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Use of Therapeutic DNA Vaccine to Control Cancer

Use of Therapeutic DNA Vaccine to Control Cancer. Ming-Derg Lai, Ph.D. Department of Biochemistry and Molecular Biology College of Medicine National Cheng Kung University. What is gene therapy?.

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Use of Therapeutic DNA Vaccine to Control Cancer

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  1. Use of Therapeutic DNA Vaccine to Control Cancer Ming-Derg Lai, Ph.D. Department of Biochemistry and Molecular Biology College of Medicine National Cheng Kung University

  2. What is gene therapy? Induction of antitumor immunity with combination of HER2/neu DNA vaccine and interleukin 2 gene-modified tumor vaccine.Clin. Cancer Res. 2000 Nov;6(11):4381-8

  3. Gene transfer • Delivery - Viral - Nonviral • Retrovirus • Adenovirus • Adeno-associated virus (AAV) • Herpes virus • Lipofection • Direct injection of DNA (Naked DNA vaccine)

  4. DNA Vaccine • A DNA Vaccine is essentially a DNA sequence that can be used as a vaccine • This sequence of DNA comes from apiece of pathogen or tumor antigen. • The sequence is then injected into a cell of a person, the protein is produced, and an immunity is created against the infectious organism or tumor

  5. Feature of DNA vaccine

  6. Development of the Therapeutic HER2/Neu DNA Vaccine to Inhibit Mouse TumorNaturally Overexpressing Endogenous Neu

  7. HER-2/neu

  8. HER-2/neu /Oncogene • HER-2/neu is overexpressed in many human cancers (Ovarian, bladder, breast, lung..) • Overexpression of HER-2/neu is correlated with the aggressiveness and poor prognosis of cancer

  9. Prevention HER-2/neu DNA vaccine

  10. HER-2/neu-expressing Tumor model Mouse tumor cells natively overexpressing mouse HER-2/neu

  11. HER-2/neu-expressing tumor model

  12. <20 %

  13. Fusion with activating gene

  14. Activating gene

  15. Fusion with activating gene

  16. Kaplan-Meier analysis of mouse survival

  17. HER-2/neu specific antibody HER-2/neu protein ELISA 10Antibody(serum) 20Antibody

  18. HER-2/neu specific Cytotoxic T lymphocytes Test-Negative Specific lysis : % Total-Negative

  19. Summary I • Neu DNA vaccine is effective in controlling tumor progression. • Fusion of IL-2 and neu produced strongest anti-tumor effects.

  20. 發展Neu DNA疫苗於癌症治療(新低壓基因槍之研發) 主持人：賴明德教授 (Ming-Derg Lai) 執行人：林季千博士 (Chi-Chen Lin) 國立成功大學醫學院 生物化學暨分子生物研究所

  21. 研究計劃 • 背景：本計劃是發展利用Neu DNA疫苗治療癌症之相關作用機轉及傳送DNA 之技術。利用發展癌症治療性疫苗(therapeutic DNA vaccine)，我們和生物鎵公司(Bioware Inc.)合作發展實驗室用及醫療用基因槍(gene gun). 目前商業用基因槍價格昂貴且產生極大噪音。 • 目的：發展低壓基因槍供實驗室及醫院使用。 • 重要性：台灣自製具[低噪音][低氦量]之多功能基因槍，具有龐大商機。

  22. 低壓基因槍 Sample Loading • 此次的實驗更不同與以往所使用之高壓式傳統基因槍 (400 psi， 氦氣，Bio-Rad)， 我們所使用的是經由改良過之低壓式基因槍(40-50 psi，氦氣， Bio-Ware)。 此基因槍可明顯降低噪音。(US Patent: 6436709 B1).

  23. 主要成果 • 證明台灣製基因槍和目前商業用基因槍具有相同治療效果。 • 證明台灣製基因槍具有投遞其他分子如裸露DNA之能力。

  24. 測試比較基因槍療效 1 X 106 MBT-2 腫瘤細胞 in 6-8 週 C3H/HeNCrJ 老鼠 腫瘤生長 及老鼠存活 天 10 17 24 施打neu DNA Bioware: 1. Control mice 2. One mg gold coated with 1 mg HER-2/neu plasmid DNA Commercial high-pressure gene gun 1. Control mice 2. One mg gold coated with 1 mg HER-2/neu plasmid DNA

  25. 新式低壓基因槍(Bio-ware)與傳統高壓基因槍(Commercial) 具相同癌症療效 老鼠存活率相同! 新式低壓基因槍(Bio-ware)與傳統高壓基因槍(Commercial) 之抗腫瘤能力之評估. 觀察老鼠之存活率.*代表比較於控制組別(只施打saline)P<0.05.

  26. 新式低壓基因槍(Bio-ware)與傳統高壓基因槍(Commercial) 引發相同免疫反應 產生 相同量 抗体 相似 細胞毒殺 反應 低壓及高壓基因槍引發(B) 相同強度抗體反應及(C)細胞型反應

  27. 低壓新基因槍多功能特性 • 我們此次所使用之新式基因槍，具有之另一個特性及是其可在不須經由金粒子攜帶之情況下，直接將裸露之DNA疫苗投遞之細胞體內，而這樣的特性剛好可以克服金粉所帶來之免疫偏差(Th1/Th2)。

  28. 低壓新基因槍可傳送裸露DNA 入老鼠體內表現 比較基因槍遞送金粒子coated DNA疫苗和裸露之DNA 疫苗表現量之差異. 在打完質體後第三天,分析老鼠表皮層之pCMV-luciferase 表現, 而我們是藉由分析均質化後之表皮層內Luciferase 之活性作為比較表現量之依據

  29. 當使用低壓新基因槍投遞無金粉之裸露DNA產生相同治療腫瘤效果當使用低壓新基因槍投遞無金粉之裸露DNA產生相同治療腫瘤效果 治療效果無差異 比較基因槍遞送金粒子coated DNA疫苗和裸露之DNA 疫苗之抗腫瘤能力. 觀察老鼠腫瘤體積及老鼠之存活率.

