1358302 九、發明說明： 【發明所屬之技術領域】 本發明係關於一種融合蛋白，尤指一種預防或抑制因1358302 IX. Description of the invention: [Technical field to which the invention pertains] The present invention relates to a fusion protein, especially a preventive or inhibitory factor
人類乳犬狀病 f 16型（human papillomavirus type 16，HPV 5 type 16)所誘發腫瘤之融合蛋白及其醫藥組成物。 【先前技術】 在台灣’子宮頸癌發生率一向高居婦女癌症首位。一 .般手呂頸上皮變異或是早期的子宮頸癌發生時，幾乎沒有 10任何症狀，雖然早期治療癒後效果良好，但預防勝於治療， 因此相關學者皆以找尋能有效預防子宮頸癌的方法為目 的。 目則已S登貫人類乳突病毒（human papill〇mavirus， HPV)與子宮頸癌的形成息息相關，是子宮頸癌最主要的致 15病因子。HPV的某些亞型（如16、18型）的DNA序列已在 75%〜1 〇〇%的子宮頸癌病例的癌細胞中發現，但其致癌機 .制尚未完全清楚《近來發現18和31高危險亞型 的早期病肯基因產物E6和E7蛋白，極易與Rb和ρ53基因的 產物結合並中和其抑制細胞生長的功能，這現象說明了 20 HPV在致癌時不是單獨作用的，同時也需要環境因素的協 .同，且E7蛋白在子宫頸癌細胞和癌組織細胞内持續表現.， 並在維持轉化組織惡性表型的過程中扮演著重要的角色。 腫瘤免疫反應以細胞免疫爲主，體液免疫爲辅。參加 細胞免疫的反應細胞主要有細胞毒性τ細胞（cyt〇t〇xic τ 5 ⑧ 1358302 lymphocytes, CTL)、自然殺手細胞（NK)和巨嗟細胞e CTL 被細胞介白素2(IL — 2)啟動後，藉由可被T細胞識別，位於 抗原呈現狀態之腫瘤細胞上的組織相容性複合體（major histocompatibility compolex，MHC)第一型分子的出現，而 5 釋放某些溶解酶，將腫瘤細胞殺滅，奠抑制腫瘤細胞的增 殖。CTL的保護作用在對抗如HPV導致的腫瘤時特別明 顯，因此，如能透過增加HPV抗原與MHC I型分子之複合 體出現於腫瘤細胞的途徑來誘發屬於具備HPV抗原特異性 的CTL如CD8+T細胞的增殖，則有可能直接針對抑制腫瘤 10 細胞來進行免疫預防或治療。 已有研究證實子宮頸癌可透過施打疫苗來事先預 防，而由於與形成子宮頸癌息息相關的HPV病毒結構中， E7蛋白常被發現於癌症病灶組織或是癌化前損傷之組 織，因此具有做為疫苗發展標的的潛力；除子宮頸癌之外， 15 近來科學家更發現，人類乳突病毒HPV16、HPV18的感 染，不單是子宮頸癌的危險因子，更是女性肺癌的危險因 子；研究指出，HPV16、HPV18.兩種病毒所分別製造的E6、 E7致癌蛋白，會透過‘液循環到達肺部，將人體的p53、 Rb抑癌基因蛋白·予以分解，抑癌基因一旦失去作用，癌細 20 胞就出現於肺部；然而，目前發展中的DNA疫苗雖然具 有長效特性，但是其高成本，高危險性（病毒本身容易被誘 發突變）之考量仍是限制其發展之主因；此外在應用E7蛋 白做為子宮頸癌之基因治療時，常由於'HPV病毒E6及E7 蛋白的弱抗原特性，而無法誘發良好之免疫反應，進而無A fusion protein of a tumor induced by human papillomavirus type 16, HPV 5 type 16 and a pharmaceutical composition thereof. [Prior Art] In Taiwan, the incidence of cervical cancer has always been the highest in women's cancer. 1. When the normal cervical epithelial variability or early cervical cancer occurs, there are almost no symptoms. Although the early treatment is effective, the prevention is better than the treatment. Therefore, the relevant scholars are looking for effective prevention of cervical cancer. The method is for the purpose. The human papill〇 mavirus (HPV) is closely related to the formation of cervical cancer, and is the most important cause of cervical cancer. The DNA sequence of certain subtypes of HPV (such as type 16, 18) has been found in cancer cells of 75% to 1% of cervical cancer cases, but its carcinogen system has not been fully understood. The high-risk subtypes of the early disease gene products E6 and E7 proteins easily bind to the products of the Rb and ρ53 genes and neutralize their function of inhibiting cell growth. This phenomenon indicates that 20 HPV does not act alone in carcinogenesis. At the same time, it is also necessary to cooperate with environmental factors, and E7 protein continues to perform in cervical cancer cells and cancer tissue cells, and plays an important role in maintaining the malignant phenotype of transformed tissues. The tumor immune response is mainly cellular immunity, supplemented by humoral immunity. The cells involved in cellular immunity mainly include cytotoxic tau cells (cyt〇t〇xic τ 5 8 1358302 lymphocytes, CTL), natural killer cells (NK) and giant clam cells e CTL are interleukin 2 (IL-2). After initiation, by the recognition of T cells, the presence of a major histocompatibility compolex (MHC) type I molecule on the tumor cells in the antigen-presenting state, and 5 release of certain lytic enzymes, the tumor will be Cell killing, inhibiting the proliferation of tumor cells. The protective effect of CTL is particularly evident in the fight against tumors such as HPV. Therefore, CTLs such as CD8+ that are specific for HPV antigens can be induced by increasing the presence of a complex of HPV antigens and MHC class I molecules in tumor cells. The proliferation of T cells may directly prevent or treat tumor 10 cells. Studies have confirmed that cervical cancer can be prevented in advance by vaccination, and because of the HPV virus structure associated with the formation of cervical cancer, E7 protein is often found in cancer tissue or tissue before cancer, so As a target for vaccine development; in addition to cervical cancer, 15 recent scientists have found that HPV16 and HPV18 infection are not only risk factors for cervical cancer, but also risk factors for female lung cancer; HPV16, HPV18. The E6 and E7 oncoproteins produced by the two viruses will pass through the 'liquid circulation to the lungs, and the human p53, Rb tumor suppressor protein will be decomposed. Once the tumor suppressor gene is lost, the cancer is fine. 20 cells appear in the lungs; however, although the currently developing DNA vaccine has long-lasting properties, its high cost and high risk (the virus itself is easily induced to mutate) is still the main reason for limiting its development; When E7 protein is used as gene therapy for cervical cancer, it is often unable to induce good immunity due to the weak antigenic characteristics of 'HPV virus E6 and E7 proteins. Reaction, and thus no
