442 examination of the therapeutic potential of an engineered VEGF a transcription activator in a spinal cord injury model

442 Examination of the Therapeutic Potential of an Engineered VEGF A Transcription Activator in a Spinal Cord Injury Model killing is based on targeting of adenoviral replication to cancer cells resul[.]

killing is based on targeting of adenoviral replication to cancer

cells resulting in tumor oncolysis by a natural lytic mechanism

This cancer therapy paradigm has rationalized the rapid translation

ofCRAd agents to the context of human clinical trials Despite the

demonstrated clinical safety, little clinical efficacy of CRAds was

noted Understanding of the biological factors confounding CRAd

function in the human clinical context has been limited by the lack

ofadequate CRAd monitoring techniques This warranted

develop-ment of CRAd imaging methods potentially allowing monitoring

CRAd replication,amplification,and localization in the human

clini-cal context However, the currently employed imaging modalities

function solely as readouts for a reporter transgene expression in

CRAd-infected cells and, thus,are incompatible with the cell

kill-ing-based mechanism ofCRAd function Besides,they do not allow

tracking of viral particles to provide a dynamic index of amplified

viral mass and,thus,adequately monitor CRAd replicationin vivo.

To circumvent this limitation we previously developed a novel

Ad labeling system based on incorporation of imaging reporter

motifs in the adenoviral capsid via their genetic fusion to Ad minor

structural protein IX (pIX) Onthis basiswe hypothesized that this

approach can be applied to construct a first capsid-labeled human

CRAd without perturbing the virion infectivity and/or CRAd's

anti-tumor potency Furthermore,technical fcasibilitics established by

our previous studies allowed us to further hypothesize that capsid

incorporation of dual modality reporter fusions,embodying the

capacity for SPECT/PET and highly sensitive fluorescent imaging

detection systems simultaneously could provide a flexible

frame-work to monitor viral replication by complementarymodalities that

are easily translatable to human applications To validate our dual

imaging approach we constructed and successfully rescued the first

capsid-modified CRAd carrying HSV thymidine kinase

(TK)-mo-nomeric Red Flourescent Proten (mRFP) imaging reporter fusion,

covalently linked to C-terminus ofpIX In addition, the new imaging

"delta 24"-CRAd derivative carried a genetically-modified chimera

E5/3 fiber for enhancement of'the CRAd infectivity for tumors Our

results demonstrate that the triple-component fusion pIX.TK-mRFP

is efficiently incorporated in the CRAd's caps ids, as revealed by

Western blot and fluorescence microscopy (mRFP) imaging of the

purified virions, and does not significantly compromise the CRAd

replication and cytotoxicity in culture Our results, thus,validate

in vitrofunctionality of the infectivity enhanced capsid-labeled

CRAd with dual-imaging modality and justify its further testing in

animal models of cancer Further translation of this strategy in the

human clinical context could become of an immense field-wide

significance

441 Properties of Human Dendritic Cells after

High Titer Infection with Oncolytic Adenoviruses

StephanSchicrcr,'InaMuller,' Andrea Hesse,'Jan Dorrie; Niels

Schaft,IAlexander Steinkasserer,IGerold Schuler,IEckhart

Kampgen,IDirk M.Nenelbeck.'-'

IDept ofDermatology, UniversityHospital Erlangen, Erlangen,

Germany; lHelmllOltz -University Group OncolyticAdenoviruses.

German Cancer Research Center and Heidelberg University

Hospital, Heidelberg, G ermany.

In spite ofintensivc efforts to modify oncolytic adenovirus (ads)

for improved specificity and efficacy ofcancer cell lysis,it is

becom-ing increasbecom-ingly clear that the killbecom-ing of non-infected cancer cells

in parallel to viral cell lysis will be crucial for oncolytic ads to be

effective in the clinic An attractive scenario towards this goal is the

induction of systemic anti-tumorimmunity by adenoviral oncolysis

In order to establish the basis for the development of adenoviral

oncolytic vaccination we examined the effects of oncolytic ads on

the biologyofhuman dendritic cells (DCs) DCs are the most potent

antigen presenting cells,key regulators of immune induction and

Molecular Therapy Volume15 SupplementI ~ br 2007

C opyright © T he Am erican So ci ety o r G ene " 1l1f:r:lpy

have been intensively exploited for various vaccination protocols The prime objective ofthis study was to investigate how optimized, melanoma-targetedoncolytic adsaffect the viabilityand maturation

of human monocyte-derived DC and the potency of such DCs to activateTcells Both immature DCs (iDCs) and mature DCs (mDCs) were transducible with luciferase encoding ads at high titers Fiber-chimeric ads with an ad serotype 3 derived knob domain (5/3fiber) showed an improved transduction efficacy compared with an ad with serotype 5 (5fiber) fiber After infection ofiDC and mDC with oncolytic ads (5 or 5/3 fiber) at high titers(5000vp/cell),no signifi-cant toxicity was observed Oncolytic ads showed no or strongly attenuated DNA replication in human DCs, thus confirming their replication specificity As determined by staining of cell surface maturation markers of DCs two to three days post infection with oncolytic ads (5 or 5/3fiber),we observed, dependent on the donor,

