TWI302535B - A fusion protein for inhibiting cervical cancer - Google Patents
A fusion protein for inhibiting cervical cancer Download PDFInfo
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- TWI302535B TWI302535B TW094128399A TW94128399A TWI302535B TW I302535 B TWI302535 B TW I302535B TW 094128399 A TW094128399 A TW 094128399A TW 94128399 A TW94128399 A TW 94128399A TW I302535 B TWI302535 B TW I302535B
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- fusion protein
- pharmaceutical composition
- amino acid
- acid sequence
- adjuvant
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Classifications
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- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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Description
1302535 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 人類乳突狀病毒16型所誘發腫瘤之融合蛋白及其醫藥組成 5 物。 【先前技術】 90 在台灣,子宮頸癌發生率一向高居婦女癌症首位。一 般子宮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。 目前已證實人類乳突病毒(human papillomavirus, 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HPV的16、18和31高危險亞型 的早期病毒基因産物E6和E7蛋白,極易與Rb和p53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 Hp v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蛋白在子宮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,體液免疫爲輔。參加 細胞免疫的反應細胞主要有細胞毒性τ細胞(cytotoxic τ
J302535 lymphocytes,CTL)、自然殺手細胞(NK)和巨噬細胞。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 釋放某些溶解酶,將腫瘤細胞殺滅,並抑制腫瘤細胞的增 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HP V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HP V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,會透過血液循環到達肺部,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’但是其南成本’南危險性(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E7蛋白 的弱抗原特性,而無法誘發良好之免疫反應,進而無法發 1302535 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。有研究顯示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中時,多 會被細胞認為是外源抗原,而在還沒發揮作用之前即分 10解,而減低了蛋白質疫苗之效果,因此必須發展一種傳輸 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】 本發明係揭示一種可針對特定癌症誘發細胞免疫效 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。 本發明融合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 人類乳犬狀病毋16型所誘發腫瘤之融合蛋白之醫藥組合 物,以期於細胞因人類乳突狀病毒16型之感染而發生癌變 1302535 化之型態),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、或羧曱基纖維素或類似之 分散劑。其他一般使用之界面活性劑例如Tweens或Spans 或其他類似之乳化劑或生體可用率增強劑(一般用於製造 5醫藥上可接受之固體、液體、或其他劑量形式)亦可用於調 配之目的。 口服投藥用之組合物可為任何口服上可接受之劑量 形式,包含但不限於膠囊、錠、乳劑、及水性懸浮液、分 散劑、與溶液。在口服用途之錠劑之例中,一般使用之載 10體包含乳醣及玉米澱粉。一般亦常添加潤滑劑,例如,硬 脂酸鎂。對於以膠囊形式口服投藥而言,可使用之稀釋劑 包含乳糖及玉米澱粉。當口服投藥水性分散劑或乳劑時, 可使活性成份與乳化劑或懸浮劑組合懸浮或分散於油相 中。若需要,可添加特定之甜味劑、風味劑、或著色劑。 15鼻腔喷劑或吸入劑組合物可依據醫藥配方之技藝中習知之 技術製備,及可製成生理食鹽水,使用苯甲醇或其他適合 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。 20 、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 之病患無害。其他載體之實例包含膠態二氧化石夕、硬脂酉, 鎂、纖維素、十二燒基硫酸鈉、及D&C黃色1〇號。久 本發明之融合蛋白或含本發明融合蛋白之醫藥纟且成