  30. 當使用低壓新基因槍投遞無金粉之裸露DNA產生Th1 免疫反應(治療癌症較佳) 分析基因槍遞送金粒子coated DNA疫苗和裸露之DNA 疫苗之 Th1/Th2免疫反應. 在打完3劑疫苗後之一個星期取出 大腿淋巴結細胞, 利用RT-PCR 分析 IFN-g, IL-18 (Th1) 和IL-4 , IL-10 (Th2) 細胞激素之蛋白質表現量. ＊代表P<0.001 Th1 biased Th2 biased

  31. 結論 II • 新式低壓基因槍其具有能與傳統式高壓基因槍一樣之投遞DNA和誘導免疫反應治療腫瘤之功能當使用金粉包裝之DNA. • 新式低壓基因槍可成功傳送無金粉包裝之DNA 進入生物體內。並產生免疫力治療癌症。新式低壓基因槍傳遞之無金粉包裝之裸露DNA 引發偏向細胞型免疫反應。 產業發展前景： • 具專利保護 (2) 低噪音 (3) 低氦量使用 (4) 多功能使用包括金包埋DNA 及裸露DNA 或其 他分子。

  32. A novel cancer therapy based on modulating indoleamine 2,3-dioxygenase in skin dendritic cells in vivo Ming-Derg Lai Department of Biochemistry and Molecular Biology College of Medicine National Cheng Kung University

  33. Aim • Generation of anti-tumor immunity by in vivo targeting dendritic cells. • Rationale 1. Modifying dendritic cells is a promising cancer therapy. 2. The novel gene gun appears to deliver gene into DCs in an efficient way.

  34. Target gene: indoleamine 2,3-dioxygenase (IDO) • IDO also plays an important role in immune escape in cancer. The establishment of tolerance may be mediated through either localized depletion of tryptophan or accumulation of toxic metabolites. • Overexpression of IDO was observed in many types of tumors and/or tumor draining lymph node. L-tryptophan IDO L-kynuerine 3-hydroxylanthralinic acid

  35. Hypothesis: Delivery of IDO siRNA can generate anti-tumor immunity in vivo S.C. Tumor Implantation and tumor tolerance Skin delivery of IDO siRNA IDO+ IDO+ Gene gun CD8+ T cell IDO-negative Dendritic cells migrate to Lymph node Induction of cytotoxic immune responses and tumor regression CD8+ T cell Tumor-draining Lymph node DC Tumor IDO-negative DC

  36. IDO siRNA downregulates IDO but has no effect on IDO2

  37. The effects of IDO siRNA on dendritic cells in vivo CD11c+ cells was harvested from inguinal lymph node Forty eight hours after vaccination.

  38. 1x106 MBT-2 5 mg/ml in drinking water, pH=9.9 1-MT treatment …… Day0 Day8 Day15 Day22 Day29 Day36 1st vaccination and weekly vaccination, until mice were sacrificed Cancer therapeutic effect of IDO siRNA and IDO inhibitor, 1-methyl tryptophan

  39. IDO siRNA or 1-Methyltryptophandelays tumor growth

  40. Skin delivery of IDO siRNA is more effective than systemic administration of 1-MT

  41. Table.1 T cell infiltration in C3H mice model Random 5 field counted * Compared with saline ** compared with saline and scramble

  42. IDO siRNA enhances cytotoxic T cells activity

  43. Can cells obtain anti-tumor immunity by adoptive transfer of CD11C+ Cells Day 0 Day 7 Day 9 Naïve mice vaccinated with IDO siRNA. Mice were sacrificed and inguinal lymph nodes were harvested. Second IDO siRNA vaccination. Isolation of cd11c+ cells S.C. Tumor Implantation. S.C. injection CD11c+ cells to tumor-bearing mice at day 9.

  44. Adoptive transfer CD11c+ dendritic cells from vaccinated mice may provide protection from cancer

  45. ＋ ＋ ＋ ＋ ＋ pcDNA3.1-IDO ＋ － － － － pshU6 vector IDO siRNA － ＋ － － － － － ＋ － － IDO siRNA-2 － － － ＋ － IDO siRNA-3 － － － － ＋ Scramble IDO siRNA myc β-actin What about offsite effect of IDO siRNA? Approach: analyze the therapeutic effect of two other IDO siRNAs targeting different sequences.

  46. The therapeutic effects of IDO siRNAs are correlated with their suppression effects It suggests that the therapeutic effect of IDO siRNA Is NOT due to offsite effect.

  47. IDO siRNA exert anti-cancer therapeutic effect on CT-26 tumor cells in BALC/c mice model

  48. IDO siRNA is also more effective on CT-26 colon cancer cells

  49. Table 2. T cell infiltration in BALB/c mice model Random 3 field counted * Compared with saline ** Compared with saline and scramble

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