no or a partial DC maturation With respect to the maturability of DCs with cytokines and/or LPS,no negative influence of infection with oncolytic adenovirus was documented However,we did find

an increase in the IL-I2 secretion ofLPS resp LPS/IFNg matured DCs after infection with oncolytic ads Finally DCs,infected with oncolytic ads for 3 days, matured and then loaded with peptide were still able to prime and stimulate specifically CD8'l-cells with similar

or superior efficacy compared to uninfccted DCs,dependent on the donor In conclusion, oncolytic ads do not harm viability and matu-rability of monocyte derived DCs,but also do not induce complete maturation ofthese cells Therefore, oncolytic ads might represent a promising tool for oncolysis-induced anti-tumor immune activation, however, this strategy might require additional maturation signals for DCs,for example by expression of immunostimulatory genes inserted into the genome of oncolytic ads

442 Examination of the Therapeutic Potential

of an Engineered VEGF-A Transcription Activator

in a Spinal Cord Injury Model

YangLiu,' S KayeSpratt,'Gary Lee,'Michael G.Fchlings.P"

'Cell and Molecular, Toronto Western Research Institute, Toronto,

ON, Canada; lDevelopment, Sangamo BioSciences, Richmond,

C A; 'Krembil NeuroscienceCentre, University Health Network, Toronto ON, Canada ; 'Division ofNeurosurgery, Universityof Toronto, Toronto, ON, Canada

Spinal cord injury (SCI) results in the initiation of multiple secondary injury cascades, one of which is the disruption of spinal cord blood flow and the onset of spinal cord ischemia.Vascular endothelial growth factor (VEGF) is a major regulator ofendothelial cell proliferation,angiogenesis and vascular permeability, a potent neurotrophic factor and glial cell growth factor An engineered zinc finger protein transcription (ZFP-TF) factor was designed to activate expression of all the isoforms of the endogenous VEGF gene The aim of this study was to evaluate the effect ofa recombinant adeno-viral vector containing an engineered ZFP-VEGF-A transcriptional activator (AdV-ZFP-VEGF) after SCI in adult rats.The adenovirus vectors encoding either Os-Red fluorescent protein or ZFP-

VEGF-A were delivered locally immediately after a 35g clip compression injury InAd'V-Ds-Redinjected rats, adenovirus-infected cells were observed in gray and white matter, and the vectors transduced neu-rons,astrocytes and oligodendrocytcs.Adv-ZFP-VEGF treatment led to increased levels of VEGF protein,as revealed by Western blot and RT-PCR analysis three days after SCI Expression of the ZFP-VEGF-A was also confirmed by Western blot In the injured spinal cord area ten days after SCI and treatment, using immuno-histochemistry, a significant increase in vascularization (assessed

by RECA-l immunostaining) and decreased levels of apoptosis (assessed by TUNEL) occurred in rats treated with AdV-ZFP·VEG F compared with controls Our finding suggest that this engineered ZFP-VEGF-activating transcription factor led to upregulation of