1302535 物不僅可抑制或預防因人類乳突狀病毒丨6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一 段時間的抗體濃度,提高動物體之免疫效果。 【實施方式】 實施例一、E7核苷酸以及KDEL序之列合成 自美國國立生物技術資料中心(Ncbi)資料庫中找到 抑¥16£7蛋白序列(狀一001526,8£(^10.:^0.3),共98個 胺基酸。 10 利用台灣專利申請案號92126644所揭示之方法,使 HP V16E7蛋白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,改質後核苷酸 15 序列為SEQ.ID.NO.1。 本實施例合成之核苷酸片段,分別利用共8對引子, 以 I 合 8#連鎖反應(polymerase chain reaction,PCR)進行核 普酸片段之合成,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 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模板聚合產物;完成第一次 PCR後’取1μ1的聚合產物作為第二次PCr的模板dna,同 11 1302535 5,-GTG GTG GTG CTC GAG TCA TTA K3R CAG TTC GTC TTT CAG TTC ATC TCT CAGTT-35 實施例二、載體構築
10
將實施例一中完成聚合反應後所獲得之E7產物,利用 5%聚丙烯醯氨(polyacrylamide)瓊酯膠體進行分離,並以產 物分子量為依據純化萃取出標的產物;取pET或ρΡΕ (ΔΙΠ) 載體(J.R· Chen,C.W.Liao, SJ.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 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 (ΔΙΠ)基因,以及含E7基因的融合 蛋白ΡΕ(Δ ΠΙ )·Ε7之質體ρΡΕ(Δ皿)-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 切割及純化後,接合至pET23a的Sall-Xhol位置可以獲得一 段長度為 3.78kb,含 n’-KDELRDELKDEL 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 20 Xholl及Pstl切割之6.5kb的ΡΕ(ΔΙΠ)-Ε7質體DNA上,完成 一 8.0kb大小含有融合蛋白ΡΕ(ΔΙΠ )-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 13 J302535 JM108菌株中保存。 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 BL21(DE3)pLys菌株中,接著將大腸桿菌培養於含有 5 200pg/ml抗生素ampicillin的200ml LB培養液中,直到菌液
10 濃度 OD550 達到 0.3 ;接著加入 ImM IPTG(isopropylthio-/3 _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;以冷束解;東反覆操作的方式使 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 0.3mg/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 所收集到之蛋白質包涵體(Inclusion body )溶於8ml的8M 15 urea 中 ° 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 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 2〇 ρΗ(8·0、7.0、6·5、6.0、5·4以及 3.5)之緩衝液(含6M urea、 0.3m Naa、20mM磷酸鹽緩衝液以及20mM Tris_HCl)沖下 收集之;最後以SDS-PAGE分析蛋白質純度並定量;此蛋 白質經確認包含有如SEQ.ID.N0.3所示之E7蛋白質的胺基 酸序列。 1302535 實施例四、腫瘤細胞株(TC-1)製備 將HPV16-E6、E7及r似致癌基因用以癌化C57BL/6品 系之小鼠之主要肺部上皮細胞,此癌化細胞株即為TCM, 利用習知方式來維持與生產TC-1細胞株。將此細胞株培養 5 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-麩醯氨酸(L-Glutamine) 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。 10 欲使用腫瘤細胞株之當天,以腺蛋白酶處理(trypsin) 並收集培養之細胞,以IX HBSS緩衝液清洗細胞2次,最後 再以IX 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 15
