摘要:目前已經發現的人類乳突病毒超過 100 種,研究指出與人類癌症發生有關的致癌性人類乳突病毒約有 20 種。其中人類乳突病毒第 16 型被發現與許多癌症,包括子宮頸,陰道,外陰,肛門,陰莖,口咽,肺癌和食道癌。人類乳突病毒被認為是致癌性病毒的主要原因,因為其結構由兩種致癌基因E6 及 E7 有關。E7 是一種致癌基因,其機轉是藉由 E7 的胺基酸序列第 24 及 26 位置會與抑癌基因Rb 進行結合並抑制其功能。目前市場上的人類乳突病毒疫苗主要是針對生殖道疾病中人類乳突病毒表面蛋白 L1 感染的預防性疫苗。然而針對現今人類乳突病毒發展的主要問題,便是對已經感染人類乳突病毒相關癌症患者並無法清除感染後的病毒,或者並不能避免一直重覆的感染病毒。發展針對人類乳突病毒的治療性疫苗及免疫治療將能藉由刺激免疫系統並有效識別病毒,以清除任何感染人類乳突病毒的癌細胞。這種治療的策略將能獲得突破性的發展,研究成果具有龐大的商品開發與市場潛力。 本研究室進行癌症疫苗和腫瘤免疫治療的研發已有多年的經驗,同時與業界也保持著密切的合作,具有相當的研發成果。兩種子宮頸癌癌症疫苗和免疫治療的去氧核醣核酸疫苗已獲得美國及台灣專利。蛋白質疫苗已經由科技業者著手進行臨床試驗,去氧核醣核酸疫苗也正和科技業者進行技術轉移中。 在先期的研究結果中,我們已經鑑結 CTGF/E7 的減毒性疫苗,主要是將 E7 的胺基酸序列第 24及 26 位置進行點突變而降低 E7 與 Rb 基因的結合。我們進一步開發的去毒性疫苗則是將該基因結合位置進行剔除,以避免因 E7 與 Rb 基因結合而抑制 Rb 基因的功能。此外,我們更發展新一代的嵌合式疫苗,以 IGF-1 為骨架,建立能同時活化樹突細胞及誘發具抗原性免疫反應的疫苗。初步結果發現此疫苗與 CTGF/E7 比較後,能比原本抗原性免疫反應提升超過兩倍。顯示此疫苗具有更強大的抗腫瘤活性,極具治療性疫苗開發潛力。 因此我們提出這個三年計畫,第一年的目標以 IGF-1/E7 DNA 疫苗的開發,並評估 IGF-1/wt E7能否產生更強的 E7 特異性免疫反應及抗腫瘤活性。此外也將評估建立剔除特定基因結合位置的去毒性疫苗,並以人類乳突病毒相關癌症的動物模式進行腫瘤預防及治療試驗。第二年我們設計 CTGF/E7與 IGF-1/E7 的去毒性 DNA 疫苗進行對等式的比較,並將進行包括具抗原特異性的輔助 T 細胞、具抗原特異性的毒殺 T 細胞及 E7 特異性抗體生成的分析及量化,也將進行抗體剔除試驗以確定這些 DNA去毒性疫苗的活化途徑及機制。第三年我們將進行去毒性 DNA 疫苗的再提升,將具抗原特異性的去毒性 DNA 構築在具溶解腫瘤能力的病毒載體內,進行不同的免疫活化路徑的比較分析,如 DNA-初打-DNA-加打、DNA-初打-病毒-加打、病毒-初打-DNA-加打或病毒-初打-病毒-加打等路徑,以找到最強的抗原性免疫反應及抗腫瘤活性的治療策略。 本計畫可能產出的商品包括第一,嶄新的具抗原特異性人類乳突病毒治療性疫苗。第二,去氧核醣核酸疫苗為基礎之人類乳突病毒相關癌症疫苗和免疫治療。第三、具抗原特異性的溶解腫瘤能力病毒載體為基礎之人類乳突病毒相關癌症疫苗和免疫治療。本計畫未來研發獲得的成果將對人類乳突病毒相關癌症免疫治療有極高的潛力商品化,也會有相當大的市場潛能。
Abstract: Human papillomaviruses (HPVs) are composed of more than 100 types. There are around 20 HPV types defined as oncogenic HPV types which are related to many human cancers. HPV type 16 is the most prevalence HPV type that has been identified in many cancerous tissues including cervical, vaginal, vulvar, anal, penile, oropharyngeal, lung and esophageal cancers. HPV is regarded as oncovirus because it composed of two E6 and E7 oncogenes. E7 is an oncogene because the 24th to 26th amino acids of the E7 protein encodes Rb binding motif to inactivate the tumor suppressing function. The commercial HPV vaccines targeting L1 capsid protein are currently available focusing on preventing HPV infection at anogenital diseases. However, the major problem for patients with HPV-associated cancers is because they are unable to clear the HPV after infection, or they are repeatedly infected by HPV. Developing therapeutic vaccines against HPV-associated diseases and cancers are aimed at boosting the immune system, to recognize the virus and kill any HPV-infected cancer cells. Cancer treatment will access a breakthrough development, so the results of this proposal will have enormous potential for biotechnological marketing. Our research team has focused on the development of cancer vaccine and immunotherapy for years. Besides, our research group has also maintained close cooperation with industry. Two kinds of chimeric DNA vaccines focus on cervical cancer immunotherapy have got the patents in USA and Taiwan. The protein vaccines are also working with technology companies for the technology transfer. Our preliminary results showed that we have generated detoxic format of CTGF/E7 vaccine which was designed by substituting 24th to 26th amino acids to prevent E7/Rb interaction. We further design several novel truncated forms of E7 to avoid the potential Rb protein binding activity. Besides, we will develop the next generation of chemeric DNA vaccines based on IGF-1, a fusion DNA vaccine not only enhance E7-specific immunity but activating dendritic cells. A head-to head study from our results indicated IGF-1/E7 DNA vaccine could generate more than 2 folds of E7-specific immunity in compared with CTGF/E7. These results suggested IGF-1 chimeric DNA vaccine could be a potential candidate as HPV-target therapeutic vaccines. So we would like to propose this three-year proposal. In the first year, we will develop IGF-1with wild-type E7 (IGF-1/wt E7) and evaluate if the IGF-1/wt E7 could generate more potent E7-specific immunity and anti-tumor effects. Besides, we will evaluate various IGF-1/E7 deleted mutants vaccines by tumor prevention and treatment experiments in HPV-related cancer animal model. In the second year, we will investigate E7-specific immunity between CTGF/detox E7 and IGF-1/detox E7 including E7-specific help and cytotoxic T cells generation, E7-specific antibody titers and antibody depletion experiments to confirm the anti-tumor mechanisms of IGF-1 chimeric DNA vaccines. In the third year, we will upgrade our immunotherapeutic strategies to construct the E7-specific DNA vaccines into viral vector to generate E7-specific viruses. We will test and compare different strategies including DNA-prime-DNA-boost, DNA-prime-vaccinia-boost, vaccinia-prime-DNA-boost and vaccinia-prime-vaccinia-boost to investigate the most potent strategy for E7-specific immunity and anti-tumor effects. The possible products of this project are: First, novel HPV-related cancer therapeutic vaccines. Second, DNA-based vaccines focus on HPV-related cancers and their derived immunotherapeutic strategies. Third, E7-specific virotherapy combined with DNA vaccines targeting to HPV-related cancers. The results and products of this project have high potential to become commercial products and market in the field of HPV-related cancers in the future.