503 safety of intravenous ad5 mda 7, a potential anti metastatic agent in gynecologic cancer

503 Safety of Intravenous Ad5 Mda 7, a Potential Anti Metastatic Agent in Gynecologic Cancer Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy[.]

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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

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

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

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