Abstract -12, V NSC B

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1 -12, - V NSC B , Abstract Cancer therapy by surgery usually failed as a result of local recurrence and/or distant metastasis because of inadequate resection and micrometastasis. Postoperative enhancement of the specific tumoractivated host antitumor immunity was believed can abrogate these miserable sequelae. Interleukin-12 (IL-12) is a heterodimeric cytokine that plays an important role in the development of cellular immunity. It was known as natural killer cell stimulatory factor (NKSF) or cytotoxic lymphocyte maturation factor (CLMF). This cytokine is secreted by antigen-presenting cells (primarily macrophages and B cells) and appears can (1) enhance the cytolytic activity of a number of effector cells including T cells, natural killer (NK) cells, lymphokine activated killer (LAK) cells, and macrophages, (2) increase proliferation of activated NK and T cells, (3) induce production of cytokines, such as interferon-γ, (4) stimulate the induction of Th1 cells, (5) upregulate a number of cell surface molecules, (6) inhibit IgE secretion, and (7) act as a synergistic factor for hemopoietic stem cells. Using the C3H/He-MBT-2 bladder tumor model and the constructed retroviral vector carrying mil-12 cdna (pclnrx-il12) to transfect wild type MBT-2 or low TGF-β secreting MBT-2/ TGF-β (-) #3 cell line, we intended to investigate if (1) MBT-2 or MBT-2 /TGF-β (-) #3 tumor cells infected with retroviral plasmid carrying mil-12 cdna [ MBT-2 / IL-12 or MBT-2 / TGF-β(-) #3 / IL-12] may produce adequate amount of IL-12, (2) the MHC class I and II molecules expression on the cell surface of them can be upgraded, (3) after treated with lethal dose X-ray irradiation [IRMBT-2 / IL-12, IRMBT-2 / TGF- β(-) #3 / IL-12 ] remain long enough metabolic active to serve as an autologous tumor vaccine, (4) the 1

2 immunostimulating effect of IRMBT-2/ IL-12 and IRMBT-2/TGF- β(-) #3/IL-12 on tumor bearing mice when immunized after tumor resection. Key words: Interleukin-12, Bladder cancer, Immunotherapy, TGF-β murine IL-12 cdna Retroviral vector (pclnrx-il12) wild type MBT-2-12, - antisense TGF-β oligoneucleotide -12 [Interleukin-12 [MBT-2/TGF-β(-)#3] (IL-12)] IL-12 TGF-β (macrophage) B wild type MBT-2, (cytokine) 6000 rads X- (1) [Natural Killer (NK) cells] (Autologous tumor vaccine) [Lymphokine D17TBM, Activated Killer (LAK) cells] T [Cytotoxic T Lymphocytes (CTL)] (2) NK T -γ [Interferon-γ] (3) T [Th1(Helper T cell)] (4) MHC class I, (5), synergistic IL-12 IL-12 cdna retroviral plasmid vector IL-12 cdna Target cells, wild type MBT-2, MBT-2/TGF-β (-)#3, IL-12 retroviral plamid vector pclnrx reporter gene (neomycin resistant gene) mil-12 cdna integrade Target cell clones clones IL-12 TGF-β, (Day 17 TBM in C3H/He-MBT-2) :, IL-12 IL-12 2

3 TGFβ in vitro CD8 + T NK proliferation activity in vivo CD4 + T tumorigenecity IL-12 T (T cell [5] anergy) MHC : inbred female C3H/He Mice 4-6 IL-12 week of age. MBT-2 (Murine bladder tumor) FANFT IL-12 C3H/He Mice induce (Transitional cell TGF-β carcinoma) Complete immune suppression factor [1,2.] (Fetal Calf Serum ) mm TGF-β nonessential a.a. + 1mM sod. Pyruvate mm Glutamine + 50 mm 2-Mercaptoethanol. (1) murine IL-12 cdna retroviral TGF-β plasmid vector (pclnrx-il12) : IL-12 clone into pclnrx retroviral expression vector (RetroMax, IMGENEX)- pclnrx-il12. Transforming growth factor-β1(tgfβ1) IL-12 pclnrx [3,4] MBT-2 TGF-β (6000Rads) X- Ori Metabolic active TGF-β (retroviral vector) TGFβ antisense oligonucleotide Medium(CM): RPMI % FCS pclnrx-il12 MBT-2 genome (2) Cotransfection of pclnrx-il12 oligonucleotide and pcl-eco (packaging vector) into SV40 CMV TGFβ 293 cells Amp pbr322 LTR RU5 IL-12 Neo RSV 3