6 1358302 法發生所預期的預防或治療之功效。 一般而言，腫瘤的特異性抗原必須在宿主體内經過處 理後與+MHC- I型分子結合，才能被呈現到細胞表面以啟 動CD8+T淋巴細胞。有研究顯*子宮頸癌組織中含有 5 HPV16E7基因，但缺乏能夠呈現出E7基因編碼抗原特定 的MHC-I型分子的特定複合體於抗原呈現細胞表面，使 得HPV16 E7蛋白在宿主體内不被呈現而逃過宿主的免疫 監視。此外，一般做為疫苗之蛋白質在進入生體_時多 會被細胞認為是外源抗原，而在還沒發揮作用之前即被分 10解，而減低了蛋白質疫苗之效果，因此必須發展一種特殊 傳輸系統可以有效的將蛋白質抗原完整送達細胞質内部， 同時又能誘發生體產生有效性細胞免疫反應之方法。 【發明内容】 15 本發明係㈣一種可針對特定疾《4腫瘤而誘發細 胞免疫效果之融合蛋白，尤其是不易誘發免疫反應之弱抗 原病毒，本發明可藉由有效傳輸系統以及激發細胞免疫的 過程達到抑制癌化細胞增殖之目的，而降低癌化程度甚至 達到預防癌症之功效。 轉明融纟蛋白可於接受融合蛋白之生體細胞中誘 生CTL及抗體的保護作用，進而促使被感染之細胞由於抗 原之呈現而被殲滅；本發明亦提供一種含有預防或抑制因 人類乳突狀病毒16型所誘發腫瘤之融合蛋白之醫藥組合 物x d於細胞因人類乳突狀病毒16型之感染而發生癌變 1358302 之前抑制癌化細胞增瘦，達到抑制癌症發生之目的。 本發明所揭示之預防或抑制因人類乳突狀病毒16型 所誘發腫瘤之之融合蛋白，T細胞疫苗或含該融合蛋白之 醫藥組合物，係包括：一段人類乳突狀病毒16型（human 5 papillomavirus，HPV，type 16)之蛋白胜肽片段；一段具有 結合細胞及移位功能之胜'肽片段；以及一段具羧基終端部 分之胜肽片段；其中，該人類乳突狀病毒16型蛋白胜肽片 段係E7蛋白胜肽片段或E6蛋白胜肽片段。 於本發明融合蛋白中，其中所指之腫瘤包括子宮頸癌 1〇 或肺癌；而人類乳突狀病毒16型之E7蛋白胜肽片段之核苷 酸序列較佳為SEQ.ID.NO_l ; E7蛋白胜肽片段之胺基璇序 列為SEQ.ID.N0.2。E6蛋白胜肽片段之核苦酸序歹|J為 SEQ.ID.N0.3 ; E6蛋白胜肽片段之胺基酸序列為 SEQ.ID.N0.4。 15 適用之結合細胞以及移位胜肽片段係可選自習用之任 何一種具細胞結合及移位功能之胜肽片段，較佳是來自假 單胞菌屬夕卜毒素（pseudomonas aeruginosa exotoxin A, PE) 之第一部位（domain I)與第二部位（domain II)，其中之第一 部位係PE之配體部位（ligand)，配體部位係可與一標的細胞 20 之接受體進行結合，反應或辨識，以使所帶之融合蛋白經 由細胞接受體之細胞攝粒作用（endocytosis)，而可使帶有 抗原之融合蛋白進入標的細胞内，並且經由PE之第二部位 將融合蛋白之抗原轉送入細胞質内，抗原配合羧基終端 部位之胜肽片段共同啟動免疫反應。 8 1358302 可與配體部位作用之接受體範例包括但不限於··抗體 受體’生長因子受體’淋巴細胞活素受體，細胞激素受體 何爾蒙受體’熱休克蛋白heat-shock protein等。可與 -本發明之融合蛋白上配位部分作用之之接受體，較佳係可 5 選自至少一由以下接受體所組成之群組：TGF受體，IL2受 體，IL4受體，IL6受體，1GF1受體，CD4受體，IL18受體， IL12受體’ EGF受體’ LDL受體，α2巨球蛋白受體，熱休 • 克蛋白（heat-shock protein)。 可於本發明融合蛋白作用之細胞不限制，較佳係可包 10括：T細胞’ B細胞，樹突細胞，單核細胞或巨噬細胞。 而本發明融合蛋白中，羧基終端部分之胜肽片段，亦 可選自習用之任何一種與細胞KDEL受體接合之叛基終端 序列，較佳係來自假單胞菌屬外毒素之一部分，且，該羧 基終端部分之胜肽片段係包括KDEL或RDELK之胺基酸序 15 列，為 SEQ.ID.N0.5。 > 本發明更揭示一種與E7胜肽或E6胜肽結合之抗體組 成物’其中該E7胜肽之核苷酸序列為SEq IDN〇」；利用 本發明抗體組成物，可偵測生體内E7胜肽或E6胜肽片段的 出現，並以抗原抗體方式鍵結之。 20 本發明融合蛋白可用以預防或抑制人類乳突狀病毒 16型之感染，而含本發明融合蛋白之醫藥組合物中，較佳 係更包括一免疫佐劑；所適用之免疫佐劑不限，可以是任 何一種習用於疫苗之佐劑，包括鋁膠以及油質佐劑如：福 氏佐劑（Freund’s FCA or FIA)或乳化劑二縮甘露醇單油酸 9 1358302 鹽（mannide mono-oleate emulsifier，ISA720 或 isa206 SEPPIC，France)，較佳為 ISA206佐劑。 本發明利用細菌性毒素的特性，使帶有蛋白質之細菌 毒素之配體部位與標的細胞（抗原呈現細胞）之細胞膜表面 5接受器接合’使蛋白質進入細胞，並發揮細菌毒素自然之 位移特性，再將蛋白質帶至細胞質；此時，位於細胞質之 外源蛋白質可被產製成小胜肽，並接著與組織相容性複合 體Π或I型分子（MHCII或行結合，而被呈現於抗 原呈現細胞之外部，與結合者再藉由CD4+T或 10 CD8+T細胞辨識’而誘發一連串的免疫反應，使本發明融 合蛋白達到免疫提升之功效。 20 本發明之醫藥組合物可包括根據技藝中已知之技術 使用適合之佐劑；分散劑或保濕劑（如TWeen 80)以及懸浮 劑調配無菌注射劑，例如，無菌注射水溶液或含油性懸浮 15液。無菌注射製劑亦可用於無毒性注射之稀釋劑或溶劑中 之無菌注射液或懸浮液，例如，於丁二醇中之溶液。 在可使用之可接受之載體與溶射，有甘露醇、水、胞㈣ 溶液、與等張氯化納溶液。此外，習用上使用滅菌、固定 油類作為溶劑或懸浮介質·(例如合成之單酸或二酸甘油酷 類)。脂肪酸(例如油酸與其甘油，衍生物)，以及天然醫藥 上可接受之油類(例如橄欖油或編，尤其是其多氧乙基 化之里L )’可使用於可注射製劑。此等油溶液或懸浮液亦 可含有長鏈醇稀釋劑或分散劑、或繞甲基纖維素或類似之 分散劑。其他一般使用之界面活性_如Tweens或Spans 10 或其他類似之乳化劑或生體 醫藥上可接受之固體、液體二革增強劑（一般用於製造 配之目的。 或/、他劑量形式）亦可用於調6 1358302 The expected preventive or therapeutic effect of the method. In general, tumor-specific antigens must be treated in the host to bind to +MHC-type I molecules before they can be presented to the cell surface to activate CD8+ T lymphocytes. Studies have shown that * cervical cancer tissue contains 5 HPV16E7 gene, but lacks a specific complex capable of presenting the E7 gene-encoded antigen-specific MHC-I type molecule on the surface of the antigen presenting the cell, so that the HPV16 E7 protein is not in the host Presented and escaped from the host's immune surveillance. In addition, the protein that is generally used as a vaccine is considered to be a foreign antigen when it enters the living body, and is divided into 10 solutions before it has functioned, thereby reducing the effect of the protein vaccine, so it is necessary to develop a special The delivery system can effectively deliver protein antigens completely into the cytoplasm, while at the same time inducing the body to produce a viable cellular immune response. SUMMARY OF THE INVENTION The present invention is a fusion protein capable of inducing a cellular immune effect against a specific disease "4 tumors, particularly a weak antigen virus which is not susceptible to an immune response, and the present invention can be efficiently transmitted and stimulates cellular immunity. The process achieves the purpose of inhibiting the proliferation of cancerous cells, and reduces the degree of canceration and even the effect of preventing cancer. The fused glycoprotein can induce the protective effect of CTL and antibody in the living cells receiving the fusion protein, thereby causing the infected cells to be quenched due to the appearance of the antigen; the present invention also provides a method for preventing or inhibiting human milk The pharmaceutical composition xd of the fusion protein of the tumor-inducing tumor of the circovirus type 16 inhibits the growth of the cancerous cells before the cells become cancerous by the infection of the human papillomavirus type 16 1358302, thereby suppressing the occurrence of cancer. The present invention discloses a fusion protein for preventing or inhibiting a tumor induced by human papillomavirus type 16, a T cell vaccine or a pharmaceutical composition containing the same, comprising: a human papillomavirus type 16 (human a protein peptide fragment of 5 papillomavirus, HPV, type 16); a peptide fragment having a binding cell and a translocation function; and a peptide fragment having a carboxyl terminal portion; wherein the human papillomavirus type 16 protein The peptide fragment is an E7 protein peptide fragment or an E6 protein peptide fragment. In the fusion protein of the present invention, the tumor referred to includes cervical cancer 1 or lung cancer; and the nucleotide sequence of the human papillomavirus type 16 E7 protein peptide fragment is preferably SEQ.ID.NO_1; E7 The aminopurine sequence of the protein peptide fragment is SEQ. ID. N0.2. The nucleotide sequence of the E6 protein peptide fragment is J SEQ. ID. N0.3; the amino acid sequence of the E6 protein peptide fragment is SEQ. ID. N0.4. 15 Suitable binding cells and translocation peptide fragments may be selected from any of the peptide peptides with cell binding and translocation functions, preferably from Pseudomonas aeruginosa exotoxin A, PE. a first part (domain I) and a second part (domain II), wherein the first part is a ligand of a ligand, and the ligand part is capable of binding to a receptor of a target cell 20, the reaction Or identification, so that the fusion protein carried by the cell acceptor (endocytosis), the antigen-binding fusion protein can enter the target cell, and the antigen of the fusion protein is transferred through the second part of the PE Into the cytoplasm, the antigen cooperates with the peptide fragment of the terminal part of the carboxyl group to initiate an immune response. 