CPE (conditionally replicating) than their Ad.5 counterparts However, when HeLa cells were grown surrounded by HepSV-40 cells (1:300), this relation reversed, and the HeLa cells in co-cultures infected with Ad.5/35 exhibited higher levels of transgene expression and CPE than with Ad.5 We concluded that avoiding sequestration by hepatocytes resulted in superior tumor cell transduction and lysis Next, we tested whether trapping by non-target tissues influenced tumor cell transduction and lysis in an in vivo liver metastasis model Systemic injection of tumor bearing mice with Ad.5/35 based vectors achieved transgene expression and anti-tumor effects similar to what was observed with Ad.5, despite the lower susceptibility of HeLa cells towards Ad.5/35 Reduced sequestration is a likely explanation for this observation However, the “sequestration effect” was less pronounced in vivo than in the in vitro “metastasis” model We found that the plasma levels of Ad.5/35 vector DNA were significantly higher than Ad.5 during the first 15 minutes after systemic injection However, plasma levels of both vectors quickly dropped by several orders of magnitude within 2h A significant portion of Ad.5/35 vectors therefore seems to be taken up by a yet unidentified compartment, or is degraded Experiments to determine the bio-distribution and fate of short-shafted Ad.5/35 vectors in vivo are ongoing in our lab While the results presented here imply that abolishing the natural tropism might be used to increase not only the specificity, but also the efficiency of a vector, successful targeted gene therapy will depend on better elucidating the pathways of in vivo infection and degradation of adenovirus vectors 438 Development of a Genetically-Modified Adenovirus Vector That Specifically Targets Tumors Via the Bloodstream Following Systemic Administration Masaki Akiyama,1 Peter Roelvink,2 David Einfeld,2 Selva Murugesan,2 Thomas Wickham.2 GVF; 2GV The development of genetically modified adenovirus vectors capable of targeting tumors following systemic administration is an important goal for the treatment of disseminated cancer Achieving this goal requires that the native adenovirus coat protein/receptor interactions are removed and replaced with new tumor-selective ligand/receptor interactions Towards this goal we have created a vector that is ablated for native receptor binding and that additionally contains a tumor-selective peptide motif inserted into the HI loop of fiber This vector, AdL**RTD, contains a non-RGD peptide motif that is specific for the integrin receptor avb6, which is dramatically upregulated in a number to tumors including lung, colon, ovarian and oral cancers AdL**RTD was evaluated for its ability to transduce cell lines expressing high or absent levels of avb6 When compared to AdL**, which is ablated for CAR and integrin binding and lacks a targeting motif, AdL**RTD dramatically increased transduction of H441 lung carcinoma cells which express avb6, as well as high levels CAR and other av integrins However, AdL**RTD did not increase the transduction of cells expressing only avb3/5 Molecular Therapy Vol 7, No 5, May 2003, Part of Parts Copyright © The American Society of Gene Therapy integrins Furthermore, competition studies using the RTD peptide or an anti-avb6 integrin antibody completely blocked transduction of H441 and SCC-25 target cells by AdL**RTD, confirming its target specificity The vector was next tested to determine whether it could target tumors following systemic administration AdL**RTD, or vectors with native tropism (AdL), ablated tropism (AdL**), or ablated tropism plus an avb3/5 integrin targeting motif (AdL**RGD) were administered either intraperitoneally or intravenously to mice bearing subcutaneous H441 tumors followed by analysis of the tumor and liver for transgene expression Intraperitoneal administration of AdL**RTD resulted in the highest levels of transgene expression in the tumor compared to the AdL, AdL**, or AdL**RGD vectors administered either intravenously or intraperitoneally This increased tumor gene transfer was closely correlated with the presence of the targeting peptide and the amount of vector that persisted in the bloodstream at one day postadministration In addition, there was a significant increase in the tumor to liver ratio achieved by AdL**RTD compared to AdL such that the liver and tumor expressed nearly the same levels of transgene The 1:1 tumor/liver expression ratio achieved following intraperitoneal administration of AdL**RTD was 36-fold or 550fold higher, respectively, than the tumor/liver ratios achieved following intraperitoneal or intravenous delivery of AdL These studies are the first to demonstrate the feasibility of targeting tumors through the systemic administration of genetically modified adenovirus vectors containing tumor-specific peptide ligands Stockholders in GenVec, Inc 439 Effect of Ablating CAR and Integrin Binding on the Biodistribution and Cellular Localization of Adenovirus Vectors Following Intravenous or Intraperitoneal Delivery Masaki Akiyama,1 Peter Roelvink,2 David