料 96WFA *96WFA * 類 ( ) *******003. Therapeutic Cancer DNA Vaccine: Targeting Immune Regulatory Genes in Dendritic Cells B101006

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1 行 料 96WFA *96WFA * ( ) A 類 ( 行 ) 類 ( ) / 立 *******003 狀 療 DNA Therapeutic Cancer DNA Vaccine: Targeting Immune Regulatory Genes in Dendritic Cells 行 97 年 年 B 年度 類 ( ) 2 ( 不 ) 年度 ( 不 ) 1 行 列 列 / 連 ( ) (06) ( 宅 / ) (06) 路 1 (06) a @mail.ncku.edu.tw 行

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7 度 若 年列 理 度 10% 金 金 列 立 不列 年列 1 年 金 說 年 說 年 說 C009 96WFA

8 十一 研究計畫中英文摘要 : 請就本計畫要點作一概述, 並依本計畫性質自訂關鍵詞 ( 一 ) 計畫中文摘要 ( 五百字以內 ) 我們已成功的建立利用 neu DNA 作為治療大量表現 p185 neu 之成型 MBT-2 腫瘤在 C3H 老鼠 在複合型實驗中也證實伴護分子 Hsp90 之抑制劑 geldanamycin 可加強 neu DNA vaccine 治療效果, 同時也首度證實 c-met DNA 可治療 B16 黑色素細胞癌在 C57BL/6 老鼠 由於 DNA vaccine 在未來臨床使用, 很可能是和其他治療法合併使用, 所以在此計劃中我們計劃利用 DNA vaccine 針對樹狀突細胞 (dendritic cell) 免疫調節基因, 使其能更廣泛利用於不同腫瘤上. 在樹狀突細胞中有許多免疫調節基因, 在這些基因中我們選擇了 thrombospondin (TSP)-1 and thrombospondin-2 作為研目標, 其理由有三 : 第一,TSP 是分泌至細胞外之蛋白質可直接影響樹狀突細胞微環境, 造成更大效果 ; 第二,thrombospondin 一般是認為是經由抗血管新生而抑制癌症, 此研究計劃將檢驗一個新的假說將 sirna against thrombospondin 送入樹狀突細胞可利用增強免疫力而抑制癌症 ; 第三,TSP-1 及 TSP-2 對 TGF-beta 活化能力不同, 我們可同時比較此二者在改變樹狀突細胞免疫力之不同 此研究計劃有五個主要連續性研究目標 : 第一, 利用基因槍將質體 DNA 表現針對 TSP-1 及 TSP-2 之干擾 RNA (sirna) 送入老鼠表皮, 觀察是否能調節免疫能力而治療癌症 ; 而治療癌症 三種自然腫瘤動物模式將被使用 :MBT-2 腫瘤在 C3H 老鼠,B16 黑色素胞癌在 C57BL/6 老鼠,CT-26 大腸癌細胞在 BalbC 老鼠 第二, 在研究抗癌免疫機轉時, 細胞型免疫反應, 体免疫反應, 及樹狀突 (dendritic) 細胞分泌細胞素功能將被進一步分析 第三, 由於 TSP-1 or TSP-2 具有很多不同蛋白質功能區 (functional domain) 和 CD47, CD36, heparin 等作用, 為進一步了解在樹狀突細胞中作用, 我們將送入一個 sirna 及不同 TSP-1 蛋白質功能區, 觀察送入那些功能區可逆轉免疫調節而降低治療癌症效果, 如此可從另一种研究方向了解 TSP-1 在体內功能 第四, 研究質體 DNA 表現針對 TSP-1 及 TSP-2 之干擾 RNA (sirna) 可否進一步加強 neu DNA 疫苗之癌症療效 ; 如此未來可複合使用傳統 DNA vaccine 及免疫調節型 DNA vaccine. 第五, 由於皮下形成之腫瘤其免疫反應並不同於原位癌, 所以我們將使用成大黎煥耀教授發展之肝原位癌作為另一動物腫瘤模式研究, 以更模擬真實免疫反應而確認此計劃發展之 TSP-1 sirna 之療效 此計劃之結果可能導致新的抗癌思考方向, 可利用 sirna 針對樹狀突 (dendritic) 細胞免疫調節基因來產生治療癌症腫瘤 利用基因鎗, 此傳送方法相當簡便, 而此計劃中 TSP-1 sirna 也很可能是未來治療癌症之新藥 另一方面此計劃也可增加對 TSP-1, TSP-2, 樹狀突 (dendritic) 細胞之交互作用細部機轉有更進一步了解 關鍵詞 :DNA 疫苗, 樹狀突 (dendritic) 細胞, 免疫調節, 干擾 RNA,TSP-1, TSP-2, 癌症 表 C011 共頁第頁

9 十一 研究計畫中英文摘要 : 請就本計畫要點作一概述, 並依本計畫性質自訂關鍵詞 ( 一 ) 計畫英文摘要 ( 五百字以內 ) We have demonstrated the therapeutic efficacy of neu DNA vaccine on MBT-2 tumor in its syngeneic host C3H mice. The combination therapy of neu DNA vaccine and HSP90 inhibitor further enhanced the cancer therapeutic effect. Furthermore, we are the first to demonstrate the therapeutic efficacy of c-met DNA vaccine on B16 melanoma implanted in C57BL/6 mice. Since DNA vaccine may function as a adjuvant therapy to combine with other therapy module, we wish to develop DNA vaccines can target on multiple types of tumors with any particular tumor-associated antigen or without specific tumor associated antigen. In this proposal, we wish investigate the use of immunological regulation of DNA vaccine in more broad types of animal tumor models. Among the immunoregulatory molecules in dendritic cells, we select thrombospondin (TSP)-1 and thrombospondin-2 as our target genes. There are three rationales. First, TSPs are secretory proteins that can affect dendritic cells and their microenvironment; Second, TSPs are usually considered as anti-tumor agents, we will test the novel hypothesis that sirna against TSP-1 or TSP-2 can function as anti-tumor agent by regulating dendritic cell function through delivery into skin dendritic cells; Thirdly, the TSP-2 and TSP-1 is different in the ability to activate TGF-beta, we can examine and compare their roles in dendritic cells in vivo. In this project, we will carry out five experimental approaches: 1. DNA vaccine expressing TSP-1 and TSP-2 with sirna will be examined for their therapeutic efficacy on established tumor scbcutaneously in vivo. DNA will be bombarded into skin with gene gun. MBT-2 in C3H mice will be used first. Additional two animal tumor models will be examined including B16 melanoma/c57bl6 and CT26 colon cancer/balbc. 2. Investigate the immunological mechanism responsible for delaying tumor growth in vivo. The cytokine response and tumor infiltration lymphocytes around tumor will be examined. 3. Since TSP-1 contains many functional domains which can interact with CD47, CD36, and heparin respectively, we will further define which functional domain of TSP-1 is required for immune suppression in vivo. We will construct the DNA vaccine containing the TSP-1 sirna with truncation TSP-1 containing different portions of TSP-1. This study will reveal which interaction is important for dendritic cell regulatory function. 4. We will test whether TSP-1 shrna enhances DNA vaccine targeting tumor associated antigen, such as neu. This study will facilitate the combined use of traditional DNA vaccine and immunoregulatory DNA vaccine. 5. Since the immunological response of subcutaneous tumor may not mimic that of the orthotropic tumor, we will investigate for the possible the use of TSP-1 or TSP-2 sirna on the established tumor in a murine In situ hepatoma model. The result of this proposal will lead to novel anti-cancer agents based on immunoregulatory molecules around dendritic cells, more specific speaking, TSP-1 sirna may become a new anticancer agent with easy delivery by gene gun. Furthermore, the study will reveal the detailed regulatory circuit of TSP-1 and TSP-2 in the microenvironment of skin dendritic cells. Finally, the interaction of functional domain of TSP-1 or 2 with dendritic cells may be revealed from another direction presented in this proposal in vivo. Key words: DNA vaccine, dendritic cells, immune regulation, sirna, TSP-1, TSP-2, cancer 表 C011 共頁第頁

10 十二 研究計畫內容 : ( 一 ) 近五年之研究計畫內容與主要研究成果說明 ( 連續性計畫應同時檢附上年度研究進度報告 ) 研究領域主要包括三方面 : 一 Neu DNA vaccine 研究 : 我們實驗室自從 1997 開始進入基因免疫治療從腫瘤疫苗, 再發展出 DNA vaccine for bladder cancer and other types of cancer. ( 一 ) 在 年間, 我們實驗室由 tumor vaccine 轉進 DNA vaccine 領域, 在 2000 年發表 combination of neu DNA vaccine and MBT-2 tumor vaccine 之 synergistic effect 於 Clinical Cancer Research 雜誌 此論文指出 psv-neu DNA vaccine 單獨並無治療效果, 但可明顯增強 tumor vaccine 之治療效果 由於 DNA vaccine 之製備及儲存非常經濟適合未來臨床發展, 我們進一步提出一個新觀念是否可改善 DNA vaccine 之效力而能引發足夠之免疫力對抗癌症細胞一個 self-antigen neu 經由更換較強之 CMV promoter 及去除微量內毒素 endotoxin 造成之干擾而所發展之 neu DNA vaccine, 我們首次証明 neu DNA vaccine 不只可作為 prevention 也可在動物腫瘤形成後產生療效 此論文已發表在 Molecular Therapy 在科學界當時唯有義大利 Forni's group 及我們首次證明 neu DNA vaccine 具有腫瘤治療用途 同時我們也結合各種不同 cytokine 包括 IL-2, Il-4, GM-CSF, 比較之後 N'-neu-IL-2 DNA vaccine 具有最佳治療效果,2006 年已經由學校技轉中心申請獲得中華民國 I 號專利 ( 具有腫瘤相關基因與細胞激素基因之 DNA 疫苗及其合成方法 ) 及申請獲得美國之專利 ( )), 最後希望能逐步進入臨床應用 ( 二 ) 在我們研究之 neu DNA vaccine or Met DNA vaccine, 我們進一步證實可和 HSP90 抑制劑 geldanamycin ( 國外目前在 phase II clinical trial) 藥物合併使用具有更強抗癌效果 在免疫效果上可看出免疫 T 細胞及 natural killer 細胞在腫瘤處浸潤增加 目前發表 Molecular Therapy ( 三 ) 一般在進行 DNA 疫苗須使用不同種族 (Xenogenic) 較能產生療效, 但有醫學上之顧慮, 因為如豬或老鼠的 DNA 將被打入人體 而我們仔細比較 autologous ( 同種 ) 及 xenogenicdna 經由不同途徑 ( 肌肉注射或基因槍皮膚注射 ), 之治療癌症效果, 我們發現 autologous ( 同種 )DNA 利用肌肉注射同樣產生療效 如此未來醫學使用上可視情形而不用 xenogenic DNA. 目前發表在 Vaccine 2007 Jan 8;25(4): ( 四 ) 傳統上利用基因槍送 DNA 進入細胞, 必須將 DNA coated with gold, 這種 coating 有兩個缺點 : (1) 在人體使用時擔心金粉在人體內之其他副作用 (2) 金粉會改變人體內免疫反應使傾向 Th2 humoral antibody response 由於治療癌症主要靠 cellular immunity 如 CD8+ T cell and NK cell, 若能 Divert DNA vaccine 產生之 response to Th1 response 則有益於治療癌症 我們和台灣生物鎵 (Bioware) 公司合作, 改進該公司之 low-pressure gene gun (US and Taiwan patented by this company), 並證實生物鎵公司自製之基因槍可將 naked DNA 送入老鼠皮下 dendritic cell 由於並無 coating any substance, 老鼠產生明顯之 Th1 cellular immunity 及明顯抗癌作用. 使用同劑量之 DNA (1 microgram), 不論裸露 DNA 或金粉包埋 DNA, 皆可產生同樣療效 目前正在繼續研究此基因槍之其他多功能使用發展 同時台灣生物鉀 (Bioware) 公司正在測試此該基因槍之耐用性 ( 可擊發次數 ) 以上市 二 自從 2000 年起和蘇益仁, 黎煥耀教授合作研究 hepatitis B virus pre-s mutant protein 及所產生之 endoplasmic reticulum stress 如何導致 hepatocarcinogenesis. 我的實驗室研究方向加入了肝癌及內質網壓力訊息傳遞