8 1358302 Examples of receptors that can interact with ligand sites include, but are not limited to, the antibody receptor 'growth factor receptor' lymphocyte receptor, cytokine receptor Heermon receptor heat shock protein Wait. The acceptor which can interact with the coordination moiety on the fusion protein of the present invention, preferably 5, is selected from the group consisting of at least one of the following receptors: TGF receptor, IL2 receptor, IL4 receptor, IL6 Receptor, 1GF1 receptor, CD4 receptor, IL18 receptor, IL12 receptor 'EGF receptor' LDL receptor, α2 macroglobulin receptor, heat-shock protein. The cells which can act on the fusion protein of the present invention are not limited, and preferably include: T cell 'B cells, dendritic cells, monocytes or macrophages. In the fusion protein of the present invention, the peptide fragment of the terminal portion of the carboxyl group may also be selected from any of the conventionally used retinoid terminal sequences which are conjugated to the KDEL receptor, preferably from a part of the exotoxin of Pseudomonas. The peptide fragment of the terminal moiety of the carboxyl group comprises 15 columns of amino acid sequence of KDEL or RDELK, which is SEQ. ID. N0.5. > The present invention further discloses an antibody composition in which an E7 peptide or an E6 peptide is combined, wherein the nucleotide sequence of the E7 peptide is SEq IDN〇; and the antibody composition of the present invention can be used to detect the living body The appearance of E7 peptide or E6 peptide fragment is bonded by antigen-antibody method. 20 The fusion protein of the present invention can be used to prevent or inhibit the infection of human papillomavirus type 16, and the pharmaceutical composition containing the fusion protein of the present invention preferably further comprises an immunological adjuvant; the applicable immunological adjuvant is not limited It can be any adjuvant used in vaccines, including aluminum gel and oily adjuvants such as Freund's FCA or FIA or emulsifier mannitol monooleic acid 9 1358302 salt (mannide mono-oleate) Emulsifier, ISA720 or isa206 SEPPIC, France), preferably ISA206 adjuvant. The present invention utilizes the characteristics of a bacterial toxin to bind a ligand site of a bacterial toxin with a protein to a cell membrane surface receptor 5 of a target cell (antigen presenting cell) to cause the protein to enter the cell and exert the natural displacement characteristics of the bacterial toxin. The protein is then brought to the cytoplasm; at this time, the protein outside the cytoplasm can be produced as a small peptide, and then bound to the histocompatibility complex Π or type I molecule (MHCII or line, and presented to the antigen Presenting the outside of the cell and inducing a series of immune responses by the binding partner to identify by CD4+T or 10 CD8+ T cells, the fusion protein of the present invention achieves an immunostimulating effect. 20 The pharmaceutical composition of the present invention may include A technique known in the art uses a suitable adjuvant; a dispersing or moisturizing agent (such as TWeen 80) and a suspending agent to prepare a sterile injectable preparation, for example, a sterile injectable aqueous solution or an oily suspension 15. The sterile injectable preparation can also be used for non-toxic injection. A sterile injectable solution or suspension in a diluent or solvent, for example, in a solution of butanediol. Carrier and spray, mannitol, water, cell (tetra) solution, and isotonic sodium chloride solution. In addition, conventionally used sterilization, fixed oil as a solvent or suspension medium (such as synthetic monoacid or diglyceride cool Fatty acids (such as oleic acid and its glycerol, derivatives), as well as natural pharmaceutically acceptable oils (such as olive oil or woven, especially in its polyoxyethylation L) can be used in injectable preparations These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, or a methylcellulose or similar dispersing agent. Other commonly used interfacial activities such as Tweens or Spans 10 or other similar emulsifiers Or a pharmaceutically acceptable solid or liquid dermatological enhancer (generally used for manufacturing purposes or /, in dosage form)
口服投藥用之組合物可A ^ 』马任何口服上可接受之劑量 形式，包含但不限於膠囊、 私t 錠礼劑 '及水性懸浮液、分 政诏、與溶液。在口服用途 體包含乳畴及玉米殿粉。例中’一般使用之載 脂酸鎂。對於以膠囊形式口 炅 包含乳糖及… 可使用之稀釋劑 10 3礼糖及玉米《。當口服投藥水性分散劑或乳劑時， :使活性成份與乳化劑或懸浮劑組合懸浮或分散於油相 I。若需要，可添加特定之甜味劑、風味劑、或著色劑。 V腔賀劑或吸入劑組合物可依據醫藥配方之技蓺中習知之 技術製備，及可製成生理食鹽水，使用苯甲醇:其他適人 15 之防腐劑、增加生體可用率之吸收促進劑'氟碳化合物、 及/或其他技藝中已知之溶解劑或分散劑。含十朵化合物 之組合物亦可以直腸投藥之栓劑形式投藥。 醫藥組合物中之載體必須為，，可接受”，意為與配方中 之活性成份相容（及較佳地是能使配方安定）及對接受治療 之病患無害。其他載體之實例包含膠態二氧化石夕、硬脂酸 20鎂、纖維素、十二.烷基硫酸鈉、及D&c黃色⑺號。 本發明之融合蛋白或含本發明融合蛋白之醫藥組成 物不僅可抑制或預防因人類乳突狀病毒16型感染所誘發之 疾病，更可使接受本發明融合蛋白之動物體内維持很長— 段％間的抗體漠度’提向動物體之免疫效果。Orally administrable compositions can be administered orally in an acceptable dosage form, including, but not limited to, capsules, troche, and aqueous suspensions, hydrazines, and solutions. In oral use, the body contains milk domain and corn house powder. In the example, magnesium sulphate is generally used. For capsules, it contains lactose and... thinner that can be used 10 3 sugar and corn. When the aqueous dispersion or emulsion is administered orally, the active ingredient is suspended or dispersed in the oil phase I in combination with an emulsifier or suspension. A specific sweetener, flavor, or colorant may be added if desired. The V-cavitant or inhalant composition can be prepared according to the techniques known in the art of pharmaceutical formula, and can be made into physiological saline, using benzyl alcohol: other suitable 15 preservatives, and increasing absorption of bioavailability. A 'fluorocarbon, and/or other solubilizer or dispersant known in the art. Compositions containing ten compounds can also be administered in the form of a suppository for rectal administration. The carrier in the pharmaceutical composition must be, "acceptable", meaning that it is compatible with the active ingredient in the formulation (and preferably the formulation is stable) and is not deleterious to the patient being treated. Examples of other carriers include gums State sulphur dioxide, magnesium stearate 20 mg, cellulose, sodium lauryl sulfate, and D&c yellow (7). The fusion protein of the present invention or the pharmaceutical composition comprising the fusion protein of the present invention can inhibit or Prevention of diseases induced by human papillomavirus type 16 infection can further maintain the immune effect of the animal's body with the fusion protein of the present invention for a long period of time - the antibody indifference to the animal body.
