500 replication competent retrovirus vector mediated suicide gene therapy achieves significant therapeutic efficacy against human malignant mesothelioma xenografts

500 Replication Competent Retrovirus Vector Mediated Suicide Gene Therapy Achieves Significant Therapeutic Efficacy Against Human Malignant Mesothelioma Xenografts Molecular Therapy Volume 18, Supplem[.]

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CANCER-ONCOLYTIC VIRUSES II

interferon production in breast cancer cells, colon cancer cells, as well

as  broblast 3T3 cell, whereas infection of cancer cells by VSV delta

51 dismissed if these cells were pretreated by IFNa Using HDAC

inhibitors, TSA, Valproic Acid (VPA) or Phenylbutyrate (4PB), we

showed that all tested three HDAC inhibitors, can partially rescue

viral infection of and replication in both murine and human breast

cancer cells treated by IFNa Further, our results showed that TSA

or VPA can down-regulate the expression of interferon-regulatory

factor, IFR-7, by western blot assay, in a dose dependent manner in

breast cancer cells More importantly, All SCID/NOD mice in the

experiment are well tolerant to combination of VSVdel51 and TSA,

and VSVdel51 can only be detected in 4T1 tumors rather than in other

tested organs even more than two weeks after last viral injection

Collectively, our results suggest that combination of oncolytic

VSVdel51 and HDAC inhibitors have signi cant potential for the

treatment of breast cancer

496 Combination Oncolytic Virotherapy

Timothy Kottke,1 Jill Thompson,1 Konstantin Doronin,1 Sonia

Guedan Carrio,2 William Wold,3 Ramon Alemany,2 Richard Vile.1

1 Molecular Medicine, Infectious Diseases and Immunology, Mayo

Clinic, Rochester, MN; 2 Laboratori de Recerca Traslacional,

Institut Catala d’Oncologia, Barcelona, Spain; 3 Molecular

Microbiology and Immunology, St Louis University School of

Medicine, St Louis, MO.

A major barrier to replication and concomitant lysis of tumors by

oncolytic viruses is the induction of potent innate immune responses

to intratumoral viral infection In this respect, many large viruses

encode proteins which antagonize cells’ ability to induce, and/

or respond to, the Type I interferon response to infection, thereby

extending the window of opportunity for productive viral infection

and propagation However, smaller viruses encode fewer genes with

which to interfere with the cellular anti viral response and usually

have to replicate rapidly to evade the anti-viral response before it

extinguishes infection Therefore, we hypothesized that it may be

possible to combine two different oncolytic viruses for more effective

tumor treatment The  rst virus would express genes which

pre-condition the tumor micro-environment to antagonize the generation

of an anti-viral state; this would establish a more conducive,

pro-viral environment for replication of the second virus To test this

hypothesis, we co-infected a variety of tumor cells with replication

competent Adenovirus (a large DNA virus encoding several genes

which in uence both innate and adaptive anti viral immune responses)

and VSV (a smaller RNA oncolytic virus with greatly reduced coding

capacity for anti viral genes) In vitro, protective levels of IFN-α

prevented oncolysis of interferon-responsive cells (MDA468) by

adenovirus or VSV (mimicking infection of ‘normal’ cells) However,

pre-infection by adenovirus 48 hours prior to VSV signi cantly

enhanced VSV replication in, and lysis of, the cells Deletion of

either the VA or E4ORF3 genes from the pre-conditioning Adenovirus

largely abrogated this pre-conditioning effect In contrast, adenoviral

pre-infection of tumor cells, in which no interferon response is

operational, led to complete lysis of target cells by VSV at moi of

adenoviral/VSV infection which were themselves ineffective at

killing tumor cells Moreover, the AD-VAdel and Ad-E4ORF3 deleted

viruses were as effective in pre-conditioning for VSV lysis as was the

wild type virus in tumor, IFN-non-responsive cells Finally, carefully

timed intra-tumoral injection of HCT116 tumors with AdVAdel,

followed by VSV, cured over 50% of mice of subcutaneous HCT116

tumors compared to no cures in mice treated with Ad or VSV alone or

with VSV followed by Ad These data show that it will be possible to

combine oncolytic viruses for tumor treatment to improve the ef cacy

of individual viruses Safety concerns regarding the recombination of

such viruses need to be intensively addressed, although careful choice

of the combination partners should minimize such risks

497 Conditionally Replicative Adenovirus with

an Adenoviral Major Late Promoter-Driven Imaging

Cassette Predicts In Vivo Anti-Tumor Effect

Julia Davydova,1 Tatyana Gavrikova,3 Eric J Brown,1 Xianghua Luo,2 David T Curiel,3 Selwyn M Vickers,1 Masato Yamamoto.1

