167 macrophages transduced with an adenoviral vector expressing glipr1 suppress tumor growth and metastasis in a preclinical metastatic prostate cancer model

167 Macrophages Transduced with an Adenoviral Vector Expressing Glipr1 Suppress Tumor Growth and Metastasis in a Preclinical Metastatic Prostate Cancer Model 164 Enhanced Anti Tumor Effect of Modified[.]

164 Enhanced Anti-Tumor Effect of Modified

Vaccinia Virus Expressing Human-gp100 Antigen

and Rantes in Cancer Vaccine Approach

Kandan Aravindaram,IHsiu Hui Yu,IChun Wen Lan,INing Sun

Yang.'

'Agricultural Biotechnology Research Center, Academia Sinica

Taipei , Taiwan.

Among various treatment ehoices for cancer that havc emerged

in the past 100 years,recent approaches ofcancer vaccine

immuno-therapy representa promising and new strategy.In this study,we

op-timized an experimental system,by expressing a human melanoma

associated antigen (hgp-I 00) in B16 melanoma cells for C57BLl6

mouse tumor vaccine model that tests the melanoma metastases into

lung Heterologous prime and boosting tactics with recombinant

modified vaccinia virus (MVA) encoding the hgplOO showed a

significant decrease in lung metastases compared to homologous

DNA immunization.We observed that MVA vector could enhance

Th I type cytokine gamma interferon secretion by ELISPOT test

In addition, we further explored the effect on the use of chemokine

Rantes (CCL5) as an adjuvant for hgplOO antigen enhancing the

antitumor immunity.An ideal experimental approach may be to

bom-bard Rantesexpression vector by gene gun 24 h before hgp I00 DNA

vaccine at the same vaccination site enhancing gamma interferon and

reducing tumor size Prime with DNA vaccine and two boosts with

MVAi.p with gene-based vectors encoding Rantes and hgp I00 were

essential for induction ofstrong anti-hgp I00 cell mediated immunity

Rantes cDNA gene 48 h before hgp I00 vaccine or only with Rantes

cDNA was not much effective.Therefore,this study showed that

the Rantes was an effective adjuvant approach to enhance hgplOO

specific immunity induced by DNAlMVA cancer vaccine Details

of the molecular mechanisms and additional strategies to further

increase the efficiency of MVA-assisted eancer vaccine approach

will be systematically investigated in future studies

165 Heat Shock Protein 70-Antigen Fusion

Protein Expressing DNA Vaccine for Cancer

Immunotherapy

Ayumi Yamaoka,1Seiji Takemoto; Makiya Nishikawa,' Tomoya

Yata,' Yuji Ohno,'Yoshinobu Takakura.'

'Department0/Biopharmaceutics and Drug Metabolism,

Gradu-ate School0/Pharmaceutical Sciences, Kyoto University, Kyoto,

Japan.

Antigen delivery is an attractive approach to inducing cytotoxic

'I' lymphocytes (CTLs) and antibody response against tumor cells

Antigens administered in a DNA form could be directly expressed

in antigen-presenting cells (APCs),but most antigens are gener

-ally expressed in non-APCs,such as myocytcs and keratinocytes

Therefore, delivering antigens to Af'Cs is required lor inducing

high levels of antigen-specific CTLs We have shown in previous

studies that heat shock protein 70 (Hsp70)-based fusion protein

harboring MHC class I peptide is a highly effective vaccine for

inducing tumor-specific immune response This is at least partially

due to the facts that Hsp70 is actively delivered to Af'Cs through

Hsp receptors and it activates innate immunity by signaling through

CD40 and Toll-like receptors The use of poly-histidine (His) as

a endosomolytic agent was also found effective in increasing the

cytoplasmic delivery ofthe fusion proteins in a mouse dendritic cell

line DC2.4.In the present study, we have constructed plasmid DNA

encoding the fusion protein, in which His, Hsp70 and OVA257-264,

a model MHC class I antigen derived from ovalbumin (OVA) are

connected in tandem, under the control ofcytomegalovirus promoter

(pHis-Hsp70-pepl) When mice were immunized with intradermal

injections ofpHis-Hsp70-pepl followed by c1ectroporation, a strong

antigen-specific CTL response was obtained In addition,

immuniza-Molecular Therapy V olume 15 S upplement I• \I.\y 200 ;