11 25 1358302 【實施方式】 實施例一、E7核苷酸、E6核苷酸以及KDEL序列之合成 自美國國立生物技術f料中心（NCBI)資料庫中找到 • HPV16基因組DNA序列（NC_001526，SEQ.ID.NO.6)，其中 5自第83到559個鹼基爲E6基因片段ί或NCBI nucleotide V - 、 · accession No. AF4863 12, SEQ.ID.NO.34)，第 562到 858個鹼 基為 E7 基因片段（或 NCBI nucleotide accession No. , AF486342, SEQ.ID.NO.35)。 利用台灣專利申請案號92126644所揭示之方法，使 10 HPV16中E6基因或E7基因之蛋白質可藉由大腸桿菌系統 大量表現出來；改質之重點主要在將野生病毒株之核苷酸 片段，以在不影響其原本表現出之胺基酸，而又能有效的 在大腸桿菌宿主系統中表現的狀況下，進行單一核苷酸的 改質。 15 然而’由於野生型E6基因會結合人類基因組中p53抑 癌基因、而E7基因會結合pl〇5RB抑癌基因；因此E6、E7 > 這兩種基因已確定為致癌基因，研究證實此二基因片段可 與細胞之p53、p 105基因（細胞控制分裂與增殖相關之基因） 結合，並使p53、ρ 105基因去活化，造成細胞的增瘦無法抑 20 制，而使細胞的DNA在分裂的過程中更易突變、染色體錯 亂或因其他作用而發展成癌症。因此，依據美國食品與藥 品檢驗局（Food and Drug Administration，FDA USA)之規 範，必須將人類乳突狀病毒16型之野生型E6、E7基因進行 部分胺基酸之改質，才能降低致癌基因作用機會，而能安 12 1358302 全的使用於人體。 經過上述條件的改質，E7基因核苷酸序列為 SEQ.ID.N0.1，其中在E7基因N端起算之第24個胺基酸半胱 氨酸（cystein)改質為甘胺酸（glycine)，以及第26個半胱胺酸 5 改質也為甘氨酸，其對應之胺基酸序列為SEQ.ID.NO.2。 而E6基因核苷酸序列為SEQ.ID.N0.3，同樣的，將E6基因N 端起算之第70個胺基酸半胱氨酸改質為甘胺酸，以及第113 個半胱胺睃改質為甘胺酸，其對應之胺基酸序列為 SEQ.ID.N0.4。 10 本實施例合成之E6與E7核苷酸片段，分別利用多對引 子，以聚合酶連鎖反應（polymerase chain reaction, PCR)進 行核苷酸片段之合成，所有引子對之序列請見表一，表中 有底線之序列表示將與特定之片段發生互補。 首先利用無DNA模板的聚合方式，只利用F1以及R1 15 引子進行核苷酸片段，其中在此二引子的3’端部分各有15 個鹼基是設計為彼此互補的結合，再經由聚合酵素的讀寫 及補足成為一條雙股之DNA攀板聚合產物；完成第一次 PCR後，取Ιμΐ的聚合產物作為第二次PCR的模板DNA，同 時加入F 1以及R2引子各4μ1，連同所需的dNTPs、反應試劑 20 以及Pfu聚合酶等，開始進行第二次PCR ;接著依照相同方 式共進行8次的PCR，合成出改質後之核苷酸序 列一SEQ.ID.NO.1 與 SEQ.ID.NO.3。 以同樣的方式聚合出一段作為訊息胜肽的KDEL序 歹|J，其引子序列如表一中K3F與K3R ;合成出之KDEL胺基 13 1358302 酸序列為SEQ.ID_NO_5。 表一、引子對序列表11 25 1358302 [Embodiment] Example 1, the synthesis of E7 nucleotide, E6 nucleotide and KDEL sequence was found in the National Center for Biotechnology and Materials (NCBI) database. • HPV16 genomic DNA sequence (NC_001526, SEQ. ID.NO.6), wherein 5 from 83 to 559 bases are E6 gene fragments ί or NCBI nucleotide V - , · accession No. AF4863 12, SEQ.ID.NO.34), 562 to 858 bases The base is the E7 gene fragment (or NCBI nucleotide accession No., AF486342, SEQ.ID.NO.35). Using the method disclosed in Taiwan Patent Application No. 92126644, the protein of E6 gene or E7 gene in 10 HPV16 can be expressed in large quantities by the E. coli system; the focus of the modification is mainly on the nucleotide fragment of the wild virus strain. The modification of a single nucleotide is carried out without affecting the amino acid which is originally exhibited, and is effective in the expression in an E. coli host system. 15 However, 'the wild type E6 gene binds to the p53 tumor suppressor gene in the human genome, and the E7 gene binds to the pl〇5RB tumor suppressor gene; therefore, E6, E7 > these two genes have been identified as oncogenes, and studies have confirmed these two The gene fragment can bind to the p53 and p 105 genes of the cell (the genes involved in cell division and proliferation), and deactivate the p53 and ρ 105 genes, causing the cell to become thinner and inhibit the cell DNA. The process is more susceptible to mutation, chromosomal disorder or other cancers that develop into cancer. Therefore, according to the regulations of the Food and Drug Administration (FDA USA), the wild-type E6 and E7 genes of human papillomavirus type 16 must be modified with partial amino acids to reduce oncogenes. The opportunity to use, and can be used in the human body 12 1358302. After the above conditions were modified, the nucleotide sequence of the E7 gene was SEQ.ID.N0.1, in which the 24th amino acid cysteine (cystein) at the N-terminus of the E7 gene was modified to glycine ( Glycine), and the 26th cysteine 5 is also glycine, and its corresponding amino acid sequence is SEQ.ID.NO.2. The nucleotide sequence of the E6 gene is SEQ.ID.N0.3, and similarly, the 70th amino acid cysteine from the N-terminus of the E6 gene is modified to glycine, and the 113th cysteamine The hydrazine is modified to glycine, and the corresponding amino acid sequence is SEQ. ID. N0.4. 10 The E6 and E7 nucleotide fragments synthesized in this example were synthesized by polymerase chain reaction (PCR) using a plurality of pairs of primers, and the sequence of all primer pairs is shown in Table 1. A sequence of bottom lines in the table indicates that it will complement a particular fragment. First, using a DNA-free template, only the F1 and R1 15 primers are used for nucleotide fragments, wherein 15 bases in the 3' end of the two primers are designed to complement each other, and then via a polymerase. The reading, writing and complementing becomes a double-stranded DNA climbing product; after the first PCR, the 聚合μΐ polymerized product is taken as the template DNA for the second PCR, and the F 1 and R2 primers are added 4 μl each, together with the required The dNTPs, the reagent 20, and the Pfu polymerase, etc., start the second PCR; then, a total of 8 PCRs are performed in the same manner to synthesize the modified nucleotide sequence SEQ.ID.NO.1 and SEQ. .ID.NO.3. In the same way, a KDEL sequence 歹|J was synthesized as a message peptide, and its primer sequence was K3F and K3R in Table 1. The KDEL amino group 13 1358302 acid sequence synthesized was SEQ.ID_NO_5. Table 1, the primer pair sequence table