20 將所欲進行試驗之蛋白質樣品:E7、ΡΕ(ΔΠΙ)、ΡΕ(Δ ΠΙ)-Ε7、PE(AHI)_E7-KDEL3分別以1 : 10的比例稀釋於磷 酸鹽緩衝液中,使濃度為0.1mg/m卜先培養於37°C 2小時, 使用前再與10% ISA206佐劑(SEPPIC,France)以震盪方式 混合,形成四種不同之疫苗;分別取含有抗原量O.lmg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5xl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 15
1302535 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(空白組 10·0±1·4,E7組 14·0±2·1,ΡΕ(ΔΙΠ)組 12.0土 2·1,ΡΕ(ΑΙΠ )-Ε7組36.0±2.8,PE(MII)-E7-KDEL3 組564.0 ±28·0 , ρ<0·01, AVONA)。由結果可知 5 PE(AIII)-E7-KDEL3組可誘發比E7組高於40倍的具E7 專一性的CD8+T細胞前驅物。 實施例七、E7專一性抗體之檢測 如實施例五的免疫動物方法,以0.1 mg之E7、PE (A ΙΕ)、ΡΕ(ΔΙ[)-Ε7、PE(AIE)_E7-KDEL3融合蛋白進行小鼠 10 之免疫,並分別於1週與2週後追加補強一次疫苗;在最後 一次免疫進行後7天收集小鼠jk清。 於96孔盤之各孔中覆上ΙΟΟμΙ之HPV16_E7(0.5pg/ml) 融合蛋白,於4°C下培養隔夜;再以含20%胎牛血清之PBS 阻覆於培養孔中;將小鼠血清以PBS進行連續稀釋,接著 15 加入培養孔中,於37°C中培養2小時;再以含有0.05% Tween 20之PBS清洗培養孔後,加入1:2000之鍵結有過氧化 酶之兔子抗老鼠 IgG 抗體(peroxidase-conjugated rabbit anti-mouse IgG antibody,Zymed,San Francisco,CA)於培 養孔内,再於室溫下培養1小時;之後沖洗培養盤,並加入 20 1-Step Turbo TMB-ELISA (Pierce,Rockford, IL)使顏色展 開,最後以1M2H2S04終止反應;利用ELISA判讀機於 450nm之吸收光譜下進行結果之判讀。 於本實施例中,更進一步以實驗求得ΡΕ(Δ m)-E7-KDEL3可提升抗Ε7抗體的力價。如圖4所示,免疫 17
1302535 後的小鼠所生產出之抗E7的抗體’於血清中測得的數值’ 以1:100稀釋倍數來看,空白組為〇·629±0·093,E7組0.882 ±0.086,ΡΕ(ΔΠΙ)組0·69±0·06,ΡΕ(ΑΙΠ)-Ε7組0_93±0.06,而 PE(AIII)-E7-KDEL3 則高達 3·593±0·54(ρ<0·01,AVONA), 可明顯瞭解以ΡΕ (ΔΙΙΙ)-ΚΟΕΕ/Ε7融合蛋白免疫小鼠,將可 使小鼠產生高於其他組之抗Ε7抗體。 我們的實驗結果充分顯示PE(MII)-E7-KDEL3融合蛋 白具有誘發E7專一性之免疫反應,包括增加E7專一性 CD8+T淋巴細胞以及E7專一性抗體。 10 所得數據皆以平均值及標準誤差值(Mean±SEM)表 示。實驗組之間的比較數據係利用「社會科學統計套裝軟 體」(Statistical Package for Social Sciences,SPSS 9.0, SPSS Inc,Chicago, IL)進行ANOVA變異數分析,如誤差 機率低於0.05,則表示此數據具有顯著差異性。 15 實施例八、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 一性之免疫反應,如增加E7專一性CD8+T淋巴細胞之方 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。 20 實驗過程如同實施例五與六,進行試驗之蛋白質樣品 為ΡΕ(ΔΠ)-Ε7與PE(AIE)-E7_KDEL3,並分別加入或不加入 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 18 1302535 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(△羾)_E7_KDEL3以及佐劑之疫 苗,其誘發E7專一性CD8+T淋巴細胞之數量高達6〇〇,比第 二組同樣蛋白質疫苗但不含佐劑之結果至少超過5〇〇_6〇〇 5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(A1E)-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TCj· 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 10 m)-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 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。 15 實施例九、抑制腫瘤生長 利用肺部血行性移轉模式(lung hemat〇gen〇us邛代“ model)來測试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, 20再以皮下注射方式給予小鼠各〇 lmg2E7、ρΕ (ΔΙΠ)、ρΕ(Δ Π)_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。 