11 我們這個 team 在最近三年發表了數篇具質量的論文 : (a) Hepatology ;41(4):761-70, SCI=10.3(b) J Biol Chem. 2004;279(45): SCI=5.8 (c) Carcinogenesis. 2004;25(10): SCI=5.3 (d) J Hepatol ;39(5):834-42, SCI=6.0 (e) Biochem Biophys Res Commun ;336(3): (f) Mol Cancer Res. 2007;5: SCI=4.75 (g) 寫了一篇 Review article in Cancer Sci. 2006;97: SCI=3.8 我們實驗室的數據顯示 B 型病毒之產物表面抗原可經由慢性內質網壓力產生 COX-2 由此再進一步導致癌症, 發表於 J. Biol. Chem. 這個重要發現有兩層意義 :(1) 慢性內質網壓力會造成細胞對抗壓力而增加抗死亡之存活能力, 因此而導致癌症 (2) 也顯示服用 COX-2 inhibitor 或 COX inhibitor 是可能預防肝癌之產生 未來將在動物實驗中進行預防 (chemoprevention) 實驗, 若在動物實驗有效, 將尋求醫師合作進行臨床試驗 我們進一步利用 yeast two-hybrid 找到 Acid glycosidase 為 HBV large surface protein 之一個 interacting protein, 而且改變 cancer cell carbohydrate metabolism. 此也開始引發我們對 cancer metabolism 研究之興趣 以上這兩部份發現, 是我個人較為喜歡具有創新性 我們初次在國家型計劃成果報告, 或在生化學會報告此二發現, 引發很多之討論及問題, 不過經由多方實驗驗証, 最後証明我們的兩個假說 (1) DNA against self-antigen neu can function as therapeutic agent, (2) Endoplasmic reticulum stress can induce carcinogenesis via COX-2. 的確是正確的 ( 三 ) 由 publication list 中可能可以看出我的實驗室另外有一個小題目, 關於 MST3 kinase. 其原由是早期對 bladder cancer 進行 kinase 分析, 意外 clone 到一個 novel gene, 在 2002 和國衛院黃奇英研究員合作發現 nuclear translocation of MST3 during apoptosis. 發表在 J. Biol. Chem. 去年年我們又進一步確定此 kinase 有特異 cofactor preference for Zinc ion and cobalt ion. 發表在 J. Inorg. Biochem. (2005) 同時最近我們也發現 MST3 novel regulatory role in migration. 此論文目前在也已發表在 J. Biol. Chem.(2006) 這個小題目, 經由數年慢慢挖掘, 也漸更了解 MST3 功能, 由於我們已成功找出 MST3 控制 migration 之作用分子為 protein tyrosine phosphatase PTP-PEST, 目前正由基因体計劃支持進行轉殖鼠以更深入探討 MST3 在生物體內之功能 此 MST3 study 也受到注意, 收到 Cellular Signalling 雜誌 (SCI=4.8) Editor-in-Chief, Dr. Miles D. Houslay, 邀請寫 Ste-20 related kinase review article. 最近已被接受, 將於明年發表 未來研究將繼續過去 DNA vaccine and ER stress 兩方面研究 : 一 Development of neu DNA vaccine: (1) 由於 DNA vaccine 在未來臨床使用, 很可能是和其他治療法合併使用, 所以我們在本計劃中發展免疫調整 DNA 疫苗, 未來可使用於不同腫瘤癌症 (2) Combination therapy of neu DNA vaccine and other molecular targeted drug. 我們並不 focus on combination with traditional chemotherapy, 因為傳統 chemotherapy 副作用大 我們將試圖結合 DNA 疫苗及新發展之以特定基因產物為目標之藥物

12 (3) 改進 DNA vaccine 傳遞方式 : 目前基因槍須將 DNA coated with 金粉, 並不經濟, 且有其他生理作用 我們正和 Bioware 公司合作微量 DNA 基因槍傳送 naked DNA without coating! 二 Endoplasmic reticulum stress signal pathway and lipid metabolism: 我們發現 HBV large surface protein or endoplasmic reticulum stress 皆會改變脂肪代謝 同時最近癌症開始被認為同時是一種代謝疾病 Alterations of carbohydrate and lipid metabolism are frequently observed in tumor cells. 研究顯示利用針對脂肪代謝基因之藥物也發現可殺死癌細胞而不影響正常細胞 表 C012 共頁第頁

13 十二 研究計畫內容 : ( 二 ) 研究計畫之背景及目的 請詳述本研究計畫之背景 目的 重要性及國內外有關本計畫之研究情況 重要參考文獻之評述等 本計畫如為整合型研究計畫之子計畫, 請就以上各點分別述明與其他子計畫之相關性 ( 三 ) 研究方法 進行步驟及執行進度 請分年列述 :1. 本計畫採用之研究方法與原因 2. 預計可能遭遇之困難及解決途徑 3. 重要儀器之配合使用情形 4. 如為整合型研究計畫, 請就以上各點分別說明與其他子計畫之相關性 5. 如為須赴國外或大陸地區研究, 請詳述其必要性以及預期成果等 ( 四 ) 預期完成之工作項目及成果 請分年列述 :1. 預期完成之工作項目 2. 對於學術研究 國家發展及其他應用方面預期之貢獻 3. 對於參與之工作人員, 預期可獲之訓練 4. 本計畫如為整合型研究計畫之子計畫, 請就以上各點分別說明與其他子計畫之相關性 Background Plasmid DNA vaccine was discovered as a method to elicit humoral and cellular immunity against infectious diseases and cancer [1-7]. The simplicity and stability of DNA vaccines confer advantages over certain current immunological manipulations. Furthermore, DNA vaccine can be a functional adjuvant to current cancer therapy [4]. DNA vaccine is very effective against exogenous antigen, such as human papilloma virus (HPV) E7, DNA vaccine targeting HPVE7 is both a prevention and therapeutic anticancer agent [5-6]. HPV DNA vaccine represents a paradigm for the success of DNA vaccine against exogenous antigen. As for endogenous antigen, DNA vaccine targeting epidermal growth factor (EGFR) is effective for cancer overexpressing EGFR; however, the DNA vaccine must be xenogenic origin (e.g. human vs. mouse). Autologous EGFR DNA vaccine was ineffective in provoking immunity [8]. Other endogenous target antigens including c-met [9], mesothelin [10], Muc1 [11], tyrosinase [12-13], et. al. HER-2/c-erbB2/neu, One of the most prominent endogenous tumor antigens, has been used as an immunological target for DNA vaccine, and was demonstrated functional as a prophylactic agent [14-15]. Forni s group and our group successfully demonstrated that neu DNA vaccine functions as a therapeutic agent on established tumor [16-19]. Previous studies have used tumor cells transfected with exogenous neu or transgenic animal model [16-18]. Our group examined the effects of neu DNA vaccine in mice on tumor cells naturally overexpressing endogenous

14 mouse HER2/neu, and demonstrated the vaccine breaks tolerance to self-antigen neu and induced a CD8+ T cell-dependent response to eradicate established Her2/neu-expressing tumor [19]. Although DNA vaccine has been demonstrated an effective anticancer agent in several animal model, the therapeutic effect on human patients are less satisfactory [13, 20-21]. Therefore, more improvements on the DNA vaccines are awaited. These methods include immune modulation, such as conjugation with cytokine gene [19], modifying antigen presenting cells [22, 23]. Modification of delivery method may also improve the therapeutic efficacy of DNA vaccine [24]. The delivery method also affects the immune pathways. DNA vaccines are usually delivered by intramuscular injection (plus electroporation) or particle-mediated gene gun bombardment. Intramuscular injection induces a predominantly T helper1 (Th1) response, whereas a gene gun-mediated DNA immunization elicits predominantly T helper type 2 (Th2) responses [25-29]. The differences in immunological responses may be influenced by the amount of DNA and the associated CpG motifs, the nature of antigen, and the particle used in delivery [30,31]. The non-viral method can be considered as more safety and simplicity although the efficiency is generally lower than viral method [32-34]. Recently, we have developed a method to deliver non-carrier plasmid DNA into mouse epidermis by a novel low-pressure gene gun with the cooperation of Bioware Company, Taiwan. The expression of DNA-coded protein was dose-dependent as demonstrated by luciferase imaging in vivo, and the localization of expression was mainly in epidermis. The cytokine profile suggests that a Th1-bias response is provoked. Furthermore, the therapeutic function of non-carrier naked neu DNA vaccine was comparable to gold-coated neu DNA vaccine as demonstrated by significant delay of tumor progression and extension of mice life span. (Submitted, Please see attached manuscript in Appendix). Since the cancer patients are frequently immune compromised [35, 36], DNA vaccine targeting the immunoregulatory genes may provide the further therapeutic efficacy in these patients. Furthermore, the immunoregulatory DNA vaccine may be used in many different types of cancer because it may not target any specific tumor association antigens. To achieve the immunogeulatory function with DNA vaccine, the plasmid DNA shall encode genes for immunostimulatory, such as interleukin 2, or contains a small interfering RNA against immunoregulatory function [33,37]. We have previously demonstrated the IL-2-fusion gene can enhance therapeutic efficacy

15 [19]. In this proposal, we would like to employ the second approach, use of sirna, to enhance the therapeutic efficacy of DNA vaccine. There are a lot of genes involve the interaction between dendritic cells, T cells, and regulatory T cells [32, 38]. We select the thrombospondin (TSP)-1 and thrombospondin-2 as the sirna targets in this research proposal. The rationale for selecting these genes are three folds: (1) The target gene products regulate the microenvironment of dendritic cells. Because it less likely delivers the sirna genes to most of the dendritic cells, we hope the alteration of a portion of dendritic cells may affect other surrounding dendritic cells. Thrombospondins are potent anti-inflammatory molecules secreted from antigen-presenting cells (APC) and platelets, serve to generate regulatory T cells [39, 40]. TSP-1 and its binding partner CD47 are important in mediating ATP-induced immunosuppression [41]. Furthermore, TSP-1 is required for achieving bystander suppression in both plasmacytoid dendritic cells (pdc) and myeloid dendritic cells (mdc) [42]. (2) TSP-1 is known important for anti-angiogenesis, and is considered as an anti-cancer agents [43, 44]. However, TSP-1 is also an important molecule for immune suppression. In this proposal, we will test the novel hypothesis that TSP-1 sirna may function as an anticancer agents by boosting immune response with DNA vaccination. (3) TSP-1 and TSP-2 are structurally and functionally similar, but with one major difference. TSP-1 can activate TGF-beta; in contrast, TSP-2 cannot activate TGF-beta [45]. The role of TSP-2 in immune system is much less addressed in previous literature. To differentiate the role of TGF-beta in TSP-1 sirna DNA vaccination, we will use TSP-2 as a comparison and also wish to demonstrate the potential role of TSP-2 in dendritic cell activation in vivo. We will give a brief background introduction for TSP-1 and TSP-2. [Background for TSP-1 and TSP-2] Thrombospondins (TSP) are a family of multidomain, calcium-binding extracellular glycoproteins that are synthesized and secreted into the extracellular matrix of many types of cells. The TSP family can be subdivided into two subfamilies. Subgroup A, TSP-1 and TSP-2, forms homotrimer. Subgroup B, TSP3-5, forms homopentamer. All five TSPs have a carboxyterminal domain that can interact with CD47, and several copies of type 3 repeats that mediates calcium binding. In addition, they all have at least three copies epidermal growth factor (EGF)-like or type 2 (EGF-like) repeats. A distinct feature of subgroup A,TSP-1 and TSP-2, is the thrombospondin type1 (TSR) repeat which interact with CD36 and beta-1 integrins. Both TSP-1 and TSP-2 have three copies of TSR repeats. The TSR domains have been shown to inhibit angiogenesis and tumor progression