4 (3) stable clones ( IL-12 gene helper virus-free, retrovirus) retrovirus ( IL-12 gene ) supernatant, transforming growth factor-β Cancer retrovirus ( IL-12 gene ) Res. 52: 1386, supernatant MBT-2 (wild type ) and MBT-2 / TGF-β (-) # 3 cell lines, L.M., Roberts, A.B., Sporn, M.B. G418 medium Burlington, D.B., Lane, H.C. and Fauci, IL-12 gene MBT-2 MBT-2/ TGF-β (-) #3. pclnrx-il-12 transformation into DH5 alpha host cells and blunting of interferon overnight incubation, responsiveness. J. Immunol., 136: 39216, plasmid DNA, electroporation pclnrx-il12 MBT-2 MBT-2/ TGF-β (-) # 3. pclnrxil12 electroporation Johnny Shinn-Nan Lin.: Modulation of MBT-2 MBT-2/TGF-β (-) # 3 (2000µF, 200V, 129 Ω) Autologous Tumor Vaccine by G418 medium Anti-TGF-β antibody and : Interferon-α on murine Bladder MBT-2, Bladder Cancer. Anticancer Res 17:, construct , mil-12 plamid retroviral vector MBT-2, Gintaras Zaleskis, Erica S. Berleth, M. electroporation Jane Ehrke.: Effect of perioperative MBT-2 MBT-2/ TGF-β(-) # 3 MBT-2/IL-12, MBT-2/ TGF-β( )/IL-12, wild type MBT-2,, in vivo in vitro study. - : 1. Inge, T.H., Hoover, S.K., Susskind, B.M., Barrett, S.K., and Bear, H.D.: Inhibition of tumor-specific cytotoxic T-lymphocyte responses by 2. Rook, A.H., Kehrl, J.H., Wakefield, A.S.: Effects of transforming growth factor beta on the functions of natural killer cells: depressed cytolytic activity Tzong-Shin Tzai, Ai-Li Shiau, Chein-Sheng Lin, Chao-Liang Wu, Immunostimulating Effect of 4. Tzong-Shin Tzai, Robert P.Huben, Chemoimmunotherapy with 4

5 Cyclophosphamide and Autologous Tumor Vaccine in Murine Bladder MBT-2 Bladder Cancer. J. Urol. 151: , Tzong-Shin Tzai, Chu-Ing Lin, Ai-Li Shiau,Chao-Liang Wu: Antisense Oligonucleotide specific for transforming growth factor-β1 inhibits both in vitro and in vivo growth of MBT-2 murine bladder cancer. Anticancer Res. 18: , Tzong-Shin Tzai, Chao-Liang Wu, Li-Ling Liu, Ai-Li Shiau,: Postoperative immunization with TGF-β antisense oligonucleotide modified autologus tumor vaccine enhances the antitumor immunity of tumor bearing mice by upgrading the expressions of MHC class I antigen and Fas. Anticancer Res. 20: , Tzong-Shin Tzai, Ai-Li Shiau, Chao-Liang Wu, Yuh-Shyan Tsai: Postoperative administration of Interleukin-12 significantly enhances the anti-tumor immune response of MBT-2 bladder cancer bearing mice. Proc. Natl. Sci. Counc. ROC (B) 24: 2; 56-62,