Einfeld,2 Imre Kovesdi,2 C Richter King.2 GVF; 2GV Adenovirus (Ad) vectors are known to efficiently transduce various cell types via the interaction of its fiber coat protein with the Coxsackie-adenovirus receptor (CAR), and via the interaction of the penton base coat protein with integrin receptors In order to create tumor-targeted Ad vectors, we have established a base vector for subsequent redirection to tumor cells by the genetic ablation of native receptor interactions Reporter gene expression was significantly attenuated using a vector ablated for CAR and integrin binding, while high levels of liver and spleen transduction were observed with the conventional (CAR and integrin binding intact) and singly-ablated (integrin binding intact) vectors [Einfeld et al J Virol., 2001, 75, 11284-11291], although other studies have reported that doubly-ablated vectors not show attenuated transduction in vivo To more fully characterization these vectors, bio-distribution in conjunction with pharmacokinetic analysis was performed for conventional, singly-ablated and doubly-ablated vectors at different doses using two routes of administration, e.g., intraperitoneal and intravenous administration Similar to our previous observations, the ablation of CAR- and integrin binding severely reduced liver transduction following intravenous administration at low, intermediate, or high doses, with the most dramatic differences occurring at the high dose In contrast to these results, the singlyablated vector showed unexpectedly high liver transduction compared to either the conventional or doubly-ablated vectors following intraperitoneal administration, at high doses Histological analysis indicated distinctive localizations for the conventional, singly-ablated and doubly-ablated vectors The conventional vector predominantly transduced mesothelial tissue on the outside of the liver while the singly-ablated vector predominantly transduced parenchymal tissue in the liver interior Importantly, the doubly-ablated vector showed S173 CANCER TARGETED GENE THERAPY II minimal transduction of either the mesothelial or parenchymal tissue following either intravenous or intraperitoneal administration Our pharmacokinetics and bio-distribution data support the advantages of ablating both the CAR and integrin interactions in the context of making a base vector for targeted gene therapy Stockholders in GenVec, Inc 440 Anti-Tumor Efficacy and Preclinical Proofof-Concept Following Systemic Administration of an Oncolytic Adenovirus Dependent upon Two Prevalent Alterations in Human Cancer David A Stewart, Patricia C Ryan, John L Jacubczak, Lynda Hawkins, Lori M Clarke, Ying Huang, Sandrina S Phipps, Pam S Shirley, Yelena Skripchenko, Jingping Yang, Ling Xu, Paul L Hallenbeck Genetic Therapy, Inc a Novartis Company, Gaithersburg, MD, United States Treating metastatic cancer with oncolytic adenoviruses will require the development of safe and effective viruses that can be systemically delivered We have constructed an oncolytic adenovirus, OAS403, in which the essential early region genes E1a and E4 are under the control of the tumor-selective promoters E2F-1 and hTERT (human telomerase reverse transcriptase), respectively The E2F-1 promoter should be primarily activated in Rb-pathway defective tumor cells, a prevalent phenotype in most cancer types Cancer cell immortalization is associated with upregulation of telomerase activity in approximately 90% of cancers Therefore, the hTERT promoter should also be active in the majority of cancer indications OAS403 was tested in a panel of in vitro and in vivo assays to assess replication selectivity, safety and efficacy OAS403 showed tumor-selective expression of E1a and E4, tumor-selective cell killing, and tumorselective virus production when tested in vitro on a panel of tumor and non-tumor cell cultures A variety of parameters were measured in vivo following systemic administration in tumor bearing nude mice including hepatotoxicity, body weight, adenoviral genomic copy number by hexon gene PCR, E1a and E4 RNA levels by RT-PCR, and anti-tumor efficacy A single tail vein injection of OAS403 led to significant anti-tumor efficacy and improved survival when used in xenograft tumor models of hepatocellular and prostatic carcinoma Treatment was well tolerated with low hepatoxicity and no significant effects on body weight Both tumor-selective viral replication and early gene expression were demonstrated by monitoring viral genome copies and E1a and E4 expression in liver and tumor tissue In addition, we showed that oncolytic vector treatment in combination with chemotherapy is synergistic for tumor cell killing both in vitro and in vivo These results indicate that this vector may hold promise for the treatment of metastatic cancer via systemic delivery All authors employed by Genetic Therapy, Inc., a Novartis Company 441 Targeting the Cytotoxicity of Fusogenic Membrane Glycoproteins in Gliomas through Protease-Substrate Interaction Cory Allen,1 Cari McDonald,1 Kah-Whye Peng,1 A Gabriela Rosales,2 Stephen J Russell,1 Evanthia Galanis.1,3 