序列 引子代號 (序列編號） 鹼基序列 E7 HPV-E7GG-F1⑺ 5，-ACC ACC GAC CTG..TAC GGC TAG GGT CAG CTG -3， ΤΤΤ»Λ Γ nr v -jd / VJVJ- F2 (8) C 9 A A A A /~Λ A /—» A A j ••rvi vjam x丄vj hvj m οΛΛα hvj GAA ACC ACC GAC CTG TAC -3， HPV-E7GG-F3 (9) 5，- AA TTC ATG CAC GGT GAC ACC CCG ACC CTG CAC GAA TAC ATG CTG-3 ’ HPY-E7GG-F4-2 (10) 5*-GAA CTG CGT GAC GAA CTG AAA GAC GAA TTC ATG CAC GGT -3, HPV-E7GG- Rl(ll) 55-AAA GAG CTC CGG TTT CTG GGA GCA GAT CGG -3’ E6之N端 部份 N-terminal domain HPV-E6N-F1 (12) 5’-C ATC CAC GAC ATC ATC CTG GAA TGC GTT TAC TGC AAA CAG CAG CT-3， HPV-E6N-F2 (13) 5’-CCG CAG CTG TGC ACC GAA CTG CAA ACC ACC ATC CAC GAC ATC AT-3’ HPV-E6N-F3 (14) 5'- AG GAC CCG CAG GAA CGT CCG CGT AAA CTG CCG CAG CTG TGC ACC -3’ HPV-E6N-F4 (15) 5 ’-CAG AAA CGT ACC GCT ATG TTC CAG GAC CCG CAG GAA-3’ HPV-E6N-F5 (16) 5 ’-CCC GAA TTC CAT ATG GAC GTC ATG CAC CAG AAA CGT ACC GCT-3’ HPV-E6N-R1 (17) 5 ’-GAA AGC GAA GTC GTA AAC TTC ACG ACG CAG CAG CTG CTG TTT GCA-3’ HPV-E6N-R2 (18) 5 ’-CC GTC ACG GTA AAC GAT GCA CAG GTC ACG GAA AGC GAA GTC GTA -3 HPV-E6N-R3 (19) 5，-AG GCA TTT GTC GCA AAC AGC GTA CGG GTT ACC GTC ACG GTA AAC-3， HPV-E6N-R4 (20) 5，-C G.GA GAT TTT GGA GTA GAA TTT CAG GCA TTT GTC GCC A -3 ’ HPV-E6N-R5 (21) 5，_ TTT TTT CTC GAG GTA GTG ACG GTA TTC GGA GAT TTT GGA G-3 ’ HPV-E6N-R4 -2(22) 55-C GGA GAT TTT GGA GTA GAA TTT CAG GCA TTT GTC CGG A -3’ HPV-E6N-F5 -2(23) 5’-CCC GAA TTC CAT ATG GAC GTC ATG CAC CAG AAA CGT ACC GCT-3， E6之C端 部位 HPV-E6C-F1 (24) y-GC ATC AACGGT CAG AAA CCG CTG TGC CCG GAA GAA AAA CAG CGT-31 HPV-E6C-F2 (25) 5*-AAC AAA CCG CTG TGC GAC CTG CTG ATC CGT TGC ATC AAC GGT CAG AAA CCG-35 © 1358302 C-terminal domain HPV-E6C-F3 (26) 5，- TG TAC GGT ACC ACC CTA GAA CAG CAG TAC AAC AAA CCG CTG TGC -35 HPV-E6C-F4 (27) 5'-AAA GAA TTC GTC GAC TGC TAC TCT CTG TAC GGT ACC ACC -3 ’ HPV-E6C-R1 (28) 5’-TT GTG GAA ACG CTG TTT TTT GTC TAG GTG ACG CTG TTT TTC TTC -3’ HPV-E6C-R2 (29) 5’-CAT GCA CAG ACC GGT CCA ACG ACC ACG GAT GTT GTG GAA ACG CTG TTT TTT-3 HPV-E6C-R3 (30) 5，-GGT ACG GCT GCT ACG GCA GCAGCT CAT GCA ACG ACC GGT -3, HPV-E6C-R4 (31) 5’-GGG CTC GAG TAG CTG GGT TTC ACG ACG GGT ACG GCT GCT -3 ’ KDEL K3F(32) 5’- AGAATTCGTCGAC TAC CTC AAA AAA GAC GAA CTG AGA GAT GAA CTG-3’ · '· K3R(33) 5，-GTG GTG GTG CTC GAG TCA TTA CAG TTC GTC TTT CAG TTC ATC TCT CAG TT-3*Sequence primer code (sequence number) Base sequence E7 HPV-E7GG-F1(7) 5,-ACC ACC GAC CTG..TAC GGC TAG GGT CAG CTG -3, ΤΤΤ»Λ Γ nr v -jd / VJVJ- F2 (8) C 9 AAAA /~Λ A /—» AA j ••rvi vjam x丄vj hvj m οΛΛα hvj GAA ACC ACC GAC CTG TAC -3, HPV-E7GG-F3 (9) 5,- AA TTC ATG CAC GGT GAC ACC CCG ACC CTG CAC GAA TAC ATG CTG-3 ' HPY-E7GG-F4-2 (10) 5*-GAA CTG CGT GAC GAA CTG AAA GAC GAA TTC ATG CAC GGT -3, HPV-E7GG- Rl(ll) 55-AAA GAG CTC CGG TTT CTG GGA GCA GAT CGG -3' E6 N-terminal part N-terminal domain HPV-E6N-F1 (12) 5'-C ATC CAC GAC ATC ATC CTG GAA TGC GTT TAC TGC AAA CAG CAG CT- 3, HPV-E6N-F2 (13) 5'-CCG CAG CTG TGC ACC GAA CTG CAA ACC ACC ATC CAC GAC ATC AT-3' HPV-E6N-F3 (14) 5'- AG GAC CCG CAG GAA CGT CCG CGT AAA CTG CCG CAG CTG TGC ACC -3' HPV-E6N-F4 (15) 5 '-CAG AAA CGT ACC GCT ATG TTC CAG GAC CCG CAG GAA-3' HPV-E6N-F5 (16) 5 '-CCC GAA TTC CAT ATG GAC GTC ATG CAC CAG AAA CGT ACC GCT-3' HPV-E6N-R1 (17) 5 '-GAA AGC GAA GTC GTA AAC T TC ACG ACG CAG CAG CTG CTG TTT GCA-3' HPV-E6N-R2 (18) 5 '-CC GTC ACG GTA AAC GAT GCA CAG GTC ACG GAA AGC GAA GTC GTA -3 HPV-E6N-R3 (19) 5, -AG GCA TTT GTC GCA AAC AGC GTA CGG GTT ACC GTC ACG GTA AAC-3, HPV-E6N-R4 (20) 5,-C G.GA GAT TTT GGA GTA GAA TTT CAG GCA TTT GTC GCC A -3 ' HPV -E6N-R5 (21) 5,_ TTT TTT CTC GAG GTA GTG ACG GTA TTC GGA GAT TTT GGA G-3 ' HPV-E6N-R4 -2(22) 55-C GGA GAT TTT GGA GTA GAA TTT CAG GCA TTT GTC CGG A -3' HPV-E6N-F5 -2(23) 5'-CCC GAA TTC CAT ATG GAC GTC ATG CAC CAG AAA CGT ACC GCT-3, C-terminal part of E6 HPV-E6C-F1 (24) y -GC ATC AACGGT CAG AAA CCG CTG TGC CCG GAA GAA AAA CAG CGT-31 HPV-E6C-F2 (25) 5*-AAC AAA CCG CTG TGC GAC CTG CTG ATC CGT TGC ATC AAC GGT CAG AAA CCG-35 © 1358302 C -terminal domain HPV-E6C-F3 (26) 5,- TG TAC GGT ACC ACC CTA GAA CAG CAG TAC AAC AAA CCG CTG TGC -35 HPV-E6C-F4 (27) 5'-AAA GAA TTC GTC GAC TGC TAC TCT CTG TAC GGT ACC ACC -3 ' HPV-E6C-R1 (28) 5'-TT GTG GAA ACG CTG TTT TTT GTC TAG GTG ACG CTG TTT TTC T TC -3' HPV-E6C-R2 (29) 5'-CAT GCA CAG ACC GGT CCA ACG ACC ACG GAT GTT GTG GAA ACG CTG TTT TTT-3 HPV-E6C-R3 (30) 5,-GGT ACG GCT GCT ACG GCA GCAGCT CAT GCA ACG ACC GGT -3, HPV-E6C-R4 (31) 5'-GGG CTC GAG TAG CTG GGT TTC ACG ACG GGT ACG GCT GCT -3 ' KDEL K3F(32) 5'- AGAATTCGTCGAC TAC CTC AAA AAA GAC GAA CTG AGA GAT GAA CTG-3' · '· K3R(33) 5,-GTG GTG GTG CTC GAG TCA TTA CAG TTC GTC TTT CAG TTC ATC TCT CAG TT-3*
實施例二、載體構築· 將實施例一中完成聚合反應後所獲得之E7片段產 物，利用5%聚丙烯醢氨（polyacrylamide)緩S旨膠體進行分 5 離，並以產物分子量為依據純化萃取出標的產物；取pET 或ρΡΕ (ΔΠ)載體（J.R. Chen, C.W.Liao, S，J.T.Mao, and C_N. Weng，Vet. Microbiol. 80(2001)347-357)，利用限制酶同時 處理二載體以及純化後E7片段，再同樣以5%聚丙烯醯氨瓊 酯膠體進行分離純化，取0.3kb含有E7序列的片段，利用T4 10 ligase將E7片段與載體建構為一段長度為7.84kb，含綠膿桿 菌外毒素A不含酵素毒性部位之ΡΕ (ΔΜ)基因，以及含E7 基因的融合蛋白ΡΕ(△瓜）-Ε7之質體ρϊ>Ε(ΔΒΙ )-Ε7，以及一段 長度為3.83kb，含Ε7及pET23a的ρΕ7質體。 藉由K3-F K3-R引子所製得之PCR DNA以利用限制酶 15 切割及純化後，接合至pET23a的Sal 1 -Xho 1位置可以獲得一 15 ㊣ 1358302 段長度為3.78kb，含n，-KDELRDELKD'EL polypeptide基因 的pKDEL3質體。 以同樣方式，以限制酶Sail及Pstl在pKDEL3質體中將 一段長度為1.47kb含KDEL序列切出後接合至以限制酶 5 Xholl及Pstl切割之6.5kb的ΡΕ(ΔΙϋ)-Ε7質體DNA上，完成 一 8.0kb大小含有融合蛋台ΡΕ(ΔΠΙ )-E7-KDEL3基因之質體 pPE(DIII)-E7-K3。上述質體構築流程圖請參見圖1。 將上述完成構築之質體轉形（transform)入大腸桿菌 JM108菌株1IM呆存。 1〇 以相同方法步驟進行含有E6基因之質體構築，E6基因 質體構築流程圖請參見圖2，接著同樣轉形入大腸桿菌 JM108菌株令保存。 