TWI237056B - Fusion protein having enhanced in vivo activity of erythropoietin - Google Patents
Fusion protein having enhanced in vivo activity of erythropoietin Download PDFInfo
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- TWI237056B TWI237056B TW091116686A TW91116686A TWI237056B TW I237056 B TWI237056 B TW I237056B TW 091116686 A TW091116686 A TW 091116686A TW 91116686 A TW91116686 A TW 91116686A TW I237056 B TWI237056 B TW I237056B
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Landscapes
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1237056 五、發明說明（1) [技術領域] 本發明係關於一種具有經強化之體内促紅素（以下稱 為ΕΡ0，其為治療貧血之新穎藥物）活性之融合蛋白質。具 體言之，本發明係關於一種於體内具有高度經延長之半生 期及促紅素活性之融合蛋白質，該融合蛋白質係藉由將 ΕΡ0分子與某些具有延長半生期活性且係衍生自人體之肽 融合而成。 [背景技藝] ΕΡ0為一種具有分子量為3 0, 0 0 0至34, 0 0 0的糖蛋白， 其係刺激紅血球細胞生產及分化之因子。此蛋白質之作用 係藉由結合至紅血球前驅細胞上之受體而造成細胞中鈣離 子濃度增加、增加DNA生物合成以及刺激血紅素之形成 等。此EPO可用於治療腎衰竭造成之貧血、早產兒之貧 血、甲狀腺機能減退造成之貧血、營養失調造成之貧血 等。臨床上利用重組人類EPO者與日倶增。然而，由於其 半生期短，所以每星期應平均投藥3次，故使用重組人類 EPO會造成某些不便以及高成本之問題。因此，如果使EPO 於體内維持長時間之活性，則可大大地降低EPO之投藥頻 率。 EPO之效力與其半生期成正比。已知EPO於體内之半生 期係與涎酸之含量有關，而該涎酸係位於EPO之糖鏈的末 端。因此，E P 0之效力係高度取決於糖鏈之存在。由於糖 類呈現出的型式係根據細胞（為表現EPO之細胞）之種類而 不同，如果糖蛋白表現於不同細胞中，則相同的糖蛋白可
92166.ptdQ32038 第6頁 1237056 五、發明說明（2) 能具有不同的糖結構。雖然近來已證實某些細菌可帶有糖 鏈’但已知之典型的細菌（例如E · c ο 1 i )則否。E · c ο 1 i中 表現的蛋白質不包含糖鏈，因此，自E· col丨得到之Ep〇不 包含糖鏈，該ΕΡ0於試管内展現絕對的活性，但於體内則 不具活性。這是因為去糖基化之ΕΡ0會快速地自人體中排 除以及該E P 0具有極短暫的半生期。總之，該糖鏈於E p 〇之 活性中扮演非常重要的角色。 已於加強ΕΡ0之活性上進行許多研究。主要的方法為 藉由ΕΡ0基因之突變來取代某些胺基酸，例如，Amgen申請 之PCT/US94/ 0 9 2 5 7，標題為”促紅素類似物”，該專利案揭 示一種經由突變增加EPO之糖含量而增長EPO於體内之半生 期的方法。同樣地，企圖藉由形成EPO二聚物而使EPO於體 内之半生期增長。參見A. J. Sytkowski等人之J.B.C. vol. 274， No. 35， pp24773-24778。此外，其他已知方 法係藉由基因工程方法將新胺基酸、胜肽或蛋白質片段與 ΕΡ0分子融合以及增加糖含量（即EP0中涎酸的含量）來增強 ΕΡ0於體内之活性。然而，所有胺基酸、胜肽或異種蛋白 質片段不可用於此目的。於大多數例子中，此等修改可能 會造成蛋白質固有的活性降低或喪失以及當此蛋白質使用 於體内時可能會有抗原力之問題。 雖然此與EP0無關，已對融合蛋白質或嵌合型蛋白質 進行研究，例如，刺激卵泡素，其為一種性激素。參見， Furuhashi等人 1995， Mol. Endocrinol。然而，由於利用 基因工程方法來修改蛋白質具有許多風險，所以該方法尚
第7頁 92,66^039 1237056