16 [46-47]. A therapeutic agent, ABT-510, an 8 amino acid fragment in the second TSR, have been examined for its efficacy in phase II clinical study [44]. The TSR repeats of TSP-1 can activate transforming growth factor beta (TGF-beta); in contrast, the TSR repeats of TSP-2 cannot activate TGF-beta since it lack the three amino acid sequence RFK between the first and second TSRs in TSP-1 [48]. The differential ability to activate TGF-beta may be the major difference between TSP-1 and TSP-2. The N-terminal domain of TSP-1 and TSP-2 confers high affinity binding to heparin. This domain is also for endocytosis of TSP-1 and TSP-2 through a low-density lipoprotein related protein (LRP)-dependent pathway [49-50]. The current concept for the antiangiogenic effect of TSP-1 and TSP-2 involves a pro-apoptotic effect on the endothelial cells. The interaction between TSR domains and CD36 receptor mainly contribute to the apoptosis in a caspase-dependent fashion [51-52]. This interaction also inhibit the migration of endothelial cells induced by vascular endothelial growth factor (VEGF) or basic fibroblast growth factor (bfgf) [45, 53] Low-density lipoprotein related protein has also been suggested to participate in the antiangiogenic activity of TSP-2 [54]. The expression of TSP-1 has been linked to inflammation, wound healing, atherosclerosis, and arthritis [55-57]. A simplified carton for TSP-1 is shown: TSP-1 and TSP-2 may have different physiological functions. TSP-1 is a major TGF-beta activator [58], but TSP-2 cannot activate TGF-beta. TSP-1 null mice had an increased number of white blood cells, with largest increases in monocytes and eosinophils. Although normal at birth, lung inflammation with neutrophils and macrophage were observed in 4-week-old mice [59]. These results suggest that TSP-1 may play an important role in anti-inflammation, more important in lung. TSP-null mice had an increase of vascular density and reduced fibroblast adhesion. Morphological changes in connective tissue and fragile skin with reduced tensile strength were also observed. Matrix metalloproteases may be the mediator for

17 fibroblast defect in TSP-null mice [60-61]. The endogenous TSP produced by early dendritic cell activation negatively regulate IL-2 and TNF-alpha. After long-term activation, dendritic cells terminate the synthesis of these cytokines and become refractory to further stimulation. Blocking the interaction between TSP-CD47 interactions during restimulation restores the cytokine synthesis. TSP was demonstrated to be an autocrine negative regulator of dendritic cell activation [62]. Extracellular ATP can induce immunosuppression through its action on dendritic cells. Microarray analysis of ATP-stimulated human DCs revealed a dramatically increase of thrombospondin-1 and indoleamine 2,3-dioxygenase. The release of TSP-1 was demonstrated further by ELISA and exerts antiproliferative effects on CD4+ T cells through interaction with CD47 [63]. The thrombospondin/cd47 interaction was then was then demonstrated to promote the generation of regulatory T cells from CD4+CD25- T cells. This pathway may participate to the limitation of exacerbated immune responses to self or foreign antigen. The regulatory activity is contact-dependent and TGF-beta-independent [40]. The other domain of TSP-1 is also involved in inducing dendritic cell tolerance. Apoptotic cell TSP-1 can be cleaved to generate 26 kda heparin-binding domain (N-terminal) which can induce immature dendritic cells to increase phagocytic ability and to tolerate the engulfed antigen. The full-length TSP-1 also have such ability, but with a lesser extent. CD29, CD36, CD47, CD51, and CD91 may all participate in generating the dendritic cell tolerizing states [64]. Recently, TSP-1 is proved to be essential for dendritic cell bystander suppression [42]. Bystander suppression can be described as an inhibition of a memory T cells response as a result of colocalized antigen [65-66]. Altogether, TSP-1 plays an important negative role on dendritic cells, and in part due to its effects on TGF-beta. TSP-2, in contrast to TSP-1, is much less studied in the regulatory function of immune system. Although TSP-2 is incapable of activating TGF-beta, we hypothesized TSP-2 may have certain roles in immune regulation. The first is that overall structure of TSP-1 and TSP-2 are similar. TSP-2, also contains the N-terminal heparin binding domain, and may also have the ability to tolerize the dendritic cells as TSP-1 [64]. The second is that prolonged inflammation and a local deficiency of T-cell apoptosis was observed in TSP-2 null mice. The interaction between TSP/CD47 and the Bcl-2 family member BNIP3 may mediate the reduced inflammation [67].

18 {The unsolved questions about TSP-1 and TSP-2 in immune system} TSP-1 has been clearly demonstrated to play an important role in negative regulation of dendritic cells and T-cell interaction; however, the role of TSP-2 was relatively studied in its function on dendritic cells. Furthermore, TSP-1 could function as a therapeutic agent in anti-angiogenesis and arthritis. Whether the TSP-1 antagonist may function as an immune modulatory agent in dendritic cells is largely unexplored. Rationale: If the TSP-1 or TSP-2 (as a reference for comparison of TGF-beta effect) play and important role in immune suppression through dendritic cells, the delivery the TSP-1 or TSP-2 sirna into skin dendritic cells by gene gun bombardment will enhance the immune response and generate an anticancer effect on an established tumor.

19 Specific Aims 1. DNA vaccine expressing TSP-1 and TSP-2 with sirna (the plasmid containing a shrna gene will generate the sirna in vivo) will be examined for their therapeutic efficacy on established tumor subcutaneously in vivo. 2. Investigate the immunological mechanism responsible for delaying tumor growth in vivo. 3. To further define the functional domain of TSP-1 is required for immune suppression in vivo. We will deliver the DNA vaccine containing the TSP-1 sirna with truncation TSP-1 4. Can TSP-1 shrna enhance DNA vaccine targeting tumor associated antigen, such as neu. 5. Investigate for the possible the use of TSP-1 or TSP-2 sirna on the established tumor in a murine In situ hepatoma model. Significance 1. This will be the first example that a molecule against TSP-1 (TSP-1 sirna) may function as a cancer therapeutic agent through immunological modulation. 2. Our results also point to an important consideration for the systemic use of TSPs as an anti-angiogenic agent to cure cancer. The systemic use of TSPs may dampen the immune response and enhance the tumor growth. If our results suggests this direction, novel TSP agents must be designed to generate anti-angiogenesis, but not to or mildly affect the immune response. 3. The immunological mechanism will reveal the difference between TSP-1 and TSP-2 and how the TGF-beta was involved in the immune suppression in vivo. 4. The last aim will extend the use of TSP- sirna to an orthotropic animal model. The immune response is clearly different whether the tumor is implanted subcutaneously or orthotropically. Therefore, we will use a murine In situ animal model developed (Hepatology 2007; 45: )in Dr. Huan-Yao Lei Lab at National Cheng Kung University to examine the therapeutic efficacy of our DNA vaccine (TSP-1 and TSP-2 sirna). If it can function in an orthotropic animal model, we may push it into clinical trial in the future.

20 [Preliminary Results] 1. We have constructed TSP-1 shrna construct and demonstrated that it could attenuate TSP-1 expression. The c-myc-tsp-1 was cotransfected with TSP-1 shrna and the expression was almost completely inhibited. The control vector has no effect 2. To demonstrate the TSP-1 shrna can downregulate the TSP-1 in vivo, we perform the following experiments. Mice were inoculated with TSP-1 sirna with gene gun, the lymph node were harvested and the supernatants were assayed for TSP-1.

21 3. To further demonstrate the TSP-1 sirna decrease the expression of TSP-1 in dendritic cells, cd11c+ dendritic cells were harvested with magnetic beads and RNA analyzed with RT-PCR. The expression of TSP-1 is much lower in the dendritic cells isolated. 4. We then constructed the TSP-1 sirna with N -neu DNA vaccine. The plasmid can express N-terminal neu DNA vaccine and TSP-1 sirna. It inhibits the expression of c-myc-tsp-1 as shown below.

22 5. We have performed cancer therapeutic experiment on MBT-2/C3H animal tumor model. (a) The TSP-1 shrna alone can delay tumor progression, and further enhance neu DNA vaccine therapeutic efficacy. (b) Kaplan-meir analysis of mice survival demonstrates the therapeutic efficacy of TSP-1 sirna. The plasmid containing TSP-1 sirna and neu DNA vaccine had the best therapeutic efficacy.

23 6. We have used RT-PCR to detect the cytokine expression pattern. The results indicated that TGF-beta was downregulated and IFN-γ was enhanced. Foxop3 can be enhanced by neu DNA vaccine, but was attenuated by coadministration of TSP-1 sirna.

24 7. To further confirm specificity of TSP-1 shrna, we have constructed TSP-1 scramble sirna, and assayed its therapeutic efficacy. The scramble sirna did not affect N-terminal neu expression, and did not affect TSP-1 expression. 8. We then assayed their therapeutic efficacy. The results clearly indicated the TSP-1 sirna alone had therapeutic effect, and can be further enhanced by fusion with other DNA vaccine against tumor-associated antigen, such as neu.

25 9. We further confirmed the enhancement of IFN-gamma. 10. Altogether, the preliminary results have demonstrated TSP-1 shrna can function as therapeutic agent by simple inoculation on skin with gene gun. This data provides a future alternative therapy. Furthermore, the preliminary results suggest that the TSP-1 sirna may act on Treg cells (expressing FoxoP3) and alter cytokine profile.