C opyright © 1111; Amer ican Soci etyorG ene Th erapy

tion with pHis-Hsp70-pepI was effective in inhibiting the growth

of OVA-expressing E.G7 tumor and in extending the survival time ofE.G7-bearing mice.pHsp70-pepl,a plasmid vector expressing a Hsp70 fusion protein with no His,showed much less potency than pHis-Hsp70-pepI, as far as the CTL response and survival were concerned These results indicate that the control of intracellular trafficking of Hsp70-antigenfusion protein using poly-histidineis

a useful strategy for increasing antigen-specific immune response induced by DNAvaccines

166 The Opposite Effect of Lipopolysaccharide on the Antitumor Therapeutic Efficacy of DNA Vaccine

Chi-Chen Lin,'Meng-Chi Yen,'·2Ming-Derg Lai.J.2

'Department ofBiochemistry and Molecular Biology, College of Medicine, National Cheng Kung University , Tainan, Taiwan; 21n-stitute ofBasic Medicin e, College ofMedicine, National Cheng Kung University , Tainan, Taiwan.

Endotoxins,also known as Iipopolysaccharides or LPS,are com-mon potential impurity in plasmid DNA vaccines Although LPS has immunostimulatory properties through activation of Toll-like receptor 4 (TLR4),the effect of various amount ofLPS on the im-munc responses induced by DNA vaccine is not fully understood

In the present study,pCMV-N'-ncu DNA plasmid was purified with traditional or endotoxin-free plasmid preparation reagents,and was used to inoculate tumor-bearing C3H/HeN mice.The mouse tumor naturally overexpress p 185ncuantigen The anti-tumor therapeutic or the plasmid DNA prepared by endotoxin-free kit was significantly better than that of plasmid DNA containing residual endotoxin.To further investigate the effect of LPS on the therapeutic efficacy of DNA vaccine,increasing amount of LpS was added to endotoxin-free plasmid DNA,and used for intramuscular inoculationon mice with established tumor Very low dose (I microgram) of LPS sig-nificantly attenuated the therapeuticeffectofneu DNA vaccine.It also skewed the immune responses to aTh2 type as demonstrated

by high IL-4 production In contrast,high amount (100 microgram) ofLPS enhanced the therapeuticefficacy ofneu DNA vaccine.The increase of therapeutic efficacy may be correlated with enhanced cytotoxic 'I' lymphocyte response, In addition,aThl immune re-sponse with high IfN-gamma production was observed with high amount orexogenous LPS furthermore, extra highamount ofLPS (higher than 500 microgram) is not tolerable by mice The observed difference with various amounts ofLPS on DNA vaccine was dimin-ished when the tumor were grown in TLR4-defect mice Hence, our results suggest that the effect ofLPS on the therapeutic efficacy of DNA vaccine is dose-dependent The trace residual amount of'Ll'S may skew the immune response and inhibit the anti-tumoreffect

of DNA vaccine,which indicates the importance of DNA vaccine preparation in laboratory or pre-clinical use

167 Macrophages Transduced with an Adenoviral Vector Expressing Glipr1 Suppress Tumor Growth and Metastasis in a Preclinical Metastatic Prostate Cancer Model

Ken-ichi Tabata,'Masami Watanabe,' Kohei Edamura,'Guang Yang,'Jianxiang Wang; £1 MoatazAbdel Fattah,' Dov Kadmon,'

Timothy C.Thompson.P>

'Scott Department a/Urology, Baylor College ofMedicine, Houston, TX; 2Moleclliar and Cellular Biology, Baylor College0/