實施例三、蛋白質純化 將上述完成構築之質體轉形（transform)入大腸桿菌 15 BL21(DE3)pLys菌株中，接著將大腸桿菌培養於含有 200pg/ml抗生素ampicillin的200ml LB培養液中，直到菌液 浪度 OD550 遠到 0.3 ;接著加入ImM IPTG(isopropylthio-/5 -D-galactoside，Promege，USA)，繼續培養2小時，將所生 長出之細胞進行離心收集；以冷凍解凍反覆操作的方式使 20 帶有目標蛋白質的細胞之細胞膜結構略鬆，此時加入溶解 液 10ml(含有 〇.3mg/ml溶菌酶，ImM的 PMSF以及 0.06mg/ml 之DNasel)，置於室溫下20分鐘，接著再加入lml 10%Triton X-100，置於室溫下10分鐘，之後以12000xg離心10分鐘收 集蛋白質；再分別以與1M與2M之urea進行清洗；最後，將 16 1358302 所收集到之蛋白質包涵體（Inclusion body )溶於8ml的8M urea 中0 接著再以市售之pET His-Tag純化系統（Novagen， USA) ’依照實驗說明書進行如下之實驗：將溶於8M urea 5中的蛋白質包涵體倒入已先以4ml NTA-Ni2+配裝完成之 瓊酯樹脂管柱中；再將黏接於管柱中之蛋白質以不同 卩11(8.0、7.0、6.5、6.0、5_4以及3.5)之緩衝液（含61^1^&、 0.3m NaCl、20mM磷酸鹽緩衝液以及2〇mM Tris-HCl)沖下 收集之；最後以SDS-PAGE分析蛋白質純度並定量；此蛋 10 白質經確認包含有如SEQ.ID.N0.2所示之E7蛋白質的胺基 酸序列。 同樣以上述方法完成含有如SEQ.ID.N0.4所示，含E6 蛋白質的胺基酸序列之純化。 .實施例四、腫瘤細胞株（TC-1)製備 15 將HPV16-E6、E7及致癌基因用以癌化C57BL/6品 系之小鼠之主要肺部上皮細胞，此癌化細胞株即為TC-1， 利用習知方式來維持與生產TC-1細胞株。將此細胞株培養 於RPMI 1640中，同時添加10%(v/v)胎牛血清，50單位/ml 的 penicillin或是 streptomycin，L-麵醯氨酸（L-Glutamine) 20 2mM，丙酮酸納（sodium pyruvate)ImM，2mM非必須胺基 酸以及0.4 mg/ml G418，培養之條件為37°C，培養箱中維 持5%之C02。 欲使用腫瘤細胞株之當夫，以胰委白酶處理（trypsin) 並收集培養之細胞，以IX HBSS缓衝液清洗細胞2次，最後 17 1358302 再以1X HBSS稀釋細胞至欲使用之濃度。 實施例五、小鼠癌化模式之活體腫瘤試驗 將所欲進行試驗之蛋白質樣品：E7、ρε(ΔΙΠ )、ΡΕ(Δ HI )-Ε7、ΡΕ(ΔΠΙ )-E7-KDEL3分別以1 : ι〇的比例稀釋於磷 5酸鹽緩衝液中，使濃度為0.1 mg/ml ;同樣依上述方法製備 E6 蛋白質樣品—E6、ΡΕ(Δ Η )、ρε(Δ Πί )-Ε6、ΡΕ(Δ BI)-E6-KDEL3。 先培養於37°C 2小時，使用前再與1〇% ISA2〇6佐劑. (SEPPIC，France)以震盪方式遇合，形成四種不同之疫苗； 10分別取含有抗原量〇. 1 mg之上述疫苗，同時進行小鼠之免 疫，並分別於2週後追加補強一次免疫；在最後一次免疫後 一週，取5x1 〇4 TC-1腫瘤細胞注射至免疫小鼠之右腿皮下 位置以誘發腫瘤生長，同時注射一組未經免疫之小鼠，以 觀察腫瘤之自然生長情形。在誘發腫瘤生長實驗進行的第 15 7、14、20、30與60天時犧牲小鼠，除進.行腫瘤生長的目測 與觸診之外’並取出小鼠之脾臟細胞。 結果，接受 ΡΕ(Δ ΠΙ )-E7-KDEL3 以及 ΡΕ(Δ ΠΙ )-E6-KDEL3融合蛋白免疫後之小鼠，在誘發腫瘤生長試 驗期間’參與該試驗組的·小鼠均無腫瘤發生1 〇〇%，且持續 20 觀察了 60天皆為同樣現象；相反的，接受Ε7、ρΕ (Δ]]Ι)、 PE (△瓜）-Ε7、Ε6、ΡΕ (ΔΠΠ-Εό等融合蛋白，以及未接受 融合蛋白注射的小鼠組，在誘發腫瘤生長試驗後，最長只 有20天維持無腫瘤發生紀錄。由結果可以看出，含有ρΕ(Δ HI)以及KDEL3序列與Ε7抗原或Ε6抗原片段之融合蛋白， 18 1358302 在TC-1細胞株誘發小鼠癌化試驗模式中可以有效的發揮 預防腫瘤生長的免疫效果。 實施例六、細胞免疫實驗 . 如上述疫苗免疫的小鼠試驗，於一週後犧牲試驗小鼠 5 後取其脾臟巨噬細胞，屬於不同疫苗處理之脾臟巨噬細胞 約含3·5X 105之細胞液與含有一組織相容複合體（MHC class I)抗原決定位的E7胜肽（第46:57個胺基酸位置） ^ lpg/ml，一起培養16小時，再利用CD8+細胞表面標記 (surface marker)與細胞内細胞素IFN-γ的結合染色反應以 10 及流式細胞儀（FACScan)分析結果，來觀察各免疫組之間 屬於E7專一性之CD8+T細胞前驅物的生成差異情形。CD8 + 細胞表面標記（surface marker)與細胞内細胞素IFN-γ的染 色方法以及使用流式細胞儀（FACScan)的檢驗方法請參見 Cheng, et al., Hum Gene Ther, 13:553-568,2002. 15 於本實施例中，首度確認PE(ADI)-E7-KDEL3融合蛋白 對於E7專一性免疫的誘發具有影響力。如圖3所示，在注 • 射？£(八皿）-£7-尺0£1^3融合蛋白此組中，可發現1?义丫所製 造出具E7專一性的CD8+T細胞前驅物，比任何一組實驗組 來的高。由結果可知PE(MII)-E7-KD+EL3組可誘發比E7 20 組高於40倍的具E7專一性的CD8+T細胞前驅物。 同樣如圖4也可看出，ΡΕ(ΔΠΙ )-E6-KDEL3融合蛋白對 於E6專一性免疫的誘發也具有影響力。 實施例七、佐劑之使用 本實施例利用所測試之融合蛋白是否具有誘發E7專 25 一性之免疫反應，如增加E7專一性C.D8+T淋巴細胞之方 19 1358302 式，來檢測於本發明含融合蛋白之醫藥組成物中，佐劑的 使用是否影響本發明融合蛋白做為疫苗時之效果。 實驗過程如同實施例五與六，進行試驗之蛋白質樣品 .. 為1^(八皿）-£7與1^(八111)-£7-尺0丑1^3，並分別力〇入或不力口入 5 10% ISA206佐劑（SEPPIC，France);結果如圖 5所示，第一 組空白組中，並未顯示可誘發E7專一性CD8 + T淋巴細胞之 反應，同樣結果出現於第二組，不管所使用之蛋白質疫苗 是否含有E7片段，不含佐劑之疫苗均未出現誘發抗體之反 應；然而到第三組，含PE(AIE)-E7-KDEL3以及佐劑之疫 10 苗，其誘發E7專一性CD8+T淋巴細胞之數量高達600，比第 二組同樣蛋白質疫苗但不含佐劑之結果至少超過500-600 倍。同時，請參見圖6，在預防小鼠腫瘤生長之試驗中，使 用佐劑之PE(AIH)-E7-KDEL3融合蛋白，其預防小鼠腫瘤生 長之效果可長達60天，而不含佐劑者，在小鼠接受TC-1腫 15 瘤細胞10天之内，發生腫瘤的小鼠數量已經與控制組（未經 過融合蛋白之免疫）相同，也就是不含佐劑之ΡΕ(Δ • IE )-E7-KDEL3融合蛋白，不具預防小鼠腫瘤發生之效果。 本實施例結果不僅再一次證實本發明融合蛋白作為 抑制免疫反應與預防腫瘤發生疫苗之可行性，更重要的 20 是，佐劑的使用亦是本發明融合蛋白作為疫苗時不可或缺 之一種成份，同時，相同的效果亦可於帶有E6片段之蛋白 質樣品中證貫。 實施例九、抑制腫瘤生長 利用肺部血行性移轉模式（lung hematogenous spread 20 1358302 model)來測試本發明融合蛋白抑制體内腫瘤生長之效果。 ' 首先將小鼠之TC-1腫瘤細胞以5xl04細胞之濃度，經 由尾巴打入C57BL/6之小鼠（每組5隻）體内，於感染後2天， 再以皮下注射方式給予小鼠各O.lmg之E7、ΡΕ (ΔΙΠ)、ΡΕ(Δ 5 ΠΙ)-Ε7或PE(AIE)-E7-KDEL3融合蛋白，另外準備一組不注 射融合蛋白之控制組；在第30天時，犧牲小鼠並取下小鼠 之肺臟，每隻小鼠之肺臟腫瘤結節以實驗計數器計算數量。 觀察控制組，給予E7融合蛋白、E6融合蛋白、ΡΕ(Δ Π)融合蛋白、ΡΕ(ΔΠΙ)-Ε7融合蛋白、ΡΕ(ΑΠΙ)-Ε6融合蛋 10 白、PE(Am)-E7-KDEL3融合蛋白以及PE(AIE)-E6-KDEL3 融合蛋白等組之肺部腫瘤發生結果，其中可明顯看出接受 ΡΕ(ΑΠΙ )-E7-KDEL3 以及 PE(AIE )-E6-KDEL3 融合蛋白之小 鼠，肺部並未發生腫瘤，證實了本發明融合蛋白可控制因 E7或E6之表現而發生之肺部腫瘤。 15 .經由上述實施例之說明得知，本發明之融合蛋白可藉 由增進MHC I與II表現出E7或E6片段之效果，進而增加E7 或E6專一性之CD8+T細胞與CD4+T細胞前驅物的生成，而 提高了 E7或E6專一性抗體之濃度；且也具有預防或抑制因 E7或E6抗原所誘發之癌症效果；此外，接受本發明融合蛋 20 白免疫過之之小鼠，更可發現其具有比傳統疫苗更長時間 的免疫反應以及抗腫瘤之效果.，因此，本發明結合抗原位 置與結合細胞以及移位功能之胜肽片段可顯著的增加抗原 特定之免疫反應，且也提供了預防癌症之疫苗領域中另一 新的研究方向。 21 1358302 本發明所 而非僅限 上述實施例僅係為了方便說明而舉例而已 主張之權利範圍自應以申請專利範圍所述為準 於上述貫施例。 5【圖式簡單說明】. 圖1係本發明實施例二含£7胜肽片段之質體構築流程圊。 圖2係本發明實施例二含邡胜肽片段之質體構築流程圊。 φ 圖3係本發明實施例六之E7專一性CD8+T細胞前驅物的誘 發結果圖。 10 圖4係本發明實施例六之E6專一性抗體之檢測結果圖。 圖5係本發明實施例七中，佐劑的使用對於E7專一性抗體 生成結果圖。 圖6係本發明實施例七中，佐劑的使用對於預防小鼠腫瘤生 長結果圖。 15 【主要元件符號說明】 Φ 無 22Example 2: Carrier Construction The E7 fragment product obtained after the completion of the polymerization in Example 1 was subjected to separation by using 5% polypropylene polyacrylamide, and purified by extraction based on the molecular weight of the product. The product of the labeling; taking the pET or ρΡΕ (ΔΠ) vector (JR Chen, CWLiao, S, JTMao, and C_N. Weng, Vet. Microbiol. 80 (2001) 347-357), simultaneously treating the two vectors with a restriction enzyme and After purification, the E7 fragment was further isolated and purified by 5% polypropylene guanamine agarate colloid. A 0.3 kb fragment containing the E7 sequence was used, and the E7 fragment and vector were constructed to a length of 7.84 kb using a T4 10 ligase. Bacillus exotoxin A does not contain the ΡΕ (ΔΜ) gene of the toxic site of the enzyme, and the fusion protein △(△瓜)-Ε7 plastid ρϊ>Ε(ΔΒΙ)-Ε7 containing the E7 gene, and a length of 3.83 kb, including Ε7 and ρΕ7 plastids of pET23a. The PCR DNA prepared by the K3-F K3-R primer was cleaved and purified by restriction enzyme 15 and then ligated to the Sal 1 -Xho 1 position of pET23a to obtain a 15 positive 1358302 segment length of 3.78 kb, containing n, - The pKDEL3 plastid of the KDELRDELKD'EL polypeptide gene. In the same manner, a length of 1.47 kb KDEL sequence was excised in pKDEL3 plastids with restriction enzymes Sail and Pstl and ligated into 6.5 kb Ιϋ(ΔΙϋ)-Ε7 plastid DNA cleaved with restriction enzyme 5 Xholl and Pstl. Above, a plastid pPE(DIII)-E7-K3 containing the fusion egg tart (ΔΠΙ)-E7-KDEL3 gene was completed in a size of 8.0 kb. See Figure 1 for the above flow chart construction flow chart. The above-described completed plastid was transformed into Escherichia coli JM108 strain 1IM and left. 1〇 The plastid construct containing the E6 gene was carried out in the same manner. The flow chart of the E6 gene