1 Surgery, University of Minnesota, Minneapolis, MN; 2 Division of Biostatistics, University of Minnesota, Minneapolis, MN; 3 Division

of Human Gene Therapy, University of Alabama, Birmingham, AL; 4 Gastroenterological Surgery, Yokohama City University,

Yokohama, Japan.

In vivo monitoring of conditionally-replicative adenovirus (CRAd)

replication and assessment of its correlation to CRAd biological effects are necessary for the clinical development of gene therapy

Noninvasive bioimaging may provide a convenient and reliable

way to monitor in vivo viral replication and evaluate oncolytic viral

therapy ef ciency in patients However, correlating CRAd replication imaging with CRAd therapeutic effect has not yet been attempted

In this work, we apply CRAd as an imaging tool to monitor viral replication and oncolytic functionality, but also to predict the  nal antitumor effect based on earlier time point analysis We established dynamic imaging of viral replication employing a Cox2

promoter-selective CRAd with a replication-dependent luciferase (Luc) cassette

In that vector structure, the reporter gene placed in the E3 region is controlled by major late promoter, and enhanced Ad oncolysis is

mediated by overexpression of the adenoviral death protein In vitro

Luc expression showed correlation with viral replication and cytolytic

effect In vivo, athymic mice bearing A549 (Cox2-positive) and A431

(Cox2-negative)  ank tumors received intratumoral injections with Cox2CRAdE3ADPLuc and wild type replicative WtE3ADPLuc

Day 6 imaging of bioluminescence following vector administration showed a signi cant correlation with tumor volumes measured at day

36 Speci cally, the mice which showed higher Luc peaks at day 6 after treatment had remarkable tumor shrinkage at day 36 Conversely, the mice exhibiting lower levels of bioluminescence at earlier time points demonstrated a minimal therapeutic response To prove the correlation between produced bioluminescence and therapeutic effect

we performed the non-parametric Spearman’s correlation analysis which demonstrated signi cant negative association between the Luc peaks at day 6 and the tumor volumes at day 36 within each treated group and overall combined samples The correlation between the bioluminescent signal and tumor volumes was further investigated

by a linear regression model which also con rmed the signi cant relationship between early time point imaging and therapeutic outcome Of note, the anti-tumor effect based on tumor volume measurement became signi cant 18 days after viral administration, suggesting that evaluating vector tumoricidal effect based on tumor size at early time points is not accurate This indicates that early time point imaging, either bioluminescent or by another modality (e.g SSTR or hNIS transgenes), may predict therapeutic outcome

Thus, our system could detect viral replication and predict therapeutic

outcome in vivo based on early imaging Further development of this approach should provide mechanistic insight into CRAd function in

vivo and an early indication of the tumoricidal effect of oncolytic

viral therapy

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CANCER-ONCOLYTIC VIRUSES II

498 Susceptibility of Neuroblastoma Primary Tumor-Initiating Cells to Oncolytic Herpes Simplex Virus Is Determined by Nectin-1 Expression

Pin-Yi Wang,1 Mark A Currier,1 Margaret H Collins,2 Betsy A

DiPasquale,2 Loen Hansford,3 David Kaplan,3,4 Sean Lawler,5 E

Antonio Chiocca,5 Timothy P Cripe.1

1 Division of Hematology/Oncology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH; 2 Division of Pathology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH;

3 Molecular Genetics, University of Toronto, Toronto, ON, Canada;

4 Cell Biology, The Hospital for Sick Children, Toronto, ON, Canada; 5 Department of Neurological Surgery, The Ohio State University, Columbus, OH.