Medicine, HOIIStOIl T Y ; "Radtology, Baylor College ofMedicine , HOII StOll, TX

INTRODUCTION AND OBJECTIVE: We previously identi-fied the mouse and human RTVP·lIGLIPRI (Gliprl and GLIPRI ,

respectively) genes as direct p53 targets with pro-apoptotic

activi-S63

ties in various cancer cell lines, including prostate cancer We also

reported that intratumoral administration of adcnoviral vector

mediatedGliprl (AdGliprl) significantly reduced primary tumor

and spontaneous lung metastasis in a preclinical mouse model of

metastatic prostate cancer (Hum Gene Ther 14, 91-101, 2003)

These preclinical studies led to an ongoing neoadjuvant gene therapy

Phase 1/11 clinical trial in which AdGLIPR I is being tested by direct

intratumoral injection prior to radical prostatectomy (IND# 13033)

Based on our ongoing studies ofGLlPR I function, we hypothesized

that GLiPRI may promote macrophage mediated anti-tumoral

ac-tivities In the current study,we analyzed the anti-tumoral activities

ofGliprlgene modified Mfll METHODS: Peritoneal exudates Mfll

were infected withAdGliprl or control Adv/CMV/Bgal24hr before

use I-IBSS,uninfected Mfll, Bgal gene-modified Mfll (Bgal/Mfll),

or Gliprl gene-modified Mfll (Gliprl/Mdi) were injected directly

into orthotopic mouse prostate cancer (metastatic 178-2 BMA) on

day 7 after tumor cell inoculation.At day 21, primary tumors and

spontaneous lung metastasis were evaluated.For survival analysis,

animals were monitored daily and euthanized when moribund

RESULTS: There were no significant differences in macrophage

viability after transduction of AdGliprl compared with contro1

FACS analysis showed an increase in the number of cells positive

for MI-ICc1assll antigen, CD40, and CD80 in Gllpr l/Mdi compared

to uninfected Mfll or Bgal/Mfll IL-12 secretion from Gliprl/Msb in

vitro was significantly increased compared to uninfected Mfllor Bgal/

Mfll GliprllMfll induced significant suppression of primary tumor

growth (I 029mg) compared with Bgal/Mfll (2414mg) or uninfected

Mfll (2691mg) (P<O.OOO I and P=0.0002, respectively) GliprllMfll

also demonstrated significant suppression of spontaneous lung

metastasis (mean 2.0) compared with Bgal/Mfll (5.9) or uninfected

Mfll (5.9) (P=0.0204 and P=0.0204, respectively) A significant

survival advantage was demonstrated for Glipr l/Mrb compared with

control Bgal/Mfll injected animals (27.8 vs, 23.0 days,respectively,

P=0.0008) Serum IL-12 levels were significantly elevated on day

5 in Gliprl/Moinjected animals compared with control Bgal/Mfll

injected animals Splenocyte-derived cytotoxic natural killer cell

activity was enhanced on day 2,and on day 7 tumor-specific

T-lymphocyte activities were significantly increased after Gliprl/Mfll

injection, compared with control BgalfMfll CONCLUSIONS:We

have demonstrated potentially anticancer therapeutic activities of

Gliprl gene-modified macrophage in a preclinical mouse model of

metastatic prostate cancer This approach may be useful for prostate

cancer therapy

168 Allograft Vaccine for Epithelial Cancers

Yucheng Tang,IHakan Akubulut,IJonathan Maynard,ILine

Ped-ersen,IAlbert Deisseroth.'

'Gene Therapy Program, Sidney Kimmel Cancer Center, San

Diego , CA.

The success ofvaccination is reduced in the aged immune system

and in the cancer host The Deisseroth laboratory has reported that

the sc injection of the Ad-sig-TAA/ecdCD40L adenoviral vector

prime followed by two TAAleedCD40L protein boost se injections

(hereafter designated TAAfecdCD40L VPP) can overcome anergy

in TAA.Tg mice,and can induce immunological memory for over

a year to tumor associated antigens (TAA).CD40L is a potent

im-munological activating signal required for induction ofboth cellular

and humoral immune responses, which is not expressed normally

in activated CD4 helper cells in the aged test subject (both mice

and humans) thereby reducing the response to vaccination among

older individuals The E7/ecdCD40L VPP vaccine has been shown

to increase the level of E7 specific antibodies and CD8 T cells in

old (18 months) as well as young (2 months) test mice We decided

to study the effect of adding the E7fecdCD40L VPP vaccine to total

body irradiation and allografting, The allodonor for donor

lyrnpho-S64

cyte infusions (DLI) was first vaccinated with the E7/ecdCD40L VPP vaccine starting on day -35 The recipient C57BLl6J mice were then injected sc with 100,000E7 TC-I cells on day -7 1,200