plastid construction is shown in Fig. 2, and then transformed into Escherichia coli JM108 strain for preservation. Example 3, Protein Purification The above-described completed plastid was transformed into Escherichia coli 15 BL21 (DE3) pLys strain, and then Escherichia coli was cultured in 200 ml LB medium containing 200 pg/ml antibiotic ampicillin until The LD550 of the bacterial solution was as high as 0.3; then ImM IPTG (isopropylthio-/5 -D-galactoside, Promege, USA) was added, the culture was continued for 2 hours, and the grown cells were collected by centrifugation; the method of freezing and thawing was repeated. The cell membrane structure of the cells with the target protein was slightly loosened. At this time, 10 ml of the lysate (containing 〇.3 mg/ml lysozyme, 1 mM PMSF and 0.06 mg/ml DNasel) was added and left at room temperature for 20 minutes. Then add 1ml of 10% Triton X-100, leave it at room temperature for 10 minutes, then collect the protein by centrifugation at 12000xg for 10 minutes; then wash it with 1M and 2M urea respectively; finally, collect the protein collected by 16 1358302 The inclusion body was dissolved in 8 ml of 8 M urea. Then the commercially available pET His-Tag purification system (Novagen, USA) was used. The following experiment was performed according to the experimental instructions: it will be dissolved in 8 M urea. The protein inclusion bodies in 5 were poured into agarose resin column which was firstly packed with 4 ml of NTA-Ni2+; the proteins adhered to the column were different in 卩11 (8.0, 7.0, 6.5, 6.0, 5_4). And 3.5) buffer (containing 61 ^ 1 ^ &, 0.3 m NaCl, 20 mM phosphate buffer and 2 mM Tris-HCl) was collected and collected; finally, the protein purity was determined by SDS-PAGE and quantified; 10 The white matter was confirmed to contain an amino acid sequence of the E7 protein as shown in SEQ. ID. N0.2. Purification of the amino acid sequence containing the E6 protein as shown in SEQ. ID. N0.4 was also carried out in the same manner as above. Example 4 Preparation of Tumor Cell Line (TC-1) 15 HPV16-E6, E7 and oncogene were used to cancer the main lung epithelial cells of C57BL/6 strain mice, and the cancer cell line was TC. -1, using a conventional method to maintain and produce TC-1 cell lines. This cell line was cultured in RPMI 1640 with 10% (v/v) fetal bovine serum, 50 units/ml of penicillin or streptomycin, L-Glutamine 20 2 mM, sodium pyruvate (sodium pyruvate) 1 mM, 2 mM non-essential amino acid and 0.4 mg/ml G418, culture conditions were 37 ° C, and 5% of CO 2 was maintained in the incubator. To use the tumor cell strain, trypsin and collect the cultured cells, wash the cells twice with IX HBSS buffer, and finally 17 1358302 and dilute the cells with 1X HBSS to the concentration to be used. Example 5: Live tumor test in mouse cancer mode The protein samples to be tested were: E7, ρε(ΔΙΠ), ΡΕ(Δ HI )-Ε7, ΡΕ(ΔΠΙ)-E7-KDEL3 were respectively 1: ι The ratio of hydrazine was diluted in phosphate 5 buffer to a concentration of 0.1 mg/ml; E6 protein samples were also prepared as described above—E6, ΡΕ(Δ Η ), ρε(Δ Πί )-Ε6, ΡΕ (Δ BI )-E6-KDEL3. First cultured at 37 ° C for 2 hours, before use with 1 〇 % ISA2 〇 6 adjuvant. (SEPPIC, France) in an oscillating manner to form four different vaccines; 10 take the antigen content 〇. 1 mg The above vaccine was simultaneously immunized with mice, and an additional immunization was performed after 2 weeks. One week after the last immunization, 5x1 〇4 TC-1 tumor cells were injected into the subcutaneous position of the right leg of the immunized mice to induce tumors. Grow and simultaneously inject a group of unimmunized mice to observe the natural growth of the tumor. Mice were sacrificed on the 15th, 14th, 20th, 30th and 60th day of the tumor growth-inducing experiment, except for the visual and palpation of tumor growth, and the spleen cells of the mice were taken out. As a result, mice immunized with ΡΕ(Δ ΠΙ )-E7-KDEL3 and ΡΕ(Δ ΠΙ )-E6-KDEL3 fusion protein did not develop tumors in the mice participating in the test group during the induced tumor growth test 1 〇〇%, and continued for 20 days, the same phenomenon was observed for 60 days; on the contrary, Ε7, ρΕ(Δ]]Ι), PE (△ melon)-Ε7, Ε6, ΡΕ (ΔΠΠ-Εό, etc. fusion protein, and The group of mice that did not receive the fusion protein injection maintained a tumor-free record for up to 20 days after the tumor growth test was induced. It can be seen from the results that it contains ρΕ(Δ HI) and KDEL3 sequence and Ε7 antigen or Ε6 antigen fragment. The fusion protein, 18 1358302, can effectively exert the immune effect of preventing tumor growth in the mouse cancer cell test model induced by TC-1 cell line. Example 6. Cellular immunization experiment. Experiment with mice immunized with the above vaccine, one week later After spleen test mice 5, their spleen macrophages were taken, and the spleen macrophages belonging to different vaccine treatments contained about 3. 5×105 cell fluid and E7 containing a histocompatibility complex (MHC class I) epitope. Peptide (46th: 57 amino acid positions) ^ lpg/ml, cultured for 16 hours, and then combined with the intracellular cytokine IFN-γ by CD8+ cell surface marker staining reaction by 10 and flow cytometry (FACScan) As a result, the difference in the production of CD8+ T cell precursors belonging to the E7-specificity between the respective immunized groups was observed. The staining method of the CD8+ cell surface marker and the intracellular cytokine IFN-γ and the use of flow cytometry For the test method of the instrument (FACScan), see Cheng, et al., Hum Gene Ther, 13: 553-568, 2002. 