Neuroblastoma is the most common extracranial solid tumor in children Although patients with high-risk disease can usually achieve remission with conventional therapies, disease relapse is common and long-term survival is less than 40% Bone marrow metastases are enriched for tumor-initiating cells (TICs; Hansford et al., Cancer Research 67:11234, 2007), consistent with the cancer stem cell hypothesis We previously showed that neuroblastoma cell lines are killed by oncolytic Herpes simplex virus (oHSV) infection (Parikh

et al., Ped Blood Cancer 44:469, 2005) and sought to determine the susceptibility of neuroblatoma TICs to oHSV While NB12 and NB122R cells were relatively susceptible to HSV transduction and cytolysis, NB88R2 cells were resistant Resistance correlated with low expression of the known major HSV-1 receptor, nectin-1, and could be partially reversed by exogenous nectin-1 expression

Xenograft tumors derived from these TICs retained  delity of nectin-1 expression: tumors derived from NB12 cells were positive while those from NB88R2 were negative Although direct intratumoral oHSV injection ef cacy studies are still underway to con rm a differential response to virus therapy, our data suggest that nectin-1 expression may be a predictor of response to oHSV Using a tissue microarray

to screen ∼90 cases of primary neuroblastoma, we found a majority

of cases expressed nectin-1, suggesting that overall neuroblastoma

is a reasonable target disease for oHSV-based therapy Interestingly, numerous primary glioblastoma neurosphere cultures were all found

to be highly susceptible to oHSV infection but showed very low or absent nectin-1 expression These cells were positive for nectin-2 and nectin-3, suggesting that HSV-1 might utilize different cellular entry receptors to infect peripheral versus central nervous system tumors Our novel  ndings raise questions about HSV-1 receptor expression on cancer cells that may represent barriers to virus spread within a tumor and may be predictive of susceptibility to oncolytic HSV therapy

499 Activity of Oncolytic Measles Virus Strains Against Brain Tumor Stem Cells

Cory Allen,1 Mark A Schroeder,1 Jann N Sarkaria,1 Mark J

Federspiel,1 Stephen J Russell,1 Evanthia Galanis.1

1 Mayo Clinic College of Medicine, Rochester, MN.

Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults and has a dismal prognosis despite multimodality treatment Given the resistance of brain tumor stem cells (BTSC)

to chemotherapy and radiation therapy, their eradication is thought

to be essential for long term glioma remission We have previously demonstrated that measles virus (MV) derivatives have signi cant activity against glioma lines and orthotopic xenografts and are currently being tested in a phase I trial in recurrent GBM patients We sought to evaluate the antitumor activity of MV derivatives against GBM BTSC METHODS/RESULTS: Primary GBM xenografts, passaged in the  anks of nude mice, were excised, mechanically disrupted with a sterile blade and grown at 37°C in a humidi ed environment containing 5% CO2 in NeuroCult media (Stemcell

Technologies, Vancouver, BC, CA) GBM6, GBM12 and GBM44 neurospheres were expanded in this media and subsequently half

of each were further passaged in DMEM containing 10% FBS to serve as differentiated controls These were further examined for the expression of the BTSC markers CD133, Nestin and ATF-5, expression of the differention markers GFAP and beta -III-tubulin and expression of the MV receptor CD46, by immuno uorescence Cytopathic effect (CPE) of MV against both BTSC and differentiated controls was examined in vitro using trypan blue exclusion assays Cells were infected with the MV strains MV-NIS or MV-CEA (MOI 0.1 and 1) and the total number of surviving cells was counted at days 2, 4, 6, 8, &16 post infection and compared against uninfected controls BTSC expressed high levels of the MV receptor CD46 Both BTSC and corresponding differentiated cells were effectively killed at a comparable rate even at low multiplicity of infection (MOI 0.1) One-step viral growth curves showed similar growth kinetics of oncolytic MV strains in BTSC and their differentiated counterpart

To examine the impact of MV infection on BTSC survival in vivo, GBM 44 BTSC were exposed to MV at an MOI of 10 in vitro and subsequently implanted into the right caudate nucleus of nude mice with uninfected BTSC serving as controls A signi cant prolongation

of survival in mice implanted with infected BTSC was observed (p=0.0483) In addition, in vivo therapy experiments were performed

in GBM6 and GBM12 BTSC derived orthotopic xenografts in nude mice Signi cant prolongation of survival was observed in animals treated orthotopically with MV (1e6 and 1.8e6 respectively) for both GBM6 and GBM12 BTSC xenografts as compared to UV inactivated virus control treated animals (GBM6 p=0.0021, GBM12 p=0.0416) CONCLUSION: Measles virus derivatives have signi cant antitumor activity against brain tumor derived stem cells in vitro and in vivo Supported by the Mayo Brain SPORE CA 108961