26 [Research Design and Methods] Specific Aim 1: DNA vaccine expressing TSP-1 and TSP-2 with sirna (the plasmid containing a shrna gene will generate the sirna in vivo) will be examined for their therapeutic efficacy on established tumor subcutaneous in vivo. {Animal models used in DNA vaccine Experiment in Specific Aim 1} In all the following experiments, three animal tumor models will be used. The first is the MBT-2 tumor (overexpressing oncogene neu) and its syngeneic host C3H mice. This is the animal model we frequently used; the second is the B16 melanoma cells (overexpressing c-met) and its syngeneic host C57BL/6; the third is murine colon carcinoma CT26 cells and its syngeneic host Balb/C mice. All experiments will be examined in MBT-2/C3H animal model first and followed by one of the other two animal model systems. {Delivery method} Gene gun delivery of naked DNA will be the primary choice, 10 μg of DNA/per mice will be delivered into mouse epidermis. When Th1/Th2 response is investigated, then gold-coated DNA will be used alternatively in gene gun inoculation. Since dendritic cells in skin (Langerhan cells) may react differently from the antigen presenting cells present in muscle, intramuscular injection of 100μg/per mice will be used in assaying different types of dendritic cells in vivo. Because cross-presentation may play an important role for the function of DNA vaccine in muscle, we expect that the sirna against TSP-1 or TSP-2 may be less effective. However, we will examine this possibility. Vaccination protocol follows reference [9, 19] {Question} Does DNA containing shrna against TSP-1 or TSP-2 as an anticancer agent? {Hypothesis} Downregulating the expression of TSP-1 will render the dendritic cells loss of autocrine negative regulation and decrease of surrounding negative regulatory T cells. {Rationale} (1) Among the immunoregulatory enzymes, we first select TSP gene because the secretory protein can produce bystander effect. (2) If the TSP plays an important role mediating the inhibitory effect of regulatory T cells on dendritic cells, then the plasmid DNA contains a shrna against TSP will activate the dendritic cells to present tumor antigen and generate anti-tumor immune response. (3) coadministration of plasmid encoding an antigen and a sirna against bax or Bcl-2 have been successfully demonstrated by Professor TC Wu [23]. Therefore, we expect

27 the sirna against TSP may function in dendritic cells in a similar fashion. {Research Aims} (1) Whether TSP-1 shrna functional as a anticancer agent? (2) What is immunological mechanism of TSP-1 shrna? (3) Can TSP-1 shrna enhance DNA vaccine targeting tumor associated antigen, such as neu? (4) Similar approach will be worked on TSP-2, and to observe the possible involvement of TGF-beta.. {Experimental Approach 1} This standard approach will be used in all the following sirna against TSP-1 and TSP-2, we will use TSP-1 as an example (A) Establish a shrna plasmid to downregulate TSP-1 in vitro and in vivo. (1)Short-hairpin RNA (shrna) targeting three different sites on TSP-1 gene will be constructed. In our previous experience, one of the three shrna will be effective in downregulating the targeting gene TSP-1 in average. Mouse U6 promoter will be used to express shrna. (2) Transient cotransfection of pcmv-driven HA-TSP-1 gene with U6promoter-driven TSP-1 shrna, and examine which TSP-1 shrna is functional in downregulating TSP-1 expression in vitro. (3) downregulation of TSP-1 will be demonstrated in vivo. Plasmid encoding GFP and TSP-1 shrna will be delivered into mouse epidermis, and dendritic cells in lymph node will be enriched with cd11c magnetic bead. The enriched cells will be sorted with GFP and stained with anti-tsp-1 antibody. (B) Assaying the cancer therapeutic efficacy in animal tumor model. (1) C3H mice will be implanted 1 million MBT-2 tumor cells. Ten days after implantation, tumor will become palpable. The tumor-bearing mice will be inoculated with (a) DNA containing shrna against TSP-1, or (b) DNA containing U6-promoter driven scramble RNA three times at weekly interval. The tumor growth will be measured and the mouse survival will be subjected to Kaplan-meir analysis. (2) The same procedure will be repeated on either one of the two animal tumor models: B16 melanoma in C57BL/6 mice and CT26 cells in Balb/C mice. {Data collection and interpretation} (A) The data on Experimental Approach 1A are straightforward, we just need to identify a shrna is effective in reducing TSP-1 expressing in vitro and in vivo. We have cloned mouse TSP-1 gene. Transient transfection assay with TSP-1 shrna and pcmv-tsp-1 will demonstrate which TSP-1 shrna is functional. Our preliminary results have demonstrated that we have identified an effective TSP-1 shrna. For in vivo assay, The TSP-1 sirna also decrease TSP-1 content in the population of the cd11c+ cells in lymph node (data not shown). We will continue to construct a shrna

28 targeting TSP-1-2. (B) The scramble sirna will be used as negative control. The scramble sirna may have minor effect since plasmid DNA is immuno-stimulatory itself and that the small double stranded RNA may exert certain immunological response. However, the authentic TSP-1 shrna shall show statistical significant difference on cancer therapy. The therapeutic efficacy will be determined by decrease of tumor volume and Kaplan-meir curve of mice survival. Our preliminary result supported this concept, and we demonstrated the therapeutic efficacy of TSP-1 shrna in one animal tumor models (MBT-2/C3H). We will continue test the therapeutic effects on other animal tumor model.

29 Specific Aim 2: Investigate the immunological mechanism responsible for delaying tumor growth in vivo. Research Outline: Investigate which immunological response is mainly responsible for anti-tumor effects. Five types of experiment will be used to investigate the immunological mechanism. (1) dendritic cell maturation and activity: the activation of dendritic cells. cd11c+ positive cells will be enriched from lymph node. Three parameters will reveal their activity (a) the expression of CD80, CD86, MHC class II, class I. (b) The expression and activation of TGF-beta which may explain the immune difference between TSP-1 and TSP-2 (c) The cytokine profile of IFN-gamma, IL-6, IL-12, IL-18, and IL-10, (d) mixed lymphocyte reaction will be performed by either one of the following two methods. Naïve CD4+ T cells will be isolated from peripheral blood mononuclear cells using a CD4+ T cell isolation kit (Miltenyl Inc.). The isolated CD4 T cells will be depleted with anti-cd45r antibody. The naïve CD4+CD45RO- cells will be cocultured with different concentrations of mature DC. Cell proliferation will be measured by [tritium]thymidine incorporation five days later. (2) cellular immunity: examination of tumor infiltrated lymphocytes, including CD4+ T cells, CD8+ T cells, neutrophils, natural killer, macrophage. The tumor will be excised from vaccinated mice and control mice and subject to immunohistochemical analysis with antibodies against each type of lymphocyte. In addition, the activity of cytotoxic T lymphocyte will be measured by coculture effector T cells with target tumor cells (expressing luciferase). The release of luciferase will be the indicator of CTL activity. (The method was developed in our Lab, ref. 9,19). (3) humoral immunity: determine the antibody response to the whole cell tumor antigen. The serum from vaccinated mice will be used to probe the total cell lysate of tumor cell in western. Since the TSP-1 shrna may enhance immune response to various antigens, we need to analyze the serum antibody response to the whole tumor cell lysates. This method has been used in our previous publication [ref 18]. (4) Whether TSP-1 shrna shift the Th1/Th2 profile: we will examine the cytokine profile, including IFN-gamma, IL-2, 4, IL-10, IL-12, and IL-18. (5) Which type of lymphocytes mainly mediate anti-tumor immune response: we will deplete the designated lymphocytes, and examine the anti-tumor effect of TSP-1 shrna. CD4, CD8, CD25, and NK cells are primary depletion targets since these cells are known to be important for the immunological responses to MBT-2 tumor cells [9, 16, 17, 19]. In the case needed, we will deplete other type of lymphocytes,

30 such as neutrophil. Our preliminary result indicated that significant infiltration of neutrophil around tumor sites was observed. {Data collection and interpretation} (1) We expect to observe the activation of dendritic cells by the enhancement of CD80 and CD86, MHC class molecules. The increases expression of cytokine IFN-gamma and IL-12 also suggest the activation. The mixed lymphocyte reaction will demonstrate the function of these matured dendritic cells in vitro. The expression of TGF-beta should be significantly different when we compare the immune effect from TSP-1 shrna and TSP-2 shrna. We will use this as criteria to judge how TSP-2 function as a therapeutic DNA vaccine. (2) and (5) we will examine the correlation between the amount of different types of tumor infiltrated lymphocytes with the degree of tumor regression in different vaccine group of mice. This result will first reveal which type of lymphocytes may be important for anti-tumor effect. To further confirm which type of lymphocyte is indeed important for immune response, depletion of a specific type lymphocyte with antibody will reveal its role in immune rejection of tumor. In addition, Depletion of CD25 T cells will reveal whether regulatory T cells (mainly CD25+ FoxP3+) plays an important role in TSP-1 sirna mediating anticancer effect. If the result is negative, then the TSP-1 sirna may go through Treg-independent pathway to regulate anti-tumor immunity. (3) Since we did not inoculate any vaccine targeting a specific antigen, we are unable to focus on any specific tumor-associated antigen. We choose to probe the total cell lysates with antiserum from vaccinated mice based on our previous experience. If the quality of results were not good enough, we will used to vaccinated/control mice serum to probe the tumor sample by immunohistochemical analysis with serial dilution of mice serum. In this way, we may see the increase staining of tumor cells with vaccinated mice serum. (4) Expression of IL-4 and IL-10 suggests a Th2-bias response, while IL-12, IL-18, IFN-gamma suggests a Th1-bias response. Downregulation of TSP-1 may lead to cytokine profile change and the cytokines further enhance total immune activity or switch immune response Th1/Th2 pathway..

31 Specific Aim 3: To further define which functional domain of TSP-1 is required for immune suppression in vivo. We will deliver the DNA vaccine containing the TSP-1 sirna with truncation TSP-1. Rationale: The TSP-1 contains multiple function domains to interact with CD36, CD47, and heparin. We will define the functions of these domains of TSP-1 by compensation study. Research Method (A) Construction of the following plasmids (1) a plasmid containing pcmv-n -TSP-1 (expressing extracellular domain that interact with heparin, and U6 promoter-drive TSP-1 shrna (2) a plasmid containing pcmv-n -TSP-1 and U6 promoter-drive scramble TSP-1 shrna. (3) a plasmid containing pcmv-type-1- repeat-tsp-1 (expressing type 1 repeat that interact with CD36, and U6 promoter-drive TSP-1 shrna (4) a plasmid containing pcmv-type-1- repeat-tsp-1and U6 promoter-drive scramble TSP-1 shrna. (5) a plasmid containing pcmv-c-terminal-tsp-1 (expressing C-terminal portion that interact with CD47, and U6 promoter-drive TSP-1 shrna (6) a plasmid containing pcmv-c-terminal-tsp-1 and U6 promoter-drive scramble TSP-1 shrna. These 6 DNA vaccines will be examined in MBT-2/C3H mice animal model to test its therapeutic efficacy. (B) is similar to that described Experimental approach for Specific Aim 2. {Data collection and interpretation} The basic assumption of this experimental design is that co-expression of the segment of TSP-1 with TSP-1 shrna will attenuate the therapeutic efficacy and increase the tumor growth if the TSP-1 mainly negatively regulate the dendritic cells through this domain. For example, if TSG/CD36 interaction is most important in mediating the dendritic tolerance, the plasmid containing pcmv-type-1- repeat-tsp-1, and U6 promoter-drive TSP-1 shrna will have no effect on tumor growth. In contrast, a plasmid containing pcmv-c-terminal-tsp-1 and U6 promoter-drive TSP-1 shrna will have similar anti-tumor effects as the U6 promoter-drive TSP-1 shrna. {Potential difficulties} The data obtained in this part are more complex than other part. The expressing of truncated TSP-1 may be considered as a compensation for the downregulated endogenous TSP-1; however, the truncated TSP-1 itself may function as antigen to induce anti-tsp-1 autoantibody. We cannot exclude this possibility; however, we have expressed IL-2, IL-4, GM-CSF, but we did not detect any anti-il-2

32 antibody in previous study. Therefore, we hope TSP-1 may react as a similar fashion to these cytokine without inducing autoantibody which may interfere with our data interpretation. On the other hand, we may use small molecule TSP mimics ABT-510 in compensation study.