結果如圖7A所示,分別為控制組(i),給予以融合蛋白 19 1302535 (ii)、ΡΕ (ΔΠΙ)融合蛋白(iii)、ρε(ΔΠΙ)-Ε7融合蛋白(ix)以及 PE(AIE)_E7-KDEL3融合蛋白(χ)之肺部腫瘤發生結果,其 中可明顯看出接受PE(ADI)-E7-KDEL3融合蛋白之小鼠,肺 部並未發生腫瘤;而圖7B更顯示肺部的重量,接受ΡΕ(Δ 5瓜)-E7-KDEL3融合蛋白之小鼠,因沒有發生肺部腫瘤,所 以重量沒有其他四組重,其肺部平均重量為214.4±11.6, 遠低於ΡΕ(ΔΙΠ )-E7融合蛋白之673·6±20·8或是控制組之
10 811.1 ±45.6(one-way ANOVA 單因子變異數分析,ρ< 0.001);上述結果證實了本發明融合蛋白可控制因Ε7之表 現而發生之肺部腫瘤。 實施例十、免疫時間之維持 本實施例目的在測試當小鼠接受不同次數之融合蛋 白的免疫後,其可持續預防腫瘤之時間;實驗方法如同實 施例五與六,小鼠以不同次數之濃度為〇1111§/1111的?£(^ 15 融合蛋白,分別進行小鼠之免疫,次數分別 為1-3次。 結果如圖8 A所示,所有未經免疫之控制組以及只免疫 過一次之小鼠,均在以腫瘤細胞TC-1感染後14天内發生腫 瘤’而接受2次免疫時,有60%之小鼠於感染後6〇天仍能維 20持不發生腫瘤;而接受3次免疫之一組,則有100%之小鼠, 均可維持60天無腫瘤發生之效果。 同樣結果亦顯示於抑制體内腫瘤生長之實驗,進行如 同貫施例九之實驗流程,小鼠於接種腫瘤細胞後,分別進 订1-3次不等的免疫步驟,同樣於第3〇天時犧牲小鼠;結果 20 1302535 發結果圖。 圖4係本發明實施例七之E7專一性抗體之檢測結果圖。 圖5係本發明實施例八中,佐劑的使用對於E7專一性抗體 生成結果圖。 5圖6係本發明實施例八中,佐劑的使用對於預防小鼠腫瘤生 長結果圖。 圖7A本發明實施例九中,使用本發明融合蛋白對於抑制肺 部腫瘤發生之結果圖,其中圖⑴為控制組、圖(丨丨)為E7融合 蛋白、圖(111)為ΡΕ (ΑΠΙ)融合蛋白、圖(ix)為pE(△皿)_E7融 10合蛋白、圖(ix)為合蛋白、圖㈨為ρΕ(Λ HI )-E7-KDEL3 融合蛋白。
圖7B係本發明實施例九中,不同融合蛋白影響肺部腫瘤發 生時,肺部之重量變化。 圖8A係本發明實施例十中,給予小鼠不同次數之融合蛋 白,其可持續預防腫瘤之時間測試結果圖。 圖8B係本發明實施例十中,給m不同次數之融合蛋 白,其可持續抑制體内腫瘤生長之時間測試結果圖。 【主要元件符號說明】 20 Μ 22
Claims (1)
- 公舌 1302535 第94128399號,97年8月修正頁 1 十、申請專利範圍: 1 · 一種預防或抑制因人類乳突狀病毒16型所誘發癌 症之融合蛋白,係包括: a. 一段人類乳突狀病毒16型(human papillomavirus, 5 HPV,type 16)之E7胜肽序列,包含一段如SEQ ID NO: 3所 示的胺基酸序列; b. —段假單胞菌屬外毒素(pseudomonas exotoxin)胜 肽片段,其只缺乏區域III (domain III);以及10 15c. 一段羧基終端部分,包括一段胺基酸序列,此胺基酸 序列選自由 KDEL、KDELRDELKDEL及 SEQ.ID.NO.2所組 群組其中的一者。 2. 如申請專利範圍第1項所述之融合蛋白,其中該腫 瘤為子宮頸癌或肺癌,或兩者。 3. 如申請專利範圍第1項所述之融合蛋白,其中該羧 基終端片段包括KDEL。 4.如申請專利範圍第1項所述之融合蛋白,其中該羧基終 端部分之胺基酸序列為SEQ.ID.NO.2。 5· —種醫藥組合物,其係用以預防及/或抑制因人類乳 突狀病毒16型所誘發之癌症,包括一有效劑量之申請專利 範圍第1項所述之融合蛋白。 6.如申請專利範圍第5項所述之醫藥組合物,更包括 一佐劑。 7. —種抑制因人類乳突狀病毒16型所誘發癌症之融 合蛋白,係包括: 20 1302535 a. —段人類乳突狀病毒16型(human papillomavirus, HPV,type 16)之E7胜肽序列,包含一段由具有SEQ ID N0:1之DNA所解碼之胺基酸序列; b. 一段假單胞菌屬外毒素(pseudomonas exotoxin)胜 5 肽片段,其只缺乏區域III (domain III);以及 c. 一段羧基終端部分,包括一段胺基酸序列,此胺基 酸序列選自由 KDEL、KDELRDELKDEL及 SEQ.ID.N0.2所 組群組其中的一者。10 158 ·如申請專利範圍第5項所述之醫藥組合物,其中該 羧基終端部分之胺基酸序列包含SEQ.ID.N0.2之胺基酸序 列0 9.如申請專利範圍第8項所述之醫藥組合物,其更包 括一佐劑。 10.如申請專利範圍第9項所述之醫藥組合物,其中 該佐劑為 ISA206佐劑(incomplete Seppic adjuvant 206)。 Π ·如申請專利範圍第1項所述之融合蛋白,其中該 ΡΕ胜肽包含部份I (domain I)及部分II (domain II)。 12. 如申請專利範圍第7項所述之融合蛋白,其中該 PE胜肽包含部份I (domain I)及部分II (domain II)。 13. 如申請專利範圍第1項所述之融合蛋白,其中該 羧基終端片段包括KDELRDELKDEL。 14. 如申請專利範圍第7項所述之融合蛋白,其中該 羧基終端片段包括KDELRDELKDEL。 15.如申請專利範圍第7項所述之融合蛋白,其中該 20 1302535 羧基終端部分之胺基酸序列包含SEQ.ID.N0.2之胺基酸序 列。 