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, I Hsiu Hui Yu,I Chun Wen Lan, I Ning 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 immunotherapy represent a promising and new strategy In this study, we optimized 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 bombard Rantes expression 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, Seiji Takemoto; Makiya Nishikawa,' Tomoya Yata,' Yuji Ohno,' Yoshinobu Takakura.' 'Department 0/ Biopharmaceutics and Drug Metabolism, Graduate School 0/ 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 generally 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 , immunizaMolecular Therapy Volume 15.Supplement Iã.\I.\y 200; Copyright â 1111; AmericanSociety or Gene 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 -antigen fusion protein using poly-histidine is a useful strategy for increasing antigen-specific immune response induced by DNA vaccines 166 The Opposite Effect of Lipopolysaccharide on the Antitumor Therapeutic Efficacy of DNA Vaccine Chi-Chen Lin,' Meng-Chi Yen,'·2 Ming-Derg Lai.J.2 'Department ofBiochemistry and Molecular Biology, College of Medicine, National Cheng Kung University, Tainan, Taiwan; 21nstitute ofBasic Medicin e, College ofMedicine, National Cheng Kung University, Tainan, Taiwan Endotoxins, also known as Iipopolysaccharides or LPS, are common potential impurity in plasmid DNA vaccines Although LPS has immunostimulatory properties through activation of Toll-like receptor (TLR4), the effect of various amount ofLPS on the immunc 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 185ncu antigen 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 endotoxinfree plasmid DNA, and used for intramuscular inocu lation on mice with established tumor Very low dose (I microgram) of LPS significantly attenuated the therapeutic effect ofneu 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 therapeutic efficacy ofneu DNA vaccine The increase of therapeutic efficacy may be correlated with enhanced cytotoxic 'I' lymphocyte response, In addition , aThl immune response with high IfN-gamma production was observed with high amount orexogenous LPS furthermore, extra high amount ofLPS (higher than 500 microgram) is not tolerable by mice The observed difference with various amounts ofLPS on DNA vaccine was diminished 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-tumor effect 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 College 0/ Medicine, HOIIStOIl T Y; "Radtology, Baylor College ofMedicine , HOIIStOll, TX INTRODUCTION AND OBJECTIVE: We previously identified the mouse and human RTVP·lIGLIPRI (Gliprl and GLIPRI , respectively) genes as direct p53 targets with pro-apoptotic activiS63 ties in various cancer cell lines, including prostate cancer We also reported that intratumoral administration of adcnoviral vector mediated Gliprl (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 activities In the current study, we analyzed the anti-tumoral activities ofGliprl gene 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 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 Mfll or Bgal/ Mfll GliprllMfll induced significant suppression of primary tumor growth (I 029mg) compared with Bgal/Mfll (2414mg) or uninfected Mfll (2691mg) (P