33 Specific Aim 4: Can TSP-1 shrna enhance DNA vaccine targeting tumor associated antigen, such as neu? Rationale: The aim of this experimental approach is to determine whether TSP-1 shrna enhance DNA vaccine targeting tumor associated antigen, such as neu. In this way, we can combine TSP-1 sirna with different DNA vaccines against various tumor associated antigens, which make the TSP-1 sirna have more versatile function. Research Method: (A) Construction of the following plasmids (1) a plasmid containing pcmv-n -neu (expressing extracellular domain, (please see our publication in reference 19) and U6 promoter-drive TSP-1 shrna (2) a plasmid containing pcmv-n -neu (expressing extracellular domain, please see our publication in reference 19) and U6 promoter-drive scramble TSP-1 shrna. The expression of neu and the downregulation of TSP-1 by TSP-1 shrna will be examined in vitro. These plasmids will be inoculated in a similar protocol as describe in Research methods for Specific Aim 1, and examined for therapeutic efficacy. The MBT-2/C3H animal model will be used since neu is naturally overexpressed in MBT-2 cells. (B) is similar to that described Experimental approach for Specific Aim 2. There are only three differences: Since that neu DNA vaccine is administered in this experimental design. Therefore, (3) the humoral immunity will be determined by anti-neu antibody titer with ELISA as we have performed before. Secondly, the IgG subtype will be determined, which will further support the Th1/Th2 bias determined in (4). Thirdly, in the process of measuring the cytotoxic T lymphocyte activity, the lymphocyte will be activated by recombinant neu protein since we have inoculated with neu DNA vaccine. {Data collection and interpretation} (A) pcmv-n --neu-tsp-1-sirna is expected to give the best therapeutic efficacy in tumor regression or mouse survival. Our preliminary result has supported this concept. (B) For immunological examination, the result will indicate whether TSP-1 shrna can enhance anti-neu antibody titer or enhance anti-neu CTL activity. The results should be more specific to a tumor-associated antigen, neu. {Potential difficulties} Most of the experiments have been performed in our Lab, we do not expect too much potential difficulty. Our preliminary result has demonstrated that the TSP-1 shrna is functional, and we are proceeding on the detailed immune examination and understand the mechanism. This data has been

34 repeated and similar result were obtained (please see preliminary result). We have less experience with dendritic cells, but we can get a lot of assistance on technical problem from our Department of Immunology, Dr. Huan-Yao Lei and Dr. Yee-Shin Lin.

35 Specific Aim 5: Investigate for the possible the use of TSP-1 or TSP-2 sirna on the established tumor in a murine In situ hepatoma model. Rationale: Since the tumor formed by subcutaneous implantation does not mimic the immunological environment of orthotropic tumor, we will finally test our TSP-1 sirna in a a murine In situ hepatoma model developed in Dr. Lei s Lab. If the TSP-1 sirna can be functional in this animal model, then it will be possible to enter clinical trial in the future. Research Method: (A) The protocol follows Hepatology (2007) 45: The mice will be anesthetized by pentobarbital, 50mg/Kg intraperitoneally. Mice will be subjected to intra-spleen injection of 1 million ML-14a murine hepatoma cells in 0.1 ml DMEM medium into Anesthetized mice. About 10 days after implantation, inoculation of therapeutic DNA vaccine (TSP-1 and TSP-2) will be performed three times at weekly interval. 10 days after last DNA vaccination, the livers of were removed to determine the numbers and sizes of the tumor nodules. (B) Immunological mechanism examination is similar to that described in Specific Aim 2. {Data collection and interpretation} Similar to research method for Specific Aim 1 and 2 (Potential Difficulty) We have successfully established the murine In situ hepatoma animal model in our laboratory with assistance from Dr. Lei s ( 黎煥耀 ) Lab member. We found that gene gun inoculation at 10 day after tumor implantation may cause the opening of the seam of the surgery. We will adjust the pressure of gene gun or altering the stitching method in tumor implantation surgery. We hope it will solve the issues; however, we may use intramuscular injection of DNA vaccine to avoid the pressure damage on the pre-surgery mice alternatively.

36 An overall research design is shown below:

37 預期完成之工作項目及成果 : 1. We hope complete specific Aim1 and Aim2 in first year. Actually, we have almost complete the work on TSP-1, and we are going to work on TSP-2 now. 2. We hope to complete specific Aims 3 and 4 in the second year. As shown in our preliminary result, we have accomplished part of Specific Aim We expect to complete the Aim 5 in the last year. 成果 : (1) 我們將申請專利 on TSP-1shRNA as an immunological therapeutic agent against cancer based on our preliminary results. However, complete study will further establish its use in cancer treatment. (2) We will increase our knowledge on immune regulatory function by the use of TSP-1 sirna and TSP-2 sirna. The comparison of their effects will be very important and interesting. (3) The domain study will further demonstrate the role of each domain of TSP in vivo. (4) If TSP sirna can be functional in an In situ animal tumor model, the pre-clinical trial of this agent or combination with other agents on cancer warrants investigation in the future. 人員所受訓練 : (1) Development of sirna as therapeutic agent (2) animal tumor immunology experience (3) Cancer therapeutic training

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44 Attached please find the revised manuscript in Journal of Gene Medicine. The naked DNA delivery device is described in this article. Skin delivery with non-carrier naked DNA vaccine by supersonic flow polarizes Th1 immune response against cancer Chi-Chen Lin 1, Meng-Chi Yen 1, 2, Chiu-Mei Lin 3, Shih-Shien Huang 1, Huei-Jiun Yang 1, Nan-Haw Chow 4 1, 2, 5, *, Ming-Derg Lai 1. Department of Biochemistry and Molecular Biology, College of Medicine, National Cheng Kung University 2. Institute of Basic Medicine, College of Medicine, National Cheng Kung University 3. School of Medicine, College of Medicine, Taipei Medical University, 4. Department of Pathology, College of Medicine, National Cheng Kung University 5. Center for Gene Regulation and Signal Transduction Research, National Cheng Kung University, Tainan, Taiwan. * To whom correspondence should be addressed Dr. Ming-Derg Lai Department of Biochemistry and Molecular Biology College of Medicine National Cheng Kung University Tainan, Taiwan, R.O.C. FAX: a @mail.ncku.edu.tw Keywords: neu, DNA vaccine, gene gun, cancer, biolistic, Th1 immune response Acknowledgements: This study is supported by Grants NSC B and B from National Science Council, Taiwan, Republic of China.

45 Abstract DNA vaccine is a new and powerful approach to generate immunological responses against infectious disease and cancer. Th1 immune response is usually required for generating effective anti-tumor response. A microparticulate bombardment system can induce an immune response using very low amounts of DNA. Based on nozzle aerodynamics, a low pressure gene gun was developed to decrease the noise associated with high pressure gene gun. The particles can be propelled by a supersonic flow generated in this novel nozzle design. To test whether the low pressure gene gun could inoculate DNA vaccine to generate anti-tumor Th1 immune response, we investigated the direct delivery of naked DNA without any carrier. The luciferase reporter plasmid DNA can be delivered and expressed in C3H/HeN, BALB/c, and C57BL/6 mice by low-pressure biolistic device. Plasmid DNA expression is mainly located in the epidermis. Furthermore, non-carrier naked neu DNA vaccine exerted similar anti-tumor effects as the gold-coated neu DNA vaccine on an MBT-2 established tumor in C3H mice at the dose of 1μg/per mouse. A cytokine profile examination on the mice vaccinated with naked DNA suggests a Th1-bias immune response, in contrast to the Th2-bias response induced by gold-coated DNA vaccine.

46 Introduction Significant advances in molecular biology have made it possible to sequence the genomes of primate and infectious agents. This achievement allowed us to identify the targets for gene therapy and vaccine. However, an effective and safe delivery method is always the primary concern for developing gene-based drugs. Although viral vectors have been demonstrated in high efficiency, they induce many side effects including host immune response, random integration and contamination of wild-type virus [1,2]. Non-viral gene transfer provides a safe, superior alternative. Nonviral delivery methods include the gene gun, liquid jet injection, intramuscular direct injection, electroporation and others [3-5]. Among these methods, the gene gun, also named biolistic device, a microparticulate bombardment system, has been demonstrated to deliver genes in an effective and homogeneous pattern. The gene gun delivery method was originally designed to coat the DNA on the gold particle which is accelerated by exploding gunpowder to penetrate the barrier of plant cell wall [6]. High pressure helium replaces gunpowder as the particle propellant in most particle-mediated delivery devices [7]. The method is also applied to deliver genes into mammalian cells [8], and may be superior to the lipofectamine method for certain cell lines. In addition, the successful introduction of DNA via skin has been demonstrated in vivo [9]. Plasmid DNA vaccine was discovered as a method to elicit humoral and cellular immunity [10-12]. The simplicity and stability of DNA vaccines confer advantages over certain current immunological manipulations. DNA vaccines are usually delivered by intramuscular injection or particle-mediated gene gun bombardment. Intramuscular injection induces a predominantly T helper1 (Th1) response, whereas a gene gun-mediated DNA immunization elicits predominantly T helper type 2 (Th2) responses [13-16]. The differences in immunological responses may be influenced by the amount of DNA and the associated CpG motifs, the nature of antigen, and the particle used in delivery [17-20]. Genes delivered by a gene gun offer an advantage over intramuscular injection in that a reduced amount of DNA is used. Th2 response is usually provoked by gene gun delivery, while Th1 response is usually required for removing invading microorganism and cancer cells [21]. To decrease the noise and cell damage caused by high-pressure ( Psi) gene gun, a low-pressure (30-60 Psi) gene gun was developed (US patent B1). This low pressure biolistic delivery device can bombard gold-coated DNA into animal with supersonic flow generated by rocket nozzle design [22,23]. Since supersonic air flow is capable of carrying low density particles through cell walls, we wished to

47 further investigate the supersonic flow in this gene gun as it delivered naked DNA into an animal. Our results demonstrate that naked DNA can be successfully delivered into mice and the Th1-biased immune responses provoked were effective for cancer therapy.

48 Results Biolistic gene delivery device. A photograph of the low-pressure gene delivery device is shown in Fig. 1A. Low pressure helium is supplied through the bottom inlet. DNA sample is applied on the top hole and propelled into the target tissue via the nozzle exit. The internal features are illustrated in Fig. 1B. The inside nozzle is convergent, while the outside nozzle is divergent. The trigger momentarily releases helium gas. A supersonic flow is generated when the difference between the internal and external pressures of the nozzle is greater than 1.9 atm. This high speed supersonic airflow can accelerate particles to an extremely high speed. Previous results from our group and others indicated that the supersonic flow can safely and effectively deliver gold-coated DNA into animals and generate immune responses delaying tumor growth [22,23]. Novel biolistic gene gun can deliver non-carrier naked DNA into mouse epidermis. Initially we investigated whether non-carrier naked DNA could be delivered into the skin of C3H/HeN mice, BALB/c mice, and C57BL/6 mice. The DNA plasmid encoded luciferase was used as a reporter to examine the expression in animal, and the expression of luciferase activity was detected with 1 μg of naked DNA plasmid in three strains of mice with imaging in vivo. However, the expression of luciferase activity was significantly higher for 1 μg gold-coated DNA plasmid than that for 1, 5 or 10 μg naked DNA plasmid in C3H/HeN, BALB/C and C57BL/6 mice (Fig. 2A). Quantitation of luciferase expression was shown in Fig. 2B. To further examine the localization of expression plasmid, the gold- coated pcmv-egfp DNA plasmid or naked pcmv-egfp DNA plasmid was used to inoculate C3H/HeN mice. The mice skins were sliced 48 hrs after delivery of DNA plasmid and examined by immunofluorescence microscopy. The expression of pcmv-egfp DNA plasmids were mainly detected in the epidermis layer for either gold-coated DNA or naked DNA (Fig. 2C). Several studies demonstrate that following intradermal immunization, dendritic cells are known to migrate to draining lymph nodes where they play a major role in priming and stimulating antigen-specific T cells [24-26]. Therefore, it is important to determine whether dendritic cells express the reporter genes in draining lymph nodes when non-carrier naked DNA is administered. There is no significant difference in numbers of CD11+ and GFP-positive cells in the inguinal lymph nodes after vaccination with gold-coated 1μg pcmv-egfp DNA plasmid and 1 μg naked pcmv-egfp DNA plasmid. Interestingly, we observed a greater percentage of GFP-positive CD11c+ cells in lymph nodes harvested from mice injected with 5 and 10 μg of naked DNA than in lymph nodes harvested from mice vaccinated with gold coated with 1μg pcmv-egfp DNA plasmid or 1μg naked pcmv-egfp DNA

