45 A Novel Synergistic Antitumor and Antimetastatic Effect of Cell Vaccines Combination Producing IL 12 and IL 10 Molecular Therapy �������� ��� ���� ���������������� �������� ���� ������© �����������[.]
CANCER VACCINES 41 MDA-7 Coordinately Regulates the βCatenin and PI3K Signaling Pathways in Breast and Lung Tumor Cells To Promote Apoptosis and Increase Homotypic Adhesion Abner Mhashilkar,1 Alexis Stewart,1 Kerry Sieger,1 Heng-Yin Yang,1 Kelly Hunt,2 Jack Roth,2 Rajagopal Ramesh,2 Sunil Chada.1 Introgen Therapeutics, Houston, TX, United States; 2The University of Texas, MD Anderson Cancer Center, Houston, TX, United States mda-7 (melanoma differentiation associated gene-7; also known as IL-24) is a novel tumor suppressor with cytokine properties Adenoviral delivery of mda-7 (Ad-mda7) induces apoptosis and cell death selectively in tumor cells The molecular mechanisms underlying the anti-tumor activity of Ad-mda7 in breast and lung cancer lines were investigated Microarray analyses implicated both the β-catenin and PI3K signaling pathways Ad-mda7 treatment increased protein expression from tumor suppressor genes, including E-cadherin, APC, GSK-3b and PTEN, and decreased expression of proto-oncogenes, including those involved in β-catenin and PI3K signaling Ad-mda7 treatment caused a redistribution of cellular βcatenin from the nucleus to the plasma membrane and upregulated the E-cadherin-β-catenin adhesion complex in a tumor cell-specific manner Functional consequences of β-catenin sub-cellular redistribution were evaluated Ad-mda7 treated cells exhibited reduced TCF-LEF transcriptional activity, consistent with the loss of βcatenin from the nucleus Ad-mda7 treated cells showed enhanced homotypic cell-cell adhesion and reduced migration, consistent with up-regulation of E-cadherin on the cell surface The PI3K pathway members (p85 PI3K; FAK; ILK-1; Akt and PLC-gamma) were down-regulated and PTEN expression was increased Thus, we demonstrate in breast and lung tumor cells that MDA-7 protein expression modulates cell-cell adhesion and intracellular signaling via co-ordinate regulation of the β-catenin and PI3K pathways Dr Jack A Roth is Chairman of the SAB of Introgen 42 Characterization of a Novel ApoptosisInducing Gene and Its Application in Prostate Cancer Gene Therapy Yi Lu,1 Ben Beheshti,2 Jun Zhang,1 Lisa K Jennings.1 Medicine, University of Tennessee Health Science Center, Memphis, TN; 2Princess Margaret Hospital, Ontario Cancer Institute, Toronto, ON, Canada A novel gene, rat pHyde, and its human homologue, hpHyde, have been cloned from prostate cancer cells A recombinant adenovirus containing rat pHyde cDNA gene showed significant growth inhibition on human prostate cancer cells both in vitro and in vivo, and induced a caspase-3 dependent apoptosis Database search and FISH analysis consistently indicated that hpHyde gene localizes at human chromosome 2q14 Protein sequence analysis suggests that hpHyde is a plasma membrane protein that may have calcium channel function Immunohistochemistry revealed a differential expression of hpHyde protein in normal and cancerous prostate tissues hpHyde also differentially expressed in various normal human tissues, suggesting that hpHyde may play roles in development and differentiation Taken together, these results suggest that a novel apoptosis-inducing gene, hpHyde, may play important roles in normal prostate development and prostate carcinogenesis Studies of biological and physiological functions of this novel gene may elucidate new signal transduction pathway in prostate biology and provide potential therapeutic approach for prostate cancer treatment Molecular Therapy Vol 7, No 5, May 2003, Part of Parts Copyright © The American Society of Gene Therapy 43 Enhanced Anti-Tumor Effect by Bik Mutant Yan Li,1 Yong Wen,1 Qingqing Ding,1 Binhua P Zhou,1 Mien-Chie Hung.1 Department of Molecular