500 Replication-Competent Retrovirus Vector-Mediated Suicide Gene Therapy Achieves Signi cant Therapeutic Ef cacy Against Human Malignant Mesothelioma Xenografts

Yoshiko Kawasaki,1 Norie Yamaoka,1 Nobuyuki Terada,2 Haruki Okamura,1 Noriyuki Kasahara,3 Shuji Kubo.1

1 Laboratory of Host Defenses, Institute for Advanced Medical Sciences, Hyogo College of Medicine, Nishinomiya, Hyogo, Japan; 2 Department of Pathology, Hyogo College of Medicine, Nishinomiya, Hyogo, Japan; 3 Department of Medicine, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA.

Purpose: Replication-competent retrovirus (RCR) vectors have

been shown to achieve signi cantly enhanced tumor transduction ef ciency and therapeutic ef cacy in various cancer models We and others have previously engineered RCR vectors for highly ef cient delivery of suicide genes, and as the virus is intrinsically incapable of infecting post-mitotic normal cells, retrovirus spread after intratumoral injection is highly restricted to tumor tissue, particularly in immunocompetent hosts In the present study, we hypothesized that RCR vector-mediated suicide gene therapy could

be effectively applied to the treatment of malignant mesothelioma, a highly aggressive tumor with poor prognosis

Methods: To evaluate the utility and ef ciency of RCR vectors for

gene delivery to mesothelioma cells, RCR-GFP vector, expressing the green  uorescent protein (GFP) marker gene, was  rst tested

on a panel of human malignant mesothelioma and non-malignant transformed mesothelial cell lines in vitro Transduction ef ciency and replicative spread of the RCR vector over time was monitored by

 ow cytometry and in vivo imaging Next, to evaluate the potential

of RCR vector-mediated suicide gene therapy for this malignancy,

we employed RCR-yCD, expressing the yeast cytosine deaminase (yCD) suicide gene Following administration of the prodrug

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CANCER-ONCOLYTIC VIRUSES II

5- uorocytosine (5FC), cytotoxicity against RCR-yCD infected

malignant mesothelioma cells was assessed in vitro by Alamar blue

assay, and tumor growth inhibition effects in vivo were assessed in

both subcutaneous xenograft tumor and disseminated peritoneal tumor

models of malignant mesothelioma

Results: Even at multiplicities of infection (MOI) as low as

0.01, RCR vectors successfully infected and ef ciently replicated

in human malignant mesothelioma cell lines, as compared to

non-malignant transformed mesothelial cells In mice with pre-established

subcutaneous tumor xenografts, the RCR-GFP showed robust

spread throughout entire tumor masses by Day 12 after intratumoral

administration of 1 x 104 total infectious units per 100 µl inoculum

Notably, no RCR infection was detectable in adjacent normal

tissue RCR-yCD showed ef cient transmission of the suicide gene

associated with replicative spread of the virus, resulting in ef cient

killing of malignant mesothelioma cells in a 5FC-dose dependent

manner in vitro After intratumoral injection of RCR-yCD followed

by intraperitoneal administration of 5FC prodrug, RCR

vector-mediated suicide gene therapy achieved signi cant inhibition of

subcutaneous tumor growth, and signi cantly prolonged survival in

the disseminated peritoneal model of malignant mesothelioma

Conclusions: These data indicate that RCR vector-mediated suicide

gene therapy may represent a highly useful new treatment strategy

for malignant mesothelioma

501 Oncolytic Adenovirus with Somatostatin

Motif for Selective Infection of Neuroendocrine

Tumors

Di Yu,1 Justyna Leja,1 Berith Nilsson,1 Magnus Essand.1

1 Clinical Immunology, Uppsala University, Uppsala, Sweden.

We have produced an oncolytic serotype 5 adenovirus,

Ad[CgA-E1A-miR122], where the human chromogranin A (CgA) promoter and

miR122 target sequences control E1A gene expression It selectively

replicates in and kills neuroendocrine cells, including neuroblastoma

and carcinoid cells Neuroendocrine tumors (NETs) contain a high

density of homogeneously distributed somatostatin receptors (SSTR)

on their surface and somatostatin analogues, such as octreotide, are

used for tumor imaging and treatment In order to target SSTRs and

increase the infectious capacity for NETs, we have introduced a cyclic

loop with four amino acids (FWKT) from octreotide into either the

HI-loop of the  ber knob or the hexon hypervariable region (HVR)