49 plasmid (Fig. 2D). Cancer therapy with naked DNA delivery by low-pressure Biolistic device. To examine whether non-carrier DNA vaccine had the cancer therapeutic effect, we employed an animal tumor model with MBT-2 bladder tumor cells and C3H/HeN mice [27]. Overexpression of endogenous p185 neu was observed in MBT-2 cells. We have previously demonstrated that skin delivery of gold-coated DNA encoding the extracellular portion of p185 neu (pcmv-huamn-n -neu) by low pressure gene gun had therapeutic effect on the implanted MBT-2 tumor in C3H/HeN syngeneic mice [23]. The mice were implanted 1 million MBT-2 tumor cells subcutaneously at day 1, and the tumors were palpable at approximately day 10. One μg gold-coated or non-carrier naked pcmv-human-n -neu DNA plasmid was inoculated three times at weekly interval when the mouse tumor was palpable. The tumor volume of mice was measured (Fig. 3A) and the survival curve was subjected to Kaplan-meir analysis (Fig. 3B). One μg of pcmv-human-n -neu DNA plasmid could delay tumor progression, and extend the mice life span. In addition, 1 μg of non-carrier naked DNA vaccine could achieve the same therapeutic efficacy as 1 μg of gold-coated DNA vaccine. ELISA was used to examine the antigen expression of DNA vaccine encoding the extracellular portion of p185 neu in C3H/HeN mice skin. The amount of N-terminal portion of human p185 neu produced by 1 μg of naked DNA vaccine was much lower than that produced by 1 μg gold-coated DNA vaccine (Fig. 4A). The humoral immunity induced by either non-carrier or gold-coated DNA vaccine was determined by the presence of anti- p185 neu neu antibody in mice serum. Naked DNA vaccine induced a much weaker antibody response in immunized animal than gold-coated DNA vaccine, which is consistent with the expression of pcmv-human-n -neu (Fig. 4B). Anti-tumor cellular immunity in spleen population was examined with cell-mediated cytotoxic assay using MBT-2 cells as target cells. Naked DNA vaccine and gold-coated DNA vaccine exerted similar cell-mediated cytotoxic immune responses (Fig. 4C). Therefore, we hypothesized that the non-coated naked DNA vaccine may activate a Th1-biased immune reaction and lead to cellular immune responses. The Th1/Th2 cytokine profile was examined in the mice inoculated with naked or gold-coated DNA vaccine. IFN-γ was significantly induced with naked DNA plasmid but not gold-coated DNA plasmid as demonstrated by RT-PCR. In contrast, IL-4 and IL-10 was significantly induced with gold-coated DNA vaccine, suggesting a Th2 immune response (Fig. 4D). The protein expression of IL-4 and IFN-γ was further demonstrated with ELISA. The protein expression of IL-4 and IFN-γ was generally agreed to that observed with RT-PCR; however, the difference in IFN-γ was less prominent (Fig. 4E). These results suggest that non-carrier naked DNA

50 vaccine induces a more Th1-bias immune response than gold-coated DNA vaccine based on cytokine profile. Influence of gene gun firing on DNA Plasmid Integrity Finally, we wished to determine the influence of supersonic flow generated by gene gun on the integrity of DNA plasmid, we compared the quality of the DNA before and after the shot by gene gun. The gold-coated or non-carrier naked pcmv-human-her-2/neu (size 7.5kb) DNA was loaded on the gene gun and delivered onto a filter paper at 40 psi or 60 psi by supersonic flow respectively. The DNA was recovered from the filter paper and evaluated by gel electrophoresis. The control DNA was spotted on the filter and recovered without gene gun firing. Supersonic flow by gene gun had a degree of destructive effect on the plasmid DNA at air pressures of 40 psi (gold-coated DNA plasmid) or 60 psi (naked DNA plasmid). The semi-quantitative analyses of video images revealed a 65 to 70 % shearing of naked plasmid DNA at 60 psi and 20 to 25 % shearing of gold-coated DNA plasmid at 40 psi (Figure 5). The supersonic flow indeed caused a certain degree of damage on the DNA, but about than one third of the DNA remained apparently intact. The remaining intact nucleic acid might be sufficient in yield and quality to efficiently generate enough Th1-biased immune responses in the skin.

51 Discussions In this report, we demonstrate that non-carrier naked DNA can be delivered into mouse skin by the low-pressure biolistic device via supersonic flow. The non-carrier naked DNA can induce a Th1-bias immune and function as a cancer therapeutic agent. The expression of DNA-coded protein was dose-dependent as demonstrated by luciferase imaging in vivo, and the localization of expression was mainly in epidermis. Furthermore, the therapeutic function of non-carrier naked pcmv-human-n -neu DNA vaccine was comparable to gold-coated pcmv-human-n -neu DNA vaccine as demonstrated by significant delay of tumor progression and extension of mice life span. The expression of antigen in the form of naked DNA vaccine is lower than that in the gold-coated DNA vaccine form and the antibody response induced by naked DNA vaccine delivery was comparably weak. In contrast, the cellular-mediated cytotoxicity for targeting MBT-2 cells measured by spleen cell was similar no matter the DNA vaccine is naked or coated on gold. Since the eradication of tumor is mainly mediated through CD8 + T cell in this naturally overexpressing p185 neu MBT-2 animal tumor model [23,27]. The antibody response may not play a major role in anti-tumor response, which may explain the comparable antitumor therapeutic effect between gold-coated DNA and naked DNA vaccine. The Th1/Th2 profile of immune responses was influenced by multiple factors including the method, route, amounts of DNA and the nature of the antigen. The induction of a Th1 type response is mainly attributed to the immunostimulatory DNA sequences containing the CpG motif. It was assumed that the DNA dose as low as (1 μg) was sufficient for stimulation of Th1 responses with muscle injection [28], but the same low dose (1 μg) may not be enough to induce a Th1 response in skin vaccination with gene gun. On the other hand, gold particle was demonstrated to have dominant Th2-promoting effects on DNA vaccines [28, 29]. In this report, we demonstrated that 1 μg of non-carrier naked DNA might be sufficient to produce a Th1-biased immunological response in gene gun inoculation on skin, which indicated the amount of DNA did not play a dominant role in Th1/Th2 bias immune reaction in gene gun vaccination. The carrier used to coat DNA may be more important in determining Th1/Th2 reaction. Several evidences indicated that Th1 immunity is critical for the induction of specific cell-mediated cytotoxic immune response such as tumor-specific cytotoxic T lymphocytes in tumor-bearing mice [30,31]. In this study, non-carrier DNA vaccination induce a stronger Th1-biased immunological response than gold-coated DNA vaccination, but the spleen specific cell-mediated cytotoxic immune response

52 was similar no matter the DNA is naked or coated on gold (Fig 4C). This result may be possibly explained by the fact that gold-coated DNA may lead to more efficient tansfection of skin keratinocyte which is known to affect the magnitude of immune response. The expression of tansfected genes in keratinocyte may be cross-presented or contribute directly to the induction of the antigen-specific CD8 + T cell as it has been suggested for DNA-tranfected muscle cells [32]. On the other hand, cell-mediated cytotoxic immune response against MBT-2 tumor cells was determined with spleen lymphocytes that have been activated by recombinant HER-2/neu protein, which suggesting the cytotoxic response was probably mediated by specific cytotoxic lymphocytes toward MBT-2 cells overexpressing HER-2/neu. However, we can not exclude the non-specific immune cells such as natural killer (NK) cells or macrophages in the spleen also mediate the killing of the MBT-2 cell. Hence, it is necessary to further examine whether other non-specific immune cells may play an important role in killing tumor cell and whether the DNA formulation, such as gold-coated or non-carrier naked DNA, can affect these non-specific immunologic cytotoxic effect. In the present study, 1 μg gold-coated or naked pcmv-egfp DNA plasmid can generate similar amount of GPP-positive cd11c+ dendritic cells in lymph node by skin delivery. Interestingly, mice inoculated with 10 μg naked pcmv-egfp-dna plasmid have much higher percentage of GFP-positive CD11c+ cells in lymph nodes, which was a little lower than that achieved with viral vector delivery [33,34]. The result could be attributed to two possible factors. Previous result has indicated that coadministration of anti-apoptotic plasmid with DNA vaccine can increase the percentage of dendritic cells migrated from epidermis to lymph node [35]. The non-carrier DNA may be bombarded into the dendritic cells but cause less damage than the gold-coated DNA, which may increase the survival and migration of dendritic cells into lymph node. The second possibility is that the non-carrier DNA may be in a form that easily taken up by the dendritic cells in the epidermis. In this way, the non-carrier DNA was not directly bombarded into the cells, but into the epidermis where close to the dendritic cells. The DNA encoding GFP in the epidermis might be uptaken by dendritic cells through endocytosis or related pathways, and lead to the increase of GFP-positive cells in lymph node. The gold-coated DNA may have a less capability to be taken up by the dendritic cells when they were bombarded in the epidermis region. Since it is difficult to deliver the mice with 10 μg gold-coated DNA due to coating limitation[36], we cannot exclude the possibility that the mice may have similar high percentage of GFP-positive cells if the mice were bombarded

53 with 10 μg gold-coated DNA. The detailed mechanism of this interesting phenomenon requires further investigation. The detailed mechanisms of penetration of DNA through skin barrier are currently unknown. The first possibility is that the thrust force in the exit of the gene gun may cause mouse skin transiently permeable to the large molecular substance including plasmid DNA. The second possibility is that the DNA is first adhered to the skin surface by the force of biolistic device, followed by absorption through skin barrier. The latter possibility is less likely since immediate treatment of DNase on the mice skin after bombardment did not affect luciferase expression in mice (data not shown). A recent report indicated that the naked DNA can be successfully delivered into mammalian cells with modifications on the commercial available biolistic device [37]. In that report, they suggested that the shockwave generated by gene gun could cause cell membrane transiently permeable to outside substance, which may be similar to the first mechanism we proposed here. Our results demonstrate that naked DNA can be delivered to pass through skin barrier and generate enough cellular immunity to delay tumor progression. Non-carrier naked DNA vaccines may have several advantages over gold-coated DNA vaccines currently used, including ease of development, minimal preparation costs and reduced skin damage. The delivery efficiency of naked DNA is relatively lower than that of gold-coated DNA vaccine as demonstrated by the expression of antigen in the skin and the antibody response. Therefore, non-carrier delivery will not be appropriate for the type of disease that the antibody response is essential for eradication of pathogen. However, non-carrier delivery of naked DNA vaccine may be useful in treating cancer of which eradiation is more dependent on cellular immunity. In addition, naked DNA delivery may benefit certain vaccine which does not wish to induce advertent antibody response, such as Dengue virus infection. In Dengue virus vaccination, antibody-dependent enhancement of infection by anti-virion antibodies has been implicated in the development of severe dengue hemorrhagic fever/dengue shock syndrome [38]. Our experiments further extend the use of biolistic device to deliver the non-carrier DNA into animal. The basic design of this low-pressure gene gun is the converging-diverging nozzle that was used in rocket engine. The force generated by the nozzle design is determined by three parameters: (1) the gas speed (2) variable lengths of terminal spay tube, (3) the type of gas used. Alteration of these parameters may further expand the use of low-pressure gene gun, such as naked DNA may be

54 delivered into the mouse organ when the target organ was exposed under surgery in the future. Furthermore, the supersonic flow apparently caused about three-fold more damage on the naked DNA than the gold-coated DNA, investigation on the protection of the DNA molecules may further enhance its use in laboratory or clinics.