and Cellular Oncology, The University of Texas M D Anderson Cancer Center, Houston, TX, United States Bik was initially identified as a BH3-domain-only protein that interacts with E1B 19K Systemically administrated Bik significantly inhibited the tumor growth and metastasis in human breast cancer nude mice model Recently, it has been reported that posttranslational phosphorylation can regulate the pro-apoptotic potency of Bik Here, we showed that Bik mutants (T33D, S35D and T33DS35D) were more potent than wild type (wt) Bik to inhibit cell proliferation and enhance apoptosis induction in various human cancer cells We also demonstrated the Bik mutants suppressed the tumorigenicity and tumor taking rate in mice ex vivo model Finally we demonstrated the Bik mutant-SN liposome inhibited the tumor growth and prolonged the life span in mice in vivo model Thus, the current results provide that mutant Bik gene, more potent than wt Bik, to induce cell death and can be explored to inhibit various cancers growth CANCER VACCINES 44 Enhancing DNA Vaccine Potency by CoAdministration of DNA Encoding Anti-Apoptotic Proteins Chien-fu Hung,1 Tae-Woo Kim,1 T C Wu.1 Pathology, Johns Hopkins University, Baltimore, MD Intradermal vaccination via gene gun efficiently delivers DNA vaccines into dendritic cells (DCs) of the skin, resulting in the activation and priming of antigen-specific T cells in vivo However, DCs have a limited lifespan, hindering their long-term ability to prime antigen-specific T cells We reason that a strategy that prolongs the survival of DNA-transduced DCs will enhance priming of antigenspecific T cells and DNA vaccine potency Here we show that codelivery of DNA encoding inhibitors of apoptosis (BCL-xL, BCL2, XIAP, dominant negative (dn) caspase-9, or dn caspase-8) with DNA encoding model antigens prolongs the survival of transduced DCs More importantly, vaccinated mice exhibited significant enhancement in antigen-specific CD8+ T cell immune responses, resulting in a potent antitumor effect against antigen-expressing tumors Among these anti-apoptotic factors, BCL-xL DNA demonstrated the greatest enhancement in antigen-specific immune responses and antitumor effects Thus, co-administration of DNA vaccines with DNA encoding anti-apoptotic proteins represents an innovative approach to enhance DNA vaccine potency 45 A Novel Synergistic Antitumor and Antimetastatic Effect of Cell Vaccines Combination Producing IL-12 and IL-10 Soraya K Adris,1 Veronica M Lopez,1 Alicia I Bravo,2 Yuti Chernajovsky,3 Osvaldo L Podhajcer.1 Gene Therapy Laboratory, Instituto Leloir- CONICET and University of Buenos Aires, Ciudad de Buenos Aires, Buenos Aires, Argentina; 2Eva Peron Hospital, Balcarce 900, Ciudad de Buenos Aires, Buenos Aires, Argentina; 3Queen Mary Bone and Joint Research Unit, University of London, Charterhouse Square, London, United Kingdom An important number of preclinical studies have shown that cytokine administration may be therapeutically effective for cancer treatment However, the systemic administration of different and S17 CANCER VACCINES potentially useful recombinant cytokines in clinical settings was only marginally useful and evoked serious toxicity, hampering their use and stimulating the development of preclincal studies involving delivery mechanisms providing relatively high levels, locally However, the drawback of most of these studies is that the antitumor response was primed by preliminary immunization Here, we show that local vaccination of mice harboring 20-days old LM3 breast and CT26 colon carcinomas, with inactivated autologous tumor vaccines producing the combination of the Th1 and Th2 type cytokines, interleukin-12 and interleukin-10, act synergistically inducing the regression of established primary tumors Vaccines producing a single cytokine had marginal or no therapeutic benefit We established - by using