Molecular Medicine Program, Mayo Clinic, Rochester, MN; Biostatistics, Mayo Clinic, Rochester, MN; 3Medical Oncology, Mayo Clinic, Rochester, MN Fusogenic membrane glycoproteins (FMG) are potent therapeutic transgenes with potential utility in the gene therapy of gliomas (Galanis E, et al Human Gene Therapy, 2001) Transfection of glioma cell lines with FMG expression constructs results in fusion with massive syncytia formation followed by cytotoxic cell death S174 Nevertheless, FMG transfection can also result in cytopathic effect against normal cell lines such as normal human astrocytes and fibroblasts, and therefore, targeting becomes an important consideration Matrix metalloproteinase overexpression in gliomas constitutes an important effector of human glioma invasion Here we report on use of matrix metalloproteinase (MMP) cleavable linkers to target cytotoxicity of FMG expressing adenoviral vectors against gliomas Replication defective adenoviruses (Ad) were constructed expressing the hyperfusogenic version of the Gibbon Ape Leukemia Virus envelope glycoprotein (GALV) linked to a blocking ligand (CD40 ligand) through either an MMP cleavable linker (Ad M40) or a noncleavable linker (Ad N40) Both viruses also co-expressed GFP via an internal ribosomal entry site The glioma cell lines U87, U118, characterized by zymography and MMP-2 activity assay as high and medium MMP expressors respectively and the MMP negative cell line TE671 were infected with GALV M40 and GALV N40 at different MOIs from 1-30 All cell lines were also characterized for their ability to cleave the specific MMP linker by using HPLC and for CAR expression levels Fusion was quantitated by counting both number and size of syncytia Results: Infection with Ad N40 did not result in fusion or cytotoxic cell death, despite the presence of infection as indicated by GFP positivity Fusion was restored after infection with AdM40 at an MOI as low as to an extent dependent on MMP expression in the specific cell line Use of the MMP inhibitors 1, 10 phenanthroline and N-hydroxy-5.5, dimethyl-piperazine-2-carboxamide completely abolished AdM40 induced fusion while the soybean trypsin inhibitor did not affect it, thus indicating specificity of the observed effect TUNEL assay showed that AdM40 induced cell death was associated with apoptosis Intratumoral treatment of BalbC/nude mice bearing subcutaneous U87 xenografts with AdM40 at a total dose of 1.3 x 1010 pfu resulted in statistically significant tumor regression and prolongation of survival as compared to control animals either treated with AdN40 (p=0.01 and p=0.006 respectively) or untreated animals (p=0.009 and p=0.001 respectively) Our data indicate that AdM40, a replication defective adenovirus expressing the GALV fusogenic glycoprotein attached to a blocking ligand via an MMP cleavable linker, can target the cytotoxicity of GALV in MMP overexpressing glioma lines and xenografts, while maintaining antitumor activity both in vitro and in vivo (Supported by CA8438801) 442 Hypoxia Reduces Adenoviral Replication in Cancer Cells Teona Pipiya,1 Harald Sauthoff,1 Sheila Heitner,1 Shu Chen,1 William N Rom,1 John G Hay.1 Medicine, NYU School of Medicine, New York, NY Infecting the majority of cells within a solid tumor to enable effective therapy is a hurdle for most gene therapy approaches The use of replicating viruses that could continually spread within a tumor from infected to uninfected cells is an attractive prospect However, in clinical trials to date replicating adenoviruses have had limited success Further, in animal models even wild type adenovirus may not fully eliminate xenograft tumors despite the persistence of high titers of virus within the tumor (Harrison et al, 2001, Hum Gene Ther 12, 1323-1332) These findings suggest the presence of barriers for viral replication and spread within the tumor One possible barrier is the hypoxic environment that is present within a tumor, particularly considering the epithelial sites that the adenovirus usually infects are exposed to ambient oxygen concentrations Moreover, virus that is found within persistent xenograft tumors is often observed in close proximity to blood vessels (Sauthoff et al, 2003, Hum Gene Ther In press) To establish if hypoxia could limit adenoviral replication, H1299 and A549 lung carcinoma cells were infected with wild type adenovirus Ad309 (MOI 10) and incubated Molecular Therapy Vol 7, No 5, May 2003, Part of Parts Copyright © The American Society of Gene Therapy ... support the advantages of ablating both the CAR and integrin interactions in the context of making a base vector for targeted gene therapy Stockholders in GenVec, Inc 440 Anti-Tumor Efficacy and. .. GENE THERAPY II minimal transduction of either the mesothelial or parenchymal tissue following either intravenous or intraperitoneal administration Our pharmacokinetics and bio-distribution data... tumor-selective expression of E1a and E4, tumor-selective cell killing, and tumorselective virus production when tested in vitro on a panel of tumor and non-tumor cell cultures A variety of parameters were