55 Materials and Methods Mice Female C3H/HeN, BALB/c and C57BL/6, mice were obtained from the laboratory animal center at National Cheng Kung University. All animal studies were approved by the animal welfare committee at National Cheng Kung University. Gene Gun injection One to ten μg naked DNA was dissolved in 20 μl double-distilled water added to the loading hole near the nozzle. The DNA-containing water was directly propelled into mouse shaved abdomen using helium at a 60 psi pressure with pushing the trigger (Fig. 1) of the low pressure Gene Gun (BioWare Technologies Co. Ltd., Taipei, Taiwan). To avoid the cross-contamination between each shot, water and ethanol was added sequentially to wash the loading well and was fired to clear the tube. Plasmid DNA was precipitated onto gold particles (Bio-Rad , Hercules, CA) for gene gun vaccination at the ratio of 1-2μg of DNA per mg of gold particles. The 1 mg gold particles and 1-2 μg of DNA solution (50 μl) were vortexed and sonicated for several seconds, followed by adding equal volumes of 0.05 M spermidine and 2.5 M CaCl 2 solution with vortexing. This solution was placed on ice for 10 minutes. Gold particles were collected by centrifugation at 10,000 rpm and washed three times by 100% ethanol. The particles were resuspended in 20 μl of 100% ethanol as bullets. The same low pressure gene gun was used to deliver the gold-coated DNA plasmid to the shaved abdominal region of mice at a 40 psi pressure of helium. The efficiency of delivery by gene gun The luciferase activity on mice skin was detected 48 hr after bombardment of pcmv-luciferase plasmid. Treated mice were visualized with a Night Owl imaging unit (Berthold Technologies, BW, Germany) consisting of a Peltier cooled CCD slow-scan camera mounted on a light-tight specimen chamber. Images were acquired and processed using the WinLight software (Berthold Technologies, Badwildbad, Germany). Just before imaging, mice skin was shaved; and 100 µl of D-luciferin (Synchem OHG, Altenburg, Germany) in saline was injected at a dose of 100 mg/kg. Mice then were placed under the chamber, and a gray-scale image of the mice was first taken with dimmed light. Photon emission was then integrated over a period of 10 min. Luminescence measurements are expressed as the integration of the average brightness/ pixel unit expressed as photon counts per second. The location of delivery by gene gun Mice were sacrificed 48 hrs after bombardment of pcmv-egfp-n1 (Clontech,

56 Paloalto, CA) plasmid, their abdominal skin being removed and embedded in paraffin. All skin samples were cut in sections of 5 µm, and directly observed by IX71 fluorescence microscopy (Olympus). Detection of CD11c + GFP positive cells in the inguinal lymph nodes from vaccinated Mice The protocol is modified from a previous report [35]. In brief, C3H/HeN mice were inoculated with different doses of pcmv-egfp plasmid (pegfp-n1, Clontech, Paloalto, CA) via gene gun bombardment. Inguinal lymph nodes were harvested 48 hrs after mice vaccination. CD11c + cells were further enriched from single cell suspensions of isolated inguinal lymph node by cd11c (N418) microbeads (Miltenyi Biotec, Auburn, CA). To increase the purity of the enriched CD11c+ cells, magnetic separation procedure was repeated by using a new column. The purity of the populations was at least 90% as determined by detecting with monoclonal anti-cd11c-pe antibody. Enriched CD11c + cells were analyzed by using gates based on forward and side scatter around a population of cells with size and granular characteristics of monocytes, and the percentage of GFP positive CD11c + cells in gated population were further analyzed by FACSCalibur flow cytometry (BD Bioscience, MountainView, CA) Therapeutic efficacy of DNA vaccine with/without coating Mice were injected subcutaneously in the flank with MBT-2 cells in 0.5 ml PBS (day 0). Beginning on day 10 when tumors were palpable, pcmv-human N -neu DNA vaccine [23] or prc/cmv DNA plasmid (Invitrogen Carlsbad, CA) was delivered by gene gun on shaved abdominal region of mice three times at weekly interval. Control mice received three injections of water. Tumor size was measured using a caliper twice each week. Tumor volume was calculated by the formula of a 2 rational ellipsoid: (m 1 x m 2 x ), where m 1 represents the shorter axis and m 2 the longer axis. Mice were sacrificed when the tumor volume exceeded 2500 mm 3 or the mouse was in poor condition and death was expected shortly. Significant differences were revealed by Kaplan Meier analysis of survival rates. was inoculated three times at weekly interval Determination of the expression of extracellular domain of p185 neu protein in skin A 96-well plate was coated with 0.2μg rabbit-anti-erbb-2 antibody (Neomarker, Fremont, CA) in 100μl PBS buffer (ph7.4) and incubated overnight at 4 C. Nonspecific binding was blocked with PBS containing 1% BSA, followed by three

57 washes with 0.05% Tween 20 in PBS. Skin samples were homogenized 48 hrs after pcmv-human-n -neu plasmid bombardment, while 100μl of each prepared sample was added to duplicate coated wells and incubated at 37 for 2 hr. After washing three times, mouse-anti-erbb-2 antibody (Ab-20) (Neomarker, Fremont, CA) were added and incubated at 37 for 90 min. Mouse IgG, HRP-conjugated anti-mouse IgG (Calbiochem, Darmstadt, Germany) was then added and incubated for 45 min at 37 C, 3,3,5,5 -tetramethylbenzidine (TMB) substrate (ebioscience, San Diego, CA) was used for color development, and the absorbance was measured at 450 nm with a microplate reader. Determination of serum anti-neu antibody titer Recombinant human-erbb2 protein (R&D system, Minneapolis, MN) diluted to 0.2μg/100μl, and added to 96-well flat-bottom plate. The plate was incubated overnight at 4 C, and blocked with PBS buffer containing 1% BSA. The plate was incubated at room temperature 1-2 hr, and washed with PBS containing 0.05% tween times. Mouse-anti-ErbB-2 antibody (Ab-20) (Neomarker, Fremont, CA) was used to generate the standard curve, and the background value calculated from control wells receiving an irrelevant antibody against SV40 large T antigen (Oncogene Science, Cambridge, MA). Test sera were serially diluted and added to the plates to determine the titer of human anti-p185 neu antibody. For detection of total mouse IgG, HRP-conjugated anti-mouse IgG (Calbiochem, Darmstadt, Germany) was used. Color developed by using 3,3,5,5 -tetramethylbenzidine (TMB) substrate. Absorbance was read at 450 nm with a microplate reader. Spleen Cell-mediated Cytotoxicity Assay for Targeting MBT-2 cells The protocol is modified from previous reports [23,39]. Female C3H/HeN mice (6 8 weeks old) were injected with DNA vaccine three times as described above. A week after the third DNA vaccination, spleen cells were harvested and grown in RPMI 1640 with 25 mm HEPES and L-glutamate (GibcoBRL, Rockville, MD), supplemented with penicillin (100 U/ml), streptomycin (100 μg/ml), 50 mm 2-mercaptoethanol (ME), 100 U/ml penicillin, and 10% FBS. In addition, 10 μg/ml of recombinant extracellular domain of neu protein, a.a (R&D system, Minneapolis, MN) was added. After five days of incubation, non-adherent cells were harvested as effector

58 cells and plated with MBT-2 luciferase cells as target cells [23]. Target cells of 5x 10 3 cells/well were incubated for 18 hrs in triplicate at 37 with serial dilutions (50:1, 25:1, 12.5:1) of effector cells. After 18 h, cells were recovered by centrifugation and 100 μl of supernatant was obtained. The specific lysis was assessed in the supernatant using a conventional luciferase detection system (Promega, Madison, WI). One hundred μl of the culture medium was mixed with 100 μl of the substrate luciferin (dual luciferase reporter system, Promega, Madison, WI). The mixture was then placed into an EG & G MiniLumat LB9506 luminometer (Berthold Technologies, Badwildbad, Germany). Light emission was recorded for 10s with triplicate measurements being performed for each sample. The percentage-specific lysis was calculated by the following formula: % lysis= (test. RLUspontaneous.RLU/max.RLU-spontaneous.RLU) 100 Cytokine ELISA Lymphocytes (5 x 10 6 ) obtained from peripheral lymph node 1 week after the last vaccination, were cultured with 10 μg/ml of recombinant human ErbB2 protein (R&D system, Minneapolis, MN) in a total volume of 1 ml of RPMI1640 containing 10% FBS in a 24 well-plate for 48 hr. The supernatants were harvested and assayed for the presence of cytokines using the mouse ELISA Ready-SET-Go kits (ebioscience, SanDiego, CA) according to the manufacturer s instructions. RT-PCR Total RNA was extracted from lymphocytes by TRIZOL (Invitrogen, Carlsbad, CA). cdna was synthesized from 2μg RNA using MMLV-Reverse Transcriptase according to the manufacturer s directions. Primers were as follows: IL-12p40 Forward: 5'-TGC TGG TGT CTC CAC TCA TGG C-3'; IL-12p40 Reverse: 5'-TTT CAG TGG ACC AAA TTC CAT T-3'; IFN-γ Forward: 5'-AAC GCT ACA CAC TGC ATC TTG G-3'; IFN-γ Reverse: 5'-CAA GAC TTC AAA GAG TCT GAG G-3'; IL-4 Forward: 5'-GAA TGT ACC AGG AGC CAT ATC-3';IL-4 Reverse: 5'-CTC AGT ACT ACG AGT AAT CCA-3'; IL-10 Forward: 5'-CGG GAA GAC AAT AAC TG-3'; IL-10 Reverse: 5'-CAT TTC CGA TAA GGC TTG-3'; HPRT Forward: 5'-GTT GGA TAC AGG CCA GAC TTT GTT G-3'; HPRT Reverse:5'-GAT TCA ACT TGC GCT CAT CTT AGG C-3. cdna was amplified by Protaq (Protech, Taipei, Taiwan). PCR amplifications used these primers in 50 μl volumes containing pmol of each primer, Protaq buffer (Protech, Taipei, Taiwan) 200μM each of dntp, and 5 unit of ProTaq polymerase. PCR was performed on a PCR machine (MJ Research, Watertown, MA). The PCR reaction commenced at 94 C for 2 min, followed by cycles at 94 C for 30s, 55 C for 30s, and 72 C for 30s. The PCR products were

59 subjected to electrophoresis on 1.5% agarose gels and visualized by ethidium bromide staining under UV light. Determination of DNA degradation by gene gun firing 1μg naked pcmv-her-2/neu DNA plasmids or gold-coated 1μg pcmv-her-2/neu DNA plasmids was bombarded to a circular region, which had the equal area to nozzle exit of gene gun, on 6-μm-pore-size filter paper (Advantec, Tokyo, Japan) at the pressure of 60 or 40 psi respectively. The DNA on the bombarded filter paper was recovered by immersing the filter paper in 100μl ddh 2 O for 5 minutes in a PD column (Geneaid, Taiwan) The DNA was eluted with centrifugation at rpm for 5 minutes. 0.2μg of eluted DNA was loaded into agarose gel. DNA integrity was evaluated from video image by densitometry using the Visionworks LS software (UVP, Upland, CA, US) Statistical analysis SE values from each triplicate set as well as t test comparisons were derived by using the GraphPad Prism 4 software from GraphPad Software (San Diego, CA). A p value <0.05 was considered statistically significant. The Kaplan Meier survival plots for vaccinated mice were obtained using the GraphPad Prism 4, and curves compared using the log-rank test.