cGy of total body irradiation (TBI) were administered on day 0, 7 days following injection of the tumor cells I hour following the total body irradiation, the mice were transplanted with a single intravenous injection of 10 million T cell depleted bone marrow cells from an allodonor Spleen cells (50 million) collected from

an allodonor immunized one month earlier against the E7 protein with the E7fecdCD40L VPP vaccine, were given to the allorecipi

-ent mice on day 3 (3 days following TBI and allotransplant) Thc recipient also received one sc injection of the E7fCD40L protein boost vaccination one week after allogeneic stem cell transplanta-tion We showed that the administration ofthe TBI and an allogeneic stem cell transplant 7 days post injectionof the E7 positive TC-I cancer cells, DLI from a E7/ecdCD40L VPP vaccinated donor 3 days following transplant (10 days after the E7 positive TC-I tumor injection), and a single E7fccdCD40L protein boost sc vaccination one week thereafter, resulted in a growth rate of the E7 positive tumor cells which was less than the control (injection oftumor cells followed in 7 days by TBI), or the animals in which the sc injection

of the E7 positive tumor cells was followed in 7 days by TBI and allograft, and E7fecdCD40L vaccination of the recipient This data shows that the TBI/DLI from an E7 VPP vaccinated donor/ allograft followed by E7 VPP vaccination is much more powerful than the same therapy without the DLI In addition,the addition of the DLI from an E7/ccdCD40L VPP vaccinated donor to TBI,allograft and post allograft E7fecdCD40L vaccination improves overall survival dramatically Finally, this approach makes possible the use ofyoung donors for old recipients,which may be a major advantage in re-storing a vigorous immune response to the vaccine in the elderly cancer recipient This would be feasible for elderly cancer patients

if one used the TAA/ecdCD40L vaccine with the non-mycloablativc allograft which is safe even in the older cancer patient

Virus and Dendritic Cell Immunotherapy for the Treatment of Established Murine Neuroblastomas

Christopher 1 Farrell,' Cecile M Zaupa,'Robert L.Martuza,'

Samuel D Rabkin,' William T Curry.'

'Neurosurgery; Massachusetts Gen eral Hospital, Bo ston, MA.

Genetically-engineered, conditionally-replicating oncolytic viruses are capable of killing tumor cells by direct lysis Our laboratory and others have previously demonstrated that treatment with oncolytic HSV vectors also elicits induction ofa cell specific anti-tumor immune response as demonstrated by protection against tumor rechallenge and abrogation of this response in immunode-ficient mice In this study, we examined whether this immuno-therapeutic effect could be enhanced by combining oncolytic HSV with intratumoral administration of immature, ex-vivo generated dendritic cells (iDC).Subcutaneous N 18 neuroblastoma tumors were established in immunocompetentAfJmice and when tumors reached approximately 5mm in maximal diameter,the tumors were inoculated with G47A, an oncolytic I-ISV vector with engineered inactivation of ICP6 (ribonucleotide reductase) and deletions in the ICP34.5 and ICP47 genes Two days following viral infec-tion,iDC were directly administered into the tumor followed by

a second G47A inoculation three days later Significant reductions

in tumor volumes were observed at day 12 following treatment initiation in micereceivingcombinationtherapywithG47A+iDC

in comparison to those receiving G47A alone (4.7-fold reduction) and mock-treated mice (l5-foldreduction).Treatment with iDC in the absence of oncolytic virus had no demonstrable effect on tumor volume compared to the mock-treated group Additionally,survival was significantly prolonged in the combination therapy group as

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