15 In this example, it was confirmed for the first time that the PE(ADI)-E7-KDEL3 fusion protein was specific to E7. The induction of sexual immunity has an influence. As shown in Fig. 3, in the group of injections: £(eight dishes)-£7-foot 0£1^3 fusion protein, it can be found that 1? The specific CD8+ T cell precursor was higher than that of any group of experiments. It can be seen that the PE(MII)-E7-KD+EL3 group can induce E7 specificity higher than 40 times than E7 20 group. CD8+ T cell precursor. As can also be seen in Figure 4, the ΡΕ(ΔΠΙ)-E6-KDEL3 fusion protein induces E6-specific immunity. Example 7 Use of Adjuvant This example uses whether the fusion protein tested has an immune response that induces E7, such as the addition of E7-specific C.D8+ T lymphocytes. 1358302, in order to detect whether the use of the adjuvant affects the effect of the fusion protein of the present invention as a vaccine in the pharmaceutical composition containing the fusion protein of the present invention. The experimental procedure is the same as in Examples 5 and 6. The protein sample was tested: 1^(eight dishes)-£7 and 1^(eight 111)-£7-foot 0 ugly 1^3, and respectively force in or Inadequent infusion of 5 10% ISA206 adjuvant (SEPPIC, France); the results shown in Figure 5, the first group of blank group, did not show the response induced E7-specific CD8 + T lymphocytes, the same results appeared in the first In the second group, regardless of whether the protein vaccine used contained E7 fragment, the vaccine without adjuvant did not induce antibody reaction; however, to the third group, 10 samples containing PE (AIE)-E7-KDEL3 and adjuvant The number of E7-specific CD8+ T lymphocytes induced is as high as 600, which is at least 500-600 times higher than that of the second group of the same protein vaccine but without adjuvant. At the same time, please refer to Figure 6. In the test for preventing tumor growth in mice, the adjuvant PE (AIH)-E7-KDEL3 fusion protein can prevent tumor growth in mice for up to 60 days without excipients. In the 10 days after the mice received TC-1 tumor cells, the number of tumor-bearing mice was the same as that of the control group (immunized without fusion protein), that is, without adjuvant (Δ • The IE)-E7-KDEL3 fusion protein does not have the effect of preventing tumorigenesis in mice. The results of this example not only confirm once again the feasibility of the fusion protein of the present invention as a vaccine for suppressing immune response and preventing tumorigenesis, but more importantly, the use of an adjuvant is also an indispensable component of the fusion protein of the present invention as a vaccine. At the same time, the same effect can also be demonstrated in protein samples with E6 fragments. Example 9. Inhibition of tumor growth The lung hematogenous spread 20 1358302 model was used to test the effect of the fusion protein of the present invention on inhibiting tumor growth in vivo. 'First mouse TC-1 tumor cells were injected into C57BL/6 mice (5 per group) via tails at a concentration of 5×10 4 cells, and then administered subcutaneously 2 days after infection. Each O.lmg of E7, ΡΕ (ΔΙΠ), ΡΕ(Δ 5 ΠΙ)-Ε7 or PE(AIE)-E7-KDEL3 fusion protein, additionally prepared a control group that does not inject the fusion protein; on the 30th day, The mice were sacrificed and the lungs of the mice were removed, and the lung tumor nodules of each mouse were counted in an experimental counter. Observation control group, E7 fusion protein, E6 fusion protein, ΡΕ(ΔΠ) fusion protein, ΡΕ(ΔΠΙ)-Ε7 fusion protein, ΡΕ(ΑΠΙ)-Ε6 fusion egg 10 white, PE(Am)-E7-KDEL3 fusion Protein and PE (AIE)-E6-KDEL3 fusion protein and other groups of lung tumorigenesis results, which can be clearly seen in mice receiving ΡΕ(ΑΠΙ)-E7-KDEL3 and PE(AIE)-E6-KDEL3 fusion protein, No tumor occurred in the lungs, confirming that the fusion protein of the present invention can control lung tumors that occur due to the expression of E7 or E6. 15. Through the description of the above examples, it is known that the fusion protein of the present invention can increase the E7 or E6 specificity of CD8+ T cells and CD4+ T cells by enhancing the effects of MHC I and II on E7 or E6 fragments. The precursor is produced to increase the concentration of the E7 or E6 specific antibody; and also has the effect of preventing or inhibiting the cancer induced by the E7 or E6 antigen; moreover, the mouse immunized with the fusion egg 20 of the present invention, It can be found that it has a longer-term immune response and anti-tumor effect than the conventional vaccine. Therefore, the combination of the antigen position and the binding cell and the translocation peptide fragment can significantly increase the antigen-specific immune response, and It also provides another new research direction in the field of cancer prevention vaccines. The invention is not limited to the above-described embodiments, but is intended to be illustrative only and the scope of the claims is intended to be within the scope of the appended claims. 5 [Simplified illustration of the drawings] Fig. 1 is a plastid construction process of the second embodiment of the present invention containing a peptide fragment of £7. 2 is a plastid construction process of a ruthenium-containing peptide fragment according to Example 2 of the present invention. φ Figure 3 is a graph showing the results of the induction of the E7-specific CD8+ T cell precursor of Example 6 of the present invention. 10 is a graph showing the results of detection of an E6-specific antibody of Example 6 of the present invention. Fig. 5 is a graph showing the results of the use of an adjuvant for E7-specific antibody production in Example 7 of the present invention. Fig. 6 is a graph showing the results of the use of an adjuvant for preventing tumor growth in mice in Example 7 of the present invention. 15 [Key component symbol description] Φ None 22