SI71

YEGF protein, protection/repair of blood vessels and decreased

apoptosis, These data suggest that VEGF gene therapy using a ZFP

transcription factor plasmid holds promise as a therapy for SCI and

other forms of neurotrauma

443 Systemic Dissemination of the

Replication-Competent Oncolytic Adenovirus

Telomelysin (OBP-301) Following Intratumoral

Injection

Yu-An Zhang,1,2AlexW.Tong,I,2,3Padmasini Kumar.' Michael

'Cancer Research, Cancer Immunology Research Laboratory,

Oncolys Biol'harma Inc" Tokyo, Japan

A Phase I, dose escalation study was recently initiated to

exam-ine the safety of intratumoral injection with the novel conditional

replicative adenovirus Telomelysin (0131'-301),Telomelysin is a

modified,human adenovirus type 5 that incorporates the human

telomcrase reverse transcriptase gene (hTERT) promoter sequence

in its EIA region, hence restricting replication to cancers cells

that highly express hTERT Differential viral oncolytic activity is

anticipated, since normal cells commonly lack hTERT expression

To identify the systemic dissemination pattern of 0131'-301 alter

intratumoral injection, patient plasma, urine,sputum and saliva was

collected at scheduled time points before and after injection, A biopsy

of the injected tumor was also obtained at day 28 post-treatment

To date, one patient with squamous cell carcinoma of the head and

neck and one patient with melanoma have completed qPCR analysis

of 0131'-30J Following a single intratumoral injection of Ix 1010

viral particles (vp), 0131'-301 was not detected in all fluid samples

(plasma, saliva, urine and sputum) at all scheduled time points

(pre-treatment,I hr,3 hrs,day I,day 7 and day 14 post-treatment),using

a 0131'-301 specific qPCR assay with detection sensitivity as low as

5-10 vp per reaction.Plaque forming assays arc currently underway

to quantify viral titer and the presence ofanti-adenovirus neutralizing

antibody titer at these post-treatment time points Togetherwith the

determination of hexon/El A expression at day 28 post-treatment

tumor biopsies, these findings will allow us to define systemicviral

phannacokinetic and evidence of 0131'-301 replication following

intratumoral treatment Additional patients arc recently entered into

trial Updated results will be presented

444 YB-1 as a Target for the Development of

Oncolytic Adenoviruses

Klaus Mantwill,' Regina Holzmueller,' Moritz Widmaier,I

Emanuel Rognoni,'Per S.Holm.':'

'Institute ofExperimental Oncology, Klinikum Reclus derIsar;

The transcription factor YB-I is ovcrexpressed in many solid

tumors and one of'the highest overexpressed genes in drug-resistant

tumors We discovered that nuclear localization ofYI3-1 facilitates

E l-independent replication of serotype 5 adenoviruses (Cancer

Research 64,322-328,January I,2004) and studied the effect of

the otherwise non replicative Ad d1520,which carries a deletion in

E 1A and does not express the large 289 amino acid long E 1A protein

on a broad spectrum of tumor cells in vitro and in vivo To improve

the cell killing capacity ofAd d1520, we introduced a deletionin the

antiapoptotic E I13 19K gene and expanded the tropism ofAd-Oel03

toward nv integrins by insertion of an Arg-Gly-Asp (RGD) motif

SIn

into the fiber knob (Ad-Del03 RGD) Finally we tested an YB-I triggered self promoting system by incorporating the YB-I-gene into the E3 region driven by the major late promoter (Ad-Delo-YB-I RGD) All viruses tested showed strong antitumor activity

in vitro and in vivo especially in combination with radiation- and chemotherapy Since the oncolytic activity of Ad dl520 or vectors

of similar design is not limited to tumors of a particular tissue type,

we expect a broad range of application

445 Effects of Exogenous p53 on Potency and Safety of Oncolytic Adenovirus

Victor W.vanBeusechem,'Petra13.van denDoel,'Juliette C 13I0kker,1 Winald R Gerritsen '

Amsterdam, Netherlands,

Virotherapy with oncolytic adenoviruses that selectively destroy cancercells by lytic replication is a promising new way to treat cancer, Previously, we constructed an oncolytic adenovirus derived

from AdLU4 (with a deletion in the pRb-binding E IA-CR2 domain)

expressingfunctional human p53, The new virus Ad~4 -p53 was more effective than its parent AdL124 in killing most human cancer cell lines and primal)' cancer cells tested in vitro and xenograft

tumorsin vivo Here, we investigate if functional adenovirus E11355K, which binds p53, is required for p53-mediated efficacy enhancement To this end, we introduced in the genome ofAdL124 andAd~4-p53 a mutation encoding a single amino acid substitution (R240A) in E1B55K that abrogates its capacity to bind p53, Relative cytotoxicity ofthc viruses was comparedin vitro on p53 wild type

U20S cancer cells,p53 mutant PC-3 cancer cells and non-malignant human endothelial (I-IMVEC-L)cells and fibroblasts Cells were infected with a 2-fold virus dilution titration (normalized by infec-tious units on E l-complementing 911 cells) and 7 days later cell viability was measured by WST-I conversion, Two to four indepen-dent experiments in triplicate werc done on each cell type MOl 50 values were calculated using GraphPad Prism 4 software, The two virus backbones were found similarly active against each cell type, suggesting that the E IB55K(R240A) mutation did not attenuate the virus As expected and confirming earlier observations,p53 expres-sion augmented oncolytic potency ofAd~4 against U20S and PC-3 cells (in this experimental setting approximately 5 and 6 fold, respectively).In contrast,Ad~4/55K(R240A)-p53 was not more potent in killing cancer cells than its parentAd~4/55K(R240A),

suggesting that oncolysis enhancement was abolished by the R240A mutation No clear differences between any of the viruses were observed on HMYEC-L cells.Surprisingly,AdL124-p53 was 12-fold less toxic than AdL124 to normal fibroblasts This elTect was lost if EII355K was mutated Adenovirus-encoded p53 thus seems not only to improve oncolytic adenovirus potency, but also safety, at least on fibroblasts Both effects require EIB55K capable

of binding p53,suggesting that temporal regulation ofp53 activity during oncolytic adenovirus replication by E IB55K is important This makes oncolytic adcnoviruses with intact E IB55K gene and expressing p53 promising agents for cancer treatment and warrants their further evaluation

Molecular Therapy Volum e 15.Suppl crnenr I ,\b y 2007