5 of the capsid We investigate binding properties of GFP-expressing

adenoviral vectors with FWKT motif in the  ber knob

Ad[CMV-GFP]HIFWKT and HVR-5 region Ad[CMV-GFP]HVR5FWKT The

vectors were evaluated in various NET cell lines as well as in freshly

isolated carcinoid cells and normal hepatocytes Both Ad[CMV-GFP]

HIFWKT and Ad[CMV-GFP]HVR5FWKT infect SSTR-positive,

CAR-negative neuroendocrine tumor cells with greater ef cacy

than Ad[CMV-GFP] Furthermore, Ad[CMV-GFP]HVR5FWKT can

detarget factor X binding and neutralization antibody binding to the

virus capsid and hopefully prolong the systematic circulation time

We conclude that modi cation of the capsid with the FWKT motif

can be bene cial for adenovirus-based NET therapy

502 Silencing a p53 Inhibitor in Cancer Cells

Increases Killing Potency of Oncolytic Adenovirus

Christie Vermeulen,1 Nikki Tol,1 Winald R Gerritsen,1 Victor W

van Beusechem.1

1 Dept Medical Oncology, VU University Medical Center,

Amsterdam, Netherlands.

Virotherapy of cancer using oncolytic adenoviruses has shown

promise in both preclinical and clinical settings The potency of

oncolytic adenoviruses depends on their replication ef ciency in

cancer cells and their capacity to destroy these cells by oncolysis

Previously, we found that the oncolytic potency of adenoviruses was enhanced by expressing tumor suppressor p53 from the virus genome

Not unexpectedly, the effect was minimal in cancer cells expressing high levels of the p53 negative regulator human double minute (HDM2) By abrogating the interaction between HDM2 and p53 the oncolytic adenovirus potency was elevated This suggested that oncolytic adenoviruses could be made more effective by suppressing p53 inhibitor expression in cancer cells We envision that this could

be done by incorporating a gene silencing cassette into the oncolytic adenovirus genome To develop such next generation viruses, the p53 inhibitor expression pro le in cancer cells should be determined and it should be con rmed that silencing these inhibitors increases oncolytic potency In the present study, we screened derivatives of U2OS and A549 cancer cells carrying a p53 reporter construct for 18 putative p53 inhibitors using siRNA In U2OS cells, several molecules including HDM2 were found to inhibit p53 activity In contrast, in A549 cells considerable p53 activation was observed only upon silencing the SYVN1 gene SYVN1 encodes synoviolin, an ER-resident ubiquitin ligase that is highly expressed in rheumatoid arthritis synoviocytes

Interestingly, functional screening for inhibition of adenovirus-induced cell death by the 18 putative p53 inhibitors in A549 cells also revealed only SYVN1 Using three independent cell viability assays (i.e., CellTiter-Blue conversion, BCA protein assay and microscopic cell counting), we found that silencing SYVN1 reproducibly and selectively increased adenovirus killing potency Adenovirus late gene expression, as tested using a virus with endogenous MLP-driven GFP expression, was not signi cantly affected Our results establish that SYVN1 silencing empowers adenovirus-mediated killing of A549 cells without affecting early events in the virus life cycle They also show that endogenous p53 reactivation in cancer cells correlates with oncolytic adenovirus potency Therefore, our  ndings suggest that knowledge on p53 inhibitor expression in cancer could be exploited

to develop a new class of potent oncolytic adenoviruses

503 Safety of Intravenous Ad5-Mda-7, a Potential Anti-Metastatic Agent in Gynecologic Cancer

Kenneth H Kim,1 Meredith A Preuss,1 Minghui Wang,1 Anand C

Annan,1 Justin A Barnes,1 Devanand Sarkar,2 Steven Grant,2 Paul Dent,2 Paul B Fisher,2 Ronald D Alvarez,1 David T Curiel.1