第8頁 五 、發明說明（3) 未 應 用至工業上。也就 是於大多數例子中，若 無專 業技術 則 無 法輕易地得到標的 蛋白質，相反地，藉由 新胺 基酸之 加 成 或取代可能會使蛋 白質固有的活性降低或 喪失 0 [發明揭示] 本發明人已對藉由 將新胺基酸、胜肽或蛋 白質 片段與 ΕΡ0分子融合以增加其糖含量來加強ΕΡ0於體内 之活 性之方 法 進 行廣泛地研究。結 果，本發明人發現藉由 將血 小板生 成 素 (以下稱為ΤΡ0，為 存在於人體中之蛋白質 )之魏基端 胜 肽 (以下稱為CTP)與ΕΡ0之羧基端融合而得到 ΕΡ0融合蛋 白 質 ，由於其具有許多 可增加糖化作用位置之 胺基 酸且不 會 喪 失ΕΡ0固有的活性 當施用至人體時不會造成任何抗 原 力 ，所以該等帶有羧 基端之ΕΡ0於體内具有高度經加強 之 半 生期。於是本發明 人因而完成本發明。 因此，本發明之目 的為提供一種融合蛋白 質， 該蛋白 質 於 體内具有高度經加 強之人類ΕΡ0活性及含有於ΤΡ0羧基 端 與 人類ΕΡ0融合之CTP。 本發明之另一目的 為提供可編碼出融合蛋 白質 之核苷 酸 Λ 含有該核苷酸之重 組載體以及以該重組載 體轉 形之宿 主 細 胞株。 本發明之又一目的 為提供製備融合蛋白質 之方 法，該 方 法 係藉由培養經轉形 之宿主細胞株而得到於 體内 具有南 度 經 加強之人類ΕΡ0活性之融合蛋白質。 首先，本發明係關 於一種融合蛋白質，該 蛋白 質於體 内 具 有高度經加強之人 類ΕΡ0活性及含有於ΤΡ0羧基 端與人 92166 δ 似 040 1237056 五、發明說明（4) 類ΕΡ0融合之CTP。該CTP較佳包括SEQ ID No. 1之胺基酸序 列，其相當於位置第1 76至3 5 3之胺基酸（以下稱為LTP)或 其部分，特別係SEQ ID No. 2之胺基酸序列，其相當於位 置第3 3 7至3 5 3之胺基酸（以下稱為STP)。 尤其，根據本發明之融合蛋白質可包括SEQ ID No. 3 或4之胺基酸序列。 弟一 ’本發明係關於可編碼出融合蛋白質之核苷酸、 含有該核苷酸之重組載體以及以該重組載體轉形之宿主細 胞株（較佳為CH0 (中國倉鼠之卵巢）細胞）。 第三’本發明係關於製備融合蛋白質之方法，該方法 係藉由培養經轉形之宿主細胞株而得到於體内具有高度經 加強之人類ΕΡ0活性之融合蛋白質。 以下將更詳細說明本發明。 [實行本發明之最佳型態] _ 本發明包括下列步驟：製備及選殖所欲之基因、建構 έ有所欲基因之表現載體、進行動物細胞之轉形作用、表 現、純化以及生物分析。 (1)製備基因 利用與ΕΡ0之cDNA末端互補之引子（ΕΡ1與ΕΡ2，得自 ^犬員胎兒肝臟cDNA資料庫，InvitrogenCo·)，藉由進行 ^ 知之 P C R技術（Bioneer Co.之 PCR PreMix Kit)得到 ΕΡ0 之cDNA。利用與ΤΡ0之cDNA端互補之引子（T1與T2)，經由 相同方式，而得到ΤΡ0之cDNA。
1237056 五、發明說明（5) EPl: ATGGGGGCACGAATGTCCTGCCTGGCTGG (SEQ ID No. 5) EP2； GTCCCCTGTCCTGCAGGCCT (SEQ ID No. 6)
Tl : ATGGATCTGACTGAATTGCTCCTC (SEQ ID No. 7) T2: TTACCCTTCCTGAGACAGATTCTGGGA (SEQ ID No. S) 分別將藉由PCR得到的EPO cDNA與TPO cDNA選殖至 pGEM-T(Promega Co·，為一種選殖載體）中。然後定序 pGEM-ΕΡ0以及pGEM-TPO，且將其作為後續程序之模板。 LTP，為一種於本發明中所使用之ΤΡ0的CTP基因，其 可藉由利用pGEM-TPO (作為模板）以及引子ELI及T2進行PCR 而得到。 ELI ： GAGGCCTGCAGGACAGGGGACGCCCCACCCACCACAGCTGTCCCSEQ ID NO. 9) 該引子ELI含有延伸至限制酶Stu I之位置的基因，其 同時位於ΕΡ0之3’端附近以及LTP基因之5’端部分（ΤΡ0胺基 酸之位置為第176至3 5 3 )。另一方面，利用pGEM-EPO作為 模板以及引子EP1及EP2進行PCR，可只得到ΕΡ0基因。將以 此方法獲得之ΕΡ0及LTP基因分別用限制酶Stu I處理。然 後，連接ΕΡ0及LTP基因片段以及以該連接產物作為PCR步 驟之模板，利用引子EPl 1及L 2而得到具有約1 1 1 3 bp si ELTP基因片段。 EP11: TAAGCTTATGGGGOTGCACGAATGT (SEQ ID No, 10) L2: TGGATTCTTACCCTTCCTGAGACAGATTC (SEQ ID No. 11)
第10頁 92166.^32042 1237056 五、發明說明（6) 將此基因選殖至選殖載體pGEM-T，然後進行定序 (pGEM-ELTP，第 3a圖）。 再者，可藉由合成及自黏式（self-priming)PCR步驟 得到本發明中所使用之STP基因。合成之基因片段為下列 ES卜S2、S3以及上述L2。 ESI: AGCGCAGGCCTGCAGGACAGGGGACCCