180 induction of anti tumor immunity by the baculovirus autographa californica multiple nuclear polyhedrosis virus

180 Induction of Anti Tumor Immunity by the Baculovirus Autographa Californica Multiple Nuclear Polyhedrosis Virus 178 Spontaneous Glioblastoma in Dogs Preclinical Model for High Capacity Adenoviral V[.]

178 Spontaneous Glioblastoma in Dogs:

Preclinical Model for High Capacity Adenoviral

Vector-Mediated Experimental Gene Therapy

M Candolfi,' J.Curtin,' W.S Nichols,' A K M O

Muham-mad,'M.Puntel,'O D King,'W Xiong,' K M Kroeger,1C

Liu,'O.E.Pluhar,'E A.Mc Nicl,2r,R Ohlfcst,'A.B Freese,'

P F Moore,' D Palmer;"P.Ng,6J D Young,"P R Lowenstein,'

M O Castro.'

' Gene Therapeutics Research Institute, Cedars-Sinai Medical

Labora-toryMedicine, Cedars-Sinai Medical Center; L os Angeles CA ;

'Dept of Veterinary Clinical Sciences University ofMinnesota,

Saint Paul MN; 4DepII of Neurosurg ery, University ofMinnesota,

Saint Paul, MN; :;Pathology Microbiology & Immunology School

of Veterinary Medicine University ofCalifornia Davis, CA; "Dept

ofMolecular and Human Genetics Baylor College ofMedicine,

Medical Center; Los Angeles, CA

Wcevaluated the dogspontaneousGBM as a largeanimalmodel

for translational gene therapy strategies We determined the

neu-ropathology of these tumors and the extent to which it resembles

human OBM We also determined the feasibility of infecting dog

normal brain tissue by injectingadenoviralvectors(Ads) expressing

reporter and therapeutic transgenes in the cortex of healthy Beagle

dogs Since human patients usually exhibit preexisting immunity

against Ads,we determined the anti-Ad immune status ofoutbreed

dogs Wefound thatdog OBMs exhibit characteristic features of the

humanGBM, includingnecrosis,pseudopalizading,

neovasculariza-tion,endothelialproliferationand invasionintonon-neoplasticbrain

In the brain of healthy Beagle dogs we administered Ad expressing

glioma cells in the presenceofganciclovir (GCV),and the

immune-stimulatoryFIt3L,whichattractsantigen presentingcells to the brain

and the tumor mass.Transgene expression was detected in neurons

anda strocytes7 days post-injection without adverse clinical or

neu-ropathologicalside effects.Weassessed the presenceofneutralizing

antibodiesin serum from 17 outbreddogs recruitedfor our study.We

found that 60% exhibited neutralizingantibodiesagainstAds.These

datasuggest that OBM-bearing dogs would also mimic the immune

status of human patients in clinical trials Our results suggest that

high capacity-Ads are excellent candidates for treating dog OBM,

since theirexpression is not inhibitedby anti-Adimmuneresponses

Thus,we assessed the ability ofHC-Ad vectors expressing TK and

Flt3L to infect dog OBM cells in culture.HC-Ads were effective at

transducing dog GBM cells HC-Ad-TK elicited strong cytotoxic

effects when combined with OCV.Our data indicate that dogs

bear-ing spontaneous OBM constitute an attractive animal model for

testing novel therapeutic approaches in a spontaneous tumor in the

context of a larger brain.Supported by: NIH ININDS Grants IROI

42893.01; U54 NS045309-01, and IR2 I NS047298-01 to PRL; the

Bram and Elaine Goldsmith and the Medallions Group Endowed

The Linda Tallen & David Paul Kane Foundation and the Board

ofGovernors at CSMC.

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179 A Phase I Clinical Trial of Liposome-Mediated Interferon-beta Gene Therapy for High-Grade Gliomas

Toshihiko Wakabayashi,IMasaakiMi zuno,'Atsushi Natsume,'

Masazumi Fujii,"Jun Yoshida."

'Center or Genetic and Regenerative Medicine Nagoya Uni-versity Hospital Nagoya Aichi, Japan ; " Neurosurgely, Nagoya University School ofMedicine , Nagoya Aichi, Japan.

High-gradeglioms are highly lethal neoplasms that represent about 20% of all intracranial tumors Cationicliposome-rnediated interferon-beta(IFN-~) genetransferhasbeenfound to induce re-gression ofcxperimental glioma Weperformed a pilotclinical trial toevaluatesafetyand effectivenessofthisIFN-~ gene therapyin five patients with high-grade glioma Two patients showed more than 50% reductionand others had stabledisease 10weeks after the treat-ment initiation [Geneticanalysis] In order to identifyalterations in gene expression in brain tumors 2 weeks after gene therapy trial, we uscdmicroarraytechnology and MetaCore analysis Interestingly,

using hierarchical clustering and principal component analysis,5 series of gene therapy trial were classified according to response to IFNgene therapy There weresignificantchanges ingene expression which is related to immunoresponseand apoptosis, It confirms the validity of the methods Moreover, novel patterns of altered gene expression,such as inhibitionof'neo-vascularizationwere identified, suggesting the involvement of pathways not previously described

as involved [Brain autopsy] Autopsy revealed that tumortissues showed dramatic changes after therapy in all patients Many tumor cells showednecroticchanges,and immunohistochemistry identified many CD8-positive lymphocytes and macrophages infiltrating in tumor and surrounding tissues, probably resulting from therapeutic effect Simultaneously,numberofMIB-1 positivecells was notable decreased No adverse findings associated with the clinical trial were pathologically observed [Conclusions] This study suggests the feasibilityand safetyofIFN-~ gene therapy,which may become

an important treatment options for patients with malignant glioma Additional clinical trials are warranted

180 Induction of Anti-Tumor Immunity by the Baculovirus Autographa Californica Multiple Nuclear Polyhedrosis Virus

Masayuki Kitajima,' ,4TakayukiAbc,I Naoko Miyano-Kuro-saki,'·2 Toshinori Nakayama,4Hiroshi Takaku.P

IDepartment ofLife and Environmental Science, Chiba Institute ofTechnology; Narashino, Chiba , Japan ; " High Technology Research, Chiba Institute ofTeelmology Narashino, Chiba, Ja-pan; JResearch Centerfor Emerging lrfectious Diseases , O saka University, Suita, Osaka Japan; 'Department ofImmunology,

Japan.

Background Wild type Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) can infect a variety of mammalian cell types in vitro, but it does not replicate in these cells AcMNPV induces anti-viral cytokineproduction after injection in vivo, resulting in the protection of cells from infection with vesicular stomatitis virus and influenza virus Objective We here show that AcMNPV activates immune cells via TLR9/MyD88 signal path-way We also described the effects of AcMNPV on the induction

of immune responsiveness and tumor growth in mice.Results and Conclusions First, we found that AcNPV has the ability to induce innate immune system activation through a MyD88ffLR9-depen-dent pathway.Next, we examined the effects ofAcMNPV in mice

on immune responsiveness in general,and anti-tumor immunity

in particular AeMNPV treatment also increased IFN-y levels in the serumand induced a marked elevation in IFN-y production

Molecul arTherapy Volume 15 S upplement t• \by 2007

C opyright ©The American$< )(;(1,:1)"o f Gcnc Thempy

by liver mononuclear cells (MNCs) compared to splenoeytes.The

hepatic MNCs in these animals were found to be highly cytotoxic

to NK-sensitive YAC-I and B 16 melanoma cells,but they had little

effect onEL4 cells, which are NK-resistant Intravenous injection

ofAcMNPV induced NK cell proliferation in the liver and spleen

and enhanced anti-tumor immunity in mice that had B 16 liver

metastases Furthermore, such treatment increased the survival

of C57BL/6,Ja281-!" and IFN-y",mice that had been previously

injected with B I6 tumor cells Furthermore, AcMNPV efficiently

stimulates NK cell-mediated, anti-tumor immunity the strong

im-mune response induced by AcNPV makes it a promising candidate

for a novel, adjuvant-containing vaccine vehicle against infectious

diseases I.Abe,1~ etal.,(2003) J.Immunol 171: 1133-1139 2.Abe,

T ct al.,(2005) J Virol 79:2847-2858.3.Kitajima,M et aI.,(2006)

Bioehem Biophys Res Commun.343:378-384

181 Eradication of Multifocal Glioma in a

Syngeneic Glioblastoma Multiforme with Ad-Flt3L

and Ad-HSV1-TK

Gwendalyn D King,'A K M Ghulam Muhammad,' JamesF

Curtin,' Carlos Barcia,' Mariana Puntel,' Chunyan Liu,' Sarah B

Honig,IMarianelaCandolfi,' Sonali Mondkar,I Pedro R

Lowen-stein,' Maria G Castro.'

'Gene Therapeutics Research Institute, Cedar Sinai Medical

Cen-ter/University a/California Los Angeles, Los Angeles , CA.

The disseminated nature of human glioblastoma multi forme

(GBM) makes it a particularly difficult tumor to treat Most

preclini-cal models ofGBM involve treatment ofa single tumor mass when

the human disease is characterized by infiltrating tumor cells which

migrate from the primary tumor For therapeutic outcomes to

trans-late from preclinical models into clinical trials, induction ofan

anti-tumor response capable ofeliminating multi focal disease is essential

We tested the hypothesis that adenoviral (Ad) mediated expression

ofFlt3L and I·ISVI-TKwithin GBM would mediate regression of

the primary,treated tumor mass and a secondary, untreated tumor

growing at a distant site from the primary tumor and the site of

therapeutic vector injection In either the single GBM or multifoeal

GBM models used, all saline treated,control animals succumbedto

tumors by day 22 Seventy percent of the animals bearing a single

GBM mass treated with Ad-F1t3L and Ad-TK survived long-term

Fifty percent of animals bearing both a large primary GBM and a

second GBM in the contralateral hemisphere implanted at the time

the primary tumor was being treated with Ad-Flt3L and Ad-TK also

survived long-term (p<0.05) To further test the effectiveness ofthe

response, tumors were simultaneously implanted,bilaterally and

only the right tumor mass was treated with Ad-Flt3L and Ad-TK; in

this multifocal model,80% ofthe treated animals survived long term

While no significant difference in survival was found between

unifo-cal and multi founifo-cal tumor bearing animals treated with Ad-FIt3L and

Ad-TK, both treatments were statistically different from the saline

treated group (p< 0.05) Our results demonstrate that combination

therapy with Ad-FIt3L and Ad-TK can eradicate multifocal brain

tumor disease in a syngeneic, intracranial GBM model Funded by

NINDS grant # ROI NS4456.01, R03 TW006273-01 to MGC;

NINDS grant #RO I NS42893,0544 NS04-5309,and R21 NS47298

to PRL; the Linda Tallen & David Paul Kane Foundation; the Bram

and Elaine Goldsmith Endowed Chair to PRL; the Medallions Group

Endowed Chair to MGC and the BOG at CSMC.GDK is supported

by NINDS F32 NS0503034-01

Molecular Therapy Volume15 Supplementl, ~ br 2007

C opyright © T he American So ci etyo r Gen e "1l1f :r:lpy

182 Progress in the Clinical Development of a NovellL-12 Gene Therapeutic for the Treatment of Recurrent Ovarian Cancer

Khursheed N.Anwcr,'Mack N Barnes.'MathewL Anderson.' Connie McCombs,2 Souheil Saddekni.?Sharmila Makhija.' Ivan

Maya,'Jason G Fewell,' Margit M Janat-Arnsbury,' Danny H Lewis; Ronald D.Alvarez.'

'Research & Development, Expression Genetics Inc Huntsville, AL; lOB /GrN, University0/Alabama at Birmingham Birming-ham, AL; J OB/G rN Baylor College a/Medicine Houston, TX.

We recently completed a Phase I dose escalation trial to as-sess the safety and tolerability of a novel gene therapeutic agent, EGEN-OOI, in women with recurrent ovarian cancer EGEN-OOI consists of a human IL-12 plasmid encapsulated by a polymeric gene carrier, PEG-PEl-Cholesterol A total of 13 patients were enrolled in four dose-escalating cohorts and treated with either 0.6

mg/rn" ,3mg/nr ',12 rng/rn? or 24 mg/rn! EGEN-OOI weekly for a total of four doses Administration of EGEN-OOI was generally well tolerated Common side effects included transient abdominal cramping, nausea and low grade fever, each of which typically subsided within 24 hours ofEGEN-OOI infusion Peritonitis,likely related to catheter use,was observed in three patients and resolved promptly after catheter removal and intravenous antibiotics Measur-able levels of IL-12 plasmid could be detected in peritoneal fluid samples collected throughout the course of EGEN-OOI treatment

In comparison, most blood samples either did not contain detectable amounts of plasmid DNA or contained <1% of the amount found

in the corresponding peritoneal fluid samples Treatment-related increases in IFN-y levels were observed in peritoneal fluid but not serum,indicating that EGEN-OOI induced a biological response in patients who received our agent Consistent with this hypothesis,

stable disease and reduction in serum CA-125 levels was clinically observed in some patients Full details of our completed trial of EGEN-OOI monotherapy will be presented Based on our results, we have designed a subsequent study to clinically evaluate the safety and tolerability of escalating doses of EGEN-OOI in combination with standard chemotherapy in patients recently diagnosed with recurrent, platinum-sensitive ovarian cancer

183 Interferon-alpha Gene Transfer Enhances Antitumor Activity of Allogeneic Hematopoietic Stem Cell Transplantation Against Solid Cancers

Kazunori Aoki,'Hidehiko Hara,'Akihiko Kobayashi; Kimiko Yoshida; Teruhiko Yoshida.'

'Section/or Studies on Host-Immune Response , National Cancer Center Research Institute , Tokyo, Japan; lGenetics Division National Cancer Center Re search Institut e, Tokyo, Japan.

Allogeneic hematopoietic stem cell transplantation (alloHSCT) often leads to a significant graft-versus-tumor (GVT) effect for several types of leukemia and solid cancers such as renal cancers However, the benefit of the GVT effect is often offset by the occu

r-rence of graft-versus-host disease (GVHD), which could lead to a fatal outcome Therefore, enhancement of the specific antitumor activity of alloHSCT against solid cancers is one of the major is-sues in translational cancer research Interferon-alpha (IFN) is a cytokine with pleiotropic biological functions: in addition to the direct cytotoxicity to cancer cells and anti-angiogenesis activity, IFNs also have immune modulatory effects including upregulation

of the MHC class I expression,an increase in cytotoxic T lympho-cyte activity, the generation ofT helper cells and the maturation of antigen-presenting cells.However, the general toxicities following the systemic administration of the recombinant IFN protein often impede the usc of a dosage sufficient to induce effective antitumor responses in the clinical setting Because our preclinical study

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