1 University of Alabama, Birmingham, AL; 2 Virginia Commonwealth University, Richmond, VA.

Objective: Mda-7 is a cytokine protein with many antitumoral

properties that is highly conserved across multiple species; its progressive loss of expression has been shown to correlate with cancer progression Preclinical trials involving its use within an adenoviral viral vector Ad5-mda-7, have shown cancer-cell-selective apoptosis induction, and enhancement of cancer-selective cytotoxicity paired with adjuvant therapy in various solid tumors This study evaluated safety pro le after IV injection of AdCMVmda-7, and characterizes its expression and secretion from the liver, in anticipation of ongoing

evaluation as an anti-metastasis agent Methods: Two cohorts of mice

were treated with doses of 2x10^10 vp/mL of either Ad5-mda-7 (5 mice/cohort) or Ad-CMV.CEA (3 mice/cohort) via tail vein injection, while a third control group (2 mice/cohort) remained uninjected On study days 0, 1, 3, 7, 14, and 21, blood samples were taken from each cohort of mice for chemistry panel analysis Animals were then euthanized and the following organs were removed from each animal for histopathological examination Immunohistochemistry was performed to evaluate mda-7 and CEA tissue expression in

harvested organs Results: On immunohistochemistry analysis,

mda-7 expression within the liver was noted to increase, peaking on day

3 and then returning to normal Similar mda-7 expression patterns were also noted within the kidney, also returning to normal after day

3 There was minimal to no mda-7 expression in other organs At the

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CANCER-ONCOLYTIC VIRUSES II

time of necropsy, no gross abnormalities were noted at any of the time points, and there were no microscopic abnormalities were noted

in any of the tissues In the serum analysis, comparison between the mda-7 and the CMV-CEA cohorts, the only clinically signi cant difference noted in the levels of alanine aminotransferase (ALT) and alkaline phosphatase (ALP) These values peaked on day 3, and then returned to baseline levels by the end of the study The only other signi cant difference was elevated amylase levels in mda-7 versus CMV-CEA (528 vs 428, p=0.04) Of note, there were no signi cant differences between any of the groups, indicating normalization over

time Conclusions: Ad5-mda7 has an acceptable safety pro le with

IV administration Ongoing studies involving metastatic challenge are currently underway to evaluate its role in preventing metastasis

504 Characterization of a Novel Dual-Lock Oncolytic Cadvex-Vector That Combines Ef cient Prostate-Speci c Replication with Cytokine Expression

Brian D Hoel,1 Min Luo,2 David A Einfeld,2 Danher Wang,2 John

Y Dong.2

1 Department of Microbiology & Immunology, Medical University

of South Carolina, Charleston, SC; 2 GenPhar, Inc., Mount Pleasant, SC.

Prostate cancer remains the second leading cause of cancer death among American men and advanced metastatic disease remains incurable Therefore, there is a great need to develop alternative treatment strategies to improve these outcomes One approach has been exploration of virolytic approaches that target cancer cells

We have developed a novel strategy that combines a conditionally-replicative adenovirus (CRAd) vector with an immunotherapy approach We propose to advance this CAdVex vector into clinical testing for prostate cancer

The vector design employs a “dual-lock” mechanism to control expression of both the E1 and E4orf6 genes necessary for replication

We have found that using the probasin promoter to control expression

of these genes results in enhanced replication that is speci c for prostate cancer cells When injected into human prostate cancer tumors (50-100 mm3) in nude mice, virus employing this double lock mechanism was ef cacious in blocking tumor growth In contrast,

a “single-lock” virus with only the E1 genes under probasin control exhibited only a limited effect on tumor growth These data indicate the potent activity of the double-lock CAdVex is a significant improvement for CRAd design To further enhance the ef cacy of these vectors we have engineered expression of immune enhancing cytokines and tumor antigens into the dual-lock CAdVex One of the vectors we have evaluated expresses TRAIL controlled by the probasin promoter in the dual-lock CAdVex backbone

The rationale of our approach is that intratumoral injection of this vector has the ability to eliminate prostate cancer cells by a combination of mechanisms: 1) Replication of the virus among cancer cells will induce apoptosis leading to ablation of the primary tumor