different markers such as systemic and tumor specific levels of IgG2a / IgG1 and determination of IL-4 / IFNg levels produced by spleen cells - that Th1 and Th2 responses were simultaneously activated by the combined vaccines in both tumor models, with no evidence of counter inhibition Histological analysis of the primary tumor sites demonstrated an increased recruitment of neutrophils, lymphocytes and macrophages immediately after the first vaccine, when mice were vaccinated with the combined vaccine By depletion with specific antibodies, we established that both CD4+ and CD8+ T cells were essential for the complete antitumor response By using an RNAse protection assay we identified specific chemokines that might be involved in the strong recruitment of immune cells when the combined vaccine was used for treating mice IL-10 expression by the vaccine appeared to play a dominant role since its injection in the contralateral flank together with the vaccine producing IL-12 in the ipsilateral flank abolished the effectiveness of the combined vaccine Vaccination in the flank completely eliminated spontaneous lung metastases developed by mice harboring 20-days old LM3 mammary tumors Histological analysis of metastases showed a remarkable neutrophil infiltrate in the lung parenchyma of control animals and in those vaccinated with cells producing IL-12 Conversely, mice vaccinated with cells producing IL-10 showed almost no infiltrate while a shift toward a strong lymphocytes infiltrate was observed in lung parenchyma of mice vaccinated with the combined vaccine In initial studies intended to demonstrate their potential clinical use, we generated a tricistronic retroviral vector with IRES sequences between the p35 and p40 cDNAs of IL-12 and second IRES upstream of the IL-10 cDNA Co-expression of bioactive IL-12 and IL-10 was as effective as IL-12 alone on the in vitro induction of human phytohemaglutinin blasts This study demonstrates for the first time the synergistic capacity of using this cytokine combination in a genetic vaccine for cancer gene therapy 46 AAV Vectors Encoding Fusion Protein of Human Papillomavirus (HPV) Types 16E7 and 18E7 as a Preventive and Therapeutic Vaccine for Cervical Cancers Liqiao Zhou,1 Tong Zhu,1 Bing Wang,1 Xiao Xiao.1 Melocular Gentics and Biochemistry, School of Medicine, University of Pittsburgh, Pittsburgh, PA, United States Human papillomavirus type 16 (HPV16) and type 18 (HPV18) infection has been linked to the development of cervical cancer One hallmark of persistent infection is the synthesis of the viral E7 protein in cervical epithelial cells E7 has been recognized as an ideal target for immunotherapy of cervical cancer Utilizing the E7expressing murine tumour cell line, TC-1, as a model of cervical carcinoma, an immunotherapy based on the administration of AAV vector (AAV1618E7hsp70) carrying HPV16E7 and HPV18E7 linked to Mycobacterium bovis BCG heat shock protein (hsp) 70 has been developed Because of safety concerns, we introduced separately two point mutations into the Rb-binding site of HPV16E7 and HPV18E7 oncogenes to eliminate its transformation potential For HPV16E7, the codons for Glu24 and Cys26, which are known to be S18 crucial for Rb-binding, were altered to glycine codons Likewise in HPV18E7, the equivalent codons, Glu27 and Cys29 were altered to glycine After those mutations, we cloned three genes in frame one by one, which included the mutated HPV16E7, HPV18E7 and heat shock protein (hsp) 70, which is capable of enhancing to the immune responses in vivo In order to deliver these genes into animal efficiently, we packaged the gene into recombinant adeno-associate virus (AAV) vector for subsequent study in vitro and in vivo For animal experiment, 6-8 weeks C57BL/6 mice were selected For the preventive group, thirty days after the single-dose intramuscular injection of AAVHPV1618E7hsp70 (2x1012 particles/ mice), 2x104 TC-1 cells were inoculated S.C in the neck of mice, and tumor growth was monitored twice weekly Our results demonstrated that prophylactic immunization with AAV1618E7hsp70 protected 100% percent of the mice against the challenge with TC-1 cells For the therapeutic group, days after S.C inoculation of 2x104 TC-1 cells, 2x1012 (particles/mice) of AAVHPV1618E7hsp70 were injected intramuscular The therapeutic immunization with AAV1618E7hsp70 inhibited tumor growth and induced regression of the palpable tumors in some animals Our study may provide a basis for the therapy of cervical cancer with AA vectors 47 Vaccination with Bone Marrow-Derived Dendritic Cells Modified by Recombinant Adenoviral Vectors Expressing the HER-2/neu Oncogene Inhibits Development of Tumors in a Transgenic Mouse Model of Breast Cancer Yoshio Sakai,1 Brian J Morrison,1 John D Burke,1 Jong-Myun Park,1 Guido Forni,2 Jay A Berzofsky,1 John C Morris.1 Metaboslim Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD, United States; 2Dept of Clinical and Biological Sciences, University of Turin, Turin, Italy Dendritic cells (DC’s) play a central role in generating immune responses by presentation of peptide epitopes in the context of MHC to interact with T-cells Adenoviral-mediated gene transfer of tumor antigens into DC’s offers several potential advantages: (1) Entire tumor antigens could be expressed by the vector without detailed knowledge of individual epitopes or binding affinities for a particular MHC, (2) DC’s may process the antigen naturally leading to more effective presentation of epitopes, and (3) viral proteins may act as an adjuvant Based on these hypotheses, we examined vaccination with DC’s modified by recombinant adenoviral vectors (rAd’s) expressing the HER2/neu oncogene in a transgenic mouse model of breast cancer DC’s were generated by culturing bone marrow from BALB/c mice in RPMI-1640 + GM-CSF E1-deleted rAd’s encoding truncated HER2/neu cDNA expressing the extracellular and transmembrane domains [Ad.HER2/neu(ECDtm)], ECD only [Ad.HER2/neu(ECD)], or no transgene [Ad.null] were generated by standard methods BALB/c-neu T mice transgenic for the rat HER2/neu oncogene spontaneously develop breast cancers beginning at 14-15 weeks of age and progress until all 10 mammary gland are involved Groups of to week old BALB/c-neu T mice were injected weekly x with 1x106 allogeneic DC’s infected with Ad.HER2/neu(ECDtm), Ad.HER2/neu(ECD) or Ad.null (MOI 30), or unmodified DC’s and followed for tumor occurrence Serum and splenocytes were collected from other groups of mice prior to treatment and 1-week after final vaccination Titers of anti-HER2/ neu antibodies were measured by specific antibody binding potential (SBP) ELISPOT assay for IFNg production was performed using splenocytes Mice free from tumor at 28-weeks were challenged with an injection of x 105 TUBO cells At 25-weeks, 5/5 mice treated with DC-Ad.HER2(ECDtm) and 2/5 mice treated with DCAd.HER2(ECD) were free of tumor In contrast, 5/5 mice treated with DC-Ad.null and 4/4 mice treated with unmodified DC’s developed breast cancers Looking at the total number of mammary Molecular Therapy Vol 7, No 5, May 2003, Part of Parts Copyright © The American Society of Gene Therapy ... p35 and p40 cDNAs of IL- 12 and second IRES upstream of the IL- 10 cDNA Co-expression of bioactive IL- 12 and IL- 10 was as effective as IL- 12 alone on the in vitro induction of human phytohemaglutinin... harboring 20-days old LM3 mammary tumors Histological analysis of metastases showed a remarkable neutrophil infiltrate in the lung parenchyma of control animals and in those vaccinated with cells... cells producing IL- 12 Conversely, mice vaccinated with cells producing IL- 10 showed almost no infiltrate while a shift toward a strong lymphocytes infiltrate was observed in lung parenchyma of mice