60 References: 1. VandenDriessche T, Collen D, Chuah MK. Biosafety of onco-retroviral vectors. Curr Gene Ther 2003; 3: Klein RM, Wolf ED, Wu R et al. High-velocity microprojectiles for delivering nucleic acids into living cells Biotechnology 1992; 24: Liu F, Huang L. A syringe electrode device for simultaneous injection of DNA and electrotransfer. Mol Ther 2002; 5: Chen WC, Huang L. Non-viral vector as vaccine carrier. Adv Genet 2005; 54: Foldvari M, Babiuk S, Badea I. DNA delivery for vaccination and therapeutics through the skin. Curr Drug Deliv 2006 Jan; 3: Oard JH. Physical methods for the transformation of plant cells. Biotechnol Adv 1991; 9: Yang NS, Burkholder J, Roberts B et al. In vivo and in vitro gene transfer to mammalian somatic cells by particle bombardment. Proc Natl Acad Sci U S A 1990; 87: Heiser WC. Gene transfer into mammalian cells by particle bombardment. Anal Biochem 1994; 217: Macklin MD, McCabe D, McGregor MW, et al. Immunization of pigs with a particle-mediated DNA vaccine to influenza A virus protects against challenge with homologous virus. J Virol 1998; 72: Tang DC, Devit M, Johnson SA. Genetic immunization is a simple method for eliciting an immune response. Nature 1992; 356: Ulmer JB, Donnelly JJ, Parker SE, et al. Heterologous protection against influenza by injection of DNA encoding a viral protein. Science 1993; 259: Lollini PL, De Giovanni C, Pannellini T, et al. Cancer immunoprevention. Future Oncol 2005; 1: Barry MA, Johnston SA, Biological features of genetic immunization. Vaccine 1997; 15: Feltquate DM, Heaney S, Webster RG et al. Different T helper cell types and antibody isotypes generated by saline and gene gun DNA immunization. J Immunol 1997; 158: Schirmbeck R, Reimann J. Modulation of gene-gun-mediated Th2 immunity to hpatitis B surface antigen by bacterial CpG motifs or IL-12. Intervirology 2001; 44 : Cohen AD, Boyer JD, Weiner DB. Modulating the immune response to genetic immunization. FASEB J 1998; 12: Zhou X, Zheng L, Liu L, et al. T helper 2 immunity to hepatitis B surface antigen

61 primed by gene-gun-mediated DNA vaccination can be shifted towards T helper 1 immunity by codelivery of CpG motif-containing oligodeoxynucleotides. Scand J Immunol 2003; 58: Haddad D, Liljeqvist S, Stahl S, et al. Differential induction of immunoglobulin G subclasses by immunization with DNA vectors containing or lacking a signal sequence. Immunol Lett 1998; 61: Aberle JH, Aberle SW, Allison SL, et al. A DNA immunization model study with constructs expressing the tick-borne encephalitis virus envelope protein E in different physical forms. J Immunol 1999; 163: Weiss R, Scheiblhofer S, Freund J, et al. Gene gun bombardment with gold particles displays a particular Th2-promoting signal that over-rules the Th1-inducing effect of immunostimulatory CpG motifs in DNA vaccines. Vaccine 2002; 20: Ikeda H, Chamoto K, Tsuji T, et al. The critical role of type-1 innate and acquired immunity in tumor immunotherapy. Cancer Sci 2004; 95: Cheng WF, Lee CN, Chang MC, et al. Antigen-specific CD8+ T lymphocytes generated from a DNA vaccine control tumors through the Fas-FasL pathway. Mol Ther 2005; 12: Tu CF, Lin CC, Chen MC, et al. Autologous neu DNA vaccine can be as effective as xenogenic neu DNA vaccine by altering administration route. Vaccine 2007; 25: Klinman DM, Sechler JM, Conover J, M. et al. Contribution of cells at the site of DNA vaccination to the generation of antigen-specific immunity and memory. J Immunol 1998; 160: Torres CA, Iwasaki A, Barber BH et al. Differential dependence on target site tissue for gene gun and intramuscular DNA immunizations. J Immuno 1997; 158: Ji H, Wang TL, Chen CH, et al. Targeting human papillomavirus type 16 E7 to the endosomal/lysosomal compartment enhances the antitumor immunity of DNA vaccines against murine human papillomavirus type 16 E7-expressing tumors. Hum Gene Ther. 1999; 10: Lin CC, Chou CW, Shiau AL, et al. Therapeutic HER2/Neu DNA vaccine inhibits mouse tumor naturally overexpressing endogenous neu. Mol Ther 2004; 10: Feltquate DM, Heaney S, Webster RG, et al. Different T helper cell types and antibody isotypes generated by saline and gene gun DNA immunization. J Immunol 1997; 158: Weiss R, Scheiblhofer S, Freund J et al. Gene gun bombardment with gold particles displays a particular Th2-promoting signal that over-rules the Th1-inducing

62 effect of immunostimulatory CpG motifs in DNA vaccines. Vaccine 2002; 20: Nishimura T, Iwakabe K, Sekimoto M, et al. Distinct role of antigen-specific T helper type 1 (Th1) and Th2 cells in tumor eradication in vivo. J. Exp. Med 1999; 190: Germann, T., M. K. Gately, D. S. Schoenhaut, M. Lohoff, F. Mattner, S. Fischer, S.-C. Jin, E. Schmitt, E. Rude Interleukin-12/T cell stimulating factor, a cytokine with multiple effects on T helper type 1 (Th1) but not on Th2 cells. Eur. J. Immunol. 23: Shirota H, Petrenko L, Hong C, et al. Potential of transfected muscle cells to contribute to DNA vaccine immunogenicity. J Immunol 2007; 179: Lopes L, Dewannieux M, Gileadi U, et al. Immunisation with a lentivector that targets tumour antigen expression to dendritic cells induces potent CD8+ and CD4 + T cell responses J. Virol 2007; /JVI Gasteiger G, Kastenmuller W, Ljapoci R, et al. Cross-priming of cytotoxic T cells dictates antigen requisites for modified vaccinia virus Ankara vector vaccines. J. Virol ; 2007; 81: Kim TW, Hung CF, Ling M et al. Enhancing DNA vaccine potency by coadministration of DNA encoding antiapoptotic proteins. J Clin Invest. 2003; 112: van Drunen S, den Hurk LV. Novel methods for the non-invasive administration of DNA therapeutics and vaccines. Curr Drug Deli. 2006; 3: Lian WN, Chang CH, Chen YJ et al. Intracellular delivery can be achieved by bombarding cells or tissues with accelerated molecules or bacteria without the need for carrier particles. Exp Cell Res 2007; 313: Green S, Rothman A. Immunopathological mechanisms in dengue and dengue hemorrhagic fever. Curr Opin Infect Dis 2006; 19: Giovarelli M, Musiani P, Modesti A et al. Local release of IL-10 by transfected mouse mammary adenocarcinoma cells does not suppress but enhances antitumor reaction and elicits a strong cytotoxic lymphocyte and antibody-dependent immune memory. J Immunol. 1995, 15 :

63 Figure Legend Figure 1. Low pressure biolistic device. (A) Illustration of the gene delivery device. The plasmid is applied through the upper hole. An external supply of low pressure helium (psi) is supplied from the bottom. (B) Internal features of the gene delivery device. M stands for Mach. M<1, subsonic flow; M>1, supersonic flow. Figure 2. Non-carrier DNA can be delivered into mice and expressed in mice skin. (A) Expression of luciferase in skin. Mice were inoculated with 1 μg gold-coated pcmv-luciferase plasmid DNA, 1, 5, 10 μg naked pcmv-luciferase plasmid DNA. In vivo images of luciferase activity in mice were measured at 48 hrs after inoculation with Night Owl imaging unit. (B) Histogram of the quantification of the signal. The symbol * indicates a statistically significant difference when compared with the 10 μg naked CMV-luciferase group(p < 0.05) (C) Expression of green fluorescent protein in epidermis. C3H/HeN mice were inoculated with 1 μg gold-coated pcmv-egfp DNA plasmid or 1, 5, 10 μg of non-carrier naked pcmv-egfp DNA plasmid. The mouse skin was removed 48 hrs later and paraffin-formalized for green fluorescence observation. (D) Migration of GFP-positive cd11c+ dendritic cells into lymph node. Mice were inoculated with 1 μg gold-coated pcmv-egfp DNA plasmid or 1 to 10 μg of naked pcmv-egfp plasmid DNA, and the lymph node was removed 48 hrs later. The ratio of GFP positive to total cd11c+ dendritic cells was measured with flow cytometry. The cd11c + dendritic cells population were first enriched by cd11c (N418) microbeads and followed by gating a region more consistent with monocyte size and granular characteristics. The symbol * indicates a statistically significant difference when compared with the control DNA plasmid (P < 0.05). Figure 3. Cancer therapy with delivery of non-carrier naked or gold-coated pcmv-human-n -neu DNA vaccine.(a) Time course of tumor volume progression, the average tumor volume was shown until the first mice was humanity sacrificed due to excess tumor burden (B) lifespan of C3H/HeN mice after subcutaneous challenge with MBT-2 cells were plotted. The digit in the parenthesis is the number of mice in the experiment. The symbol * indicates a statistically significant difference when compared with the control saline mice (P < 0.05) the mouse survival were measured and subjected Kaplan-Meir analysis. Fig. 4 The immune responses generated by non-carrier or gold-coated pcmv-human-n -neu DNA vaccine (A) the expression of the extracellular domain of p185 neu in skin were measured with ELISA. (B) Anti-p185 neu antibody titers in the

64 serum samples were determined with ELISA. The data represent the average titer of three mice. (C) Spleen Cell-mediated Cytotoxicity Assay for Targeting MBT-2 cells in inoculated mice. Target cells were MBT-2-luciferase cells cultured in vitro. Effector cells were lymphocytes derived from mice treated with naked or gold-coated N -neu DNA vaccine. Cytotoxicity was determined by the luciferase release. Each point represents the average of triplicate wells. (D) Pooled splenocytes from each group of mice were stimulated with p185 neu antigen. RNA was extracted and RT-PCR was performed to examine the cytokine mrna. Hypoxanthine phosphoribosyltransferase (HPRT) was used as an internal control. (E) Supernatants were collected 2 days after stimulation and the concentration of IFN- and IL-4 were measured with ELISA. The symbol * indicates a statistically significant difference when compared with the 1μg naked pcmv-human-n -neu DNA vaccine group(p < 0.05). Fig 5. Influence of supersonic flow on plasmid DNA integrity. The integrity of plasmid DNA after bombarding by gene gun was analyzed by electrophoresis through a 1% agarose gel. lane A: molecular weight marker; lane B: Naked HER-2/neu DNA plasmid before delivery by gene gun; lane C: Naked HER-2/neu DNA plasmid after delivery by gene gun (60 psi); lane D: Gold-coated HER-2/neu DNA plasmid after delivery by gene gun (40 psi). 0.2 μg of DNA was loaded on each lane.

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