2) Induction of apoptosis will be further expanded by production and release of TRAIL 3) Immune responses induced to antigens expressed

by the replicating virus will lead to killing of vector-transduced cells

4) The pro-in ammatory environment created by viral replication will enhance immune activation against tumor antigens that are poorly immunogenic in the context of the intact tumor The immune response to tumor antigens may be critical for reducing metastasis

Details of the vector design and progress in preliminary evaluation will be presented

505 A Probasin Promoter, Conditionally Replicating Adenovirus that Expresses the Sodium Iodide Symporter (NIS) for Radiovirotherapy of Prostate Cancer

Miguel A Trujillo,1 Michael J Oneal,2 Samantha McDonough,1

Rui Qin,3 John C Morris.1

1 Endocrinology, Mayo Clinic, Rochester, MN; 2 Department of Molecular Medicine, Mayo Clinic, Rochester, MN; 3 Department of Health Sciences Research, Mayo Clinic, Rochester, MN.

The sodium iodide symporter (NIS) directs the uptake and concentration of iodide in thyroid cells We have extended the use of NIS-mediated radioiodine therapy to other types of cancer, and have transferred and expressed the sodium-iodide symporter (NIS) gene

in prostate, colon, and breast cancer cells using adenoviral vectors

To improve vector ef ciency we have developed a conditionally replicating adenovirus (CRAd) in which the E1a gene is driven

by the prostate speci c promoter, Probasin and the cassette RSV promoter-human NIScDNA-bGH polyA replaces the E3 region

(CRAd Ad5PB_RSV-NIS) In vitro infection of the prostate cancer

cell line LnCaP resulted in virus replication, cytolysis, and release of infective viral particles Conversely, the prostate cancer cell line PC-3 (androgen receptor negative) and the pancreatic cancer cell line

Panc-1 were refractory to the viral cytopathic effect and did not support viral replication Radioiodine uptake was readily measurable in LnCaP cells infected with Ad5PB_RSV-NIS 24 hours post-infection,

con rming NIS expression In vivo, LnCaP tumor xenografts in nude

mice injected intratumorally with Ad5PB_RSV_NIS CRAd expressed NIS actively as evidenced by 99Tc uptake and imaging Administration

of therapeutic 131I after virus injection signi cantly increased survival probability in mice carrying xenografted LnCaP tumors compared

to virotherapy alone The data indicate that Ad5PB_RSV_NIS replication is stringently restricted to androgen positive prostate cancer cells and results in effective NIS expression and uptake of radioiodine This construct may allow multimodal therapy, combining cytolytic virotherapy with radioiodine treatment, to be developed as

a novel treatment for prostate cancer

506 Adenovirus Vectors Transductionally Retargeted to Her2/neu for Prostate Tumour Treatment

Maria K Magnusson,1 Robert Kraaij,2 Corrina M A De Ridder,2

Leif Lindholm.3

1 Dep for Microbiology and Immunology, University of Gothenburg, Inst for Biomedicine, Gothenburg, Sweden; 2 Department of Urology, Erasmus MC, Rotterdam, Netherlands; 3 Got-A-Gene AB, Kullavik, Sweden.

Preclinical and clinical studies have demonstrated that adenovirus (Ad) gene transfer vectors have great potential for the treatment of tumor diseases However, several signi cant hurdles currently limit the ef ciency of gene transfer with adenovirus-based vectors One

of these hurdles is that the virus infects many different cells in the body while cells relevant for targeting (such as cancer cells) are often refractory to infection This fact necessitates the development of Ad vectors that are targeted to selected cells

We have previously reported a replication competent Ad vector speci c for Her2/neu by inserting a tandem repeat of the Her2/neu reactive Af bodyTM molecule (ZH) in the HI-loop of a CAR binding ablated  ber genetically modi ed to contain sequences for  exible linkers between the ZH and the knob sequences (Ad5/FibZH/1) ZH

is an Af bodyTM molecule speci c for the extra-cellular domain

of human epidermal growth factor receptor 2 (Her2/neu) that is over-expressed in many carcinomas To further de-target the single ablated virus Ad5/FibZH/1 we changed the RGD motif in the penton base to EGD and substituted the KKTK motif in the third shaft