16 ·如申請專利範圍第7項所述之融合蛋白,其中該 羧基終端片段包括KDEL。 17. 種抑制因人類乳突狀病毒16型所誘發腫瘤之 醫藥組成物,包括一有效劑量之申請專利範圍第7項所述之 融合蛋白。 18. 如 括一佐劑。 19. -醫藥組成物 之融合蛋白 20. _ 醫藥組成物 之融合蛋白 21. _ 醫藥組成物 之融合蛋白 22. _ 醫藥組成物 之融合蛋白 10 15 申請專利範圍第17項所述之醫藥組成物,更包 種抑制因人類乳突狀病毒16型所誘發腫瘤之 ’包括-有效劑量之巾請專利範圍第12項所述 〇 種抑制因人類乳突狀病毒16型所誘發腫瘤之 ’包括-有效劑量之申請專利範圍第14項所述 〇 種抑制因人類乳突狀 扃t 16型所誘發腫瘤之 ’包括一有效劑量之申蟢直 τ明專利乾圍第15項所述 〇 種抑制因人類乳突狀病表 ,+ id曰 扃毋16型所誘發腫瘤之 匕括一有效劑S之申★主直 甲明專利範圍第16項所述 20 1302535 Π年Ί月修正替換頁 「I敬坦脾 εΊωαΜ_ζω丨(Invwd ZSIIIVwd mfi谢 ο ο ο § ο ο ο ο ο ο ς0\χζ'£ ι+8αο m\^t lr ¥ 130253509 9 ® (B)鵪 ¥^w^鸾4墚 Ιόι 0 寸ocoocvl 0T- 0 § 09 09 0寸 OCNI 0
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DE60038622D1 (de) * | 1999-10-20 | 2008-05-29 | Univ Johns Hopkins Med | Chimäre immunogene zusammensetzungen und dafür kodierende nukleinsäure |
DE10059630A1 (de) | 2000-12-01 | 2002-06-06 | Medigene Ag | Arzneimittel zur Vermeidung oder Behandlung von durch humanen Papillomavirus-Typ 18-hervorgerufenem Tumor |
US20060147906A1 (en) | 2002-03-22 | 2006-07-06 | Amynon Biotech Gmbh | Anti-hpv-16 e7 antibodies and their use |
US7223408B2 (en) | 2002-10-03 | 2007-05-29 | Wyeth Holdings Corporation | Human papillomavirus polypeptides and immunogenic compositions |
US7335361B2 (en) * | 2003-06-09 | 2008-02-26 | Animal Technology Institute Taiwan | Fusion antigen used as vaccine |
-
2004
- 2004-11-04 JP JP2004320487A patent/JP4474264B2/ja not_active Expired - Fee Related
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2005
- 2005-08-18 RU RU2005126236/15A patent/RU2005126236A/ru not_active Application Discontinuation
- 2005-08-18 US US11/206,138 patent/US7378100B2/en active Active
- 2005-08-19 CA CA2516511A patent/CA2516511C/en not_active Expired - Fee Related
- 2005-08-19 TW TW094128399A patent/TWI302535B/zh active
- 2005-08-19 KR KR1020050075999A patent/KR100877759B1/ko active IP Right Grant
- 2005-08-22 AU AU2005203772A patent/AU2005203772C1/en not_active Ceased
Also Published As
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CA2516511A1 (en) | 2006-02-20 |
KR100877759B1 (ko) | 2009-01-12 |
JP2006056869A (ja) | 2006-03-02 |
AU2005203772A1 (en) | 2006-03-09 |
AU2005203772B2 (en) | 2007-08-23 |
US20060039919A1 (en) | 2006-02-23 |
CA2516511C (en) | 2012-05-15 |
KR20060053135A (ko) | 2006-05-19 |
US7378100B2 (en) | 2008-05-27 |
JP4474264B2 (ja) | 2010-06-02 |
AU2005203772C1 (en) | 2008-11-13 |
RU2005126236A (ru) | 2007-02-27 |
TW200607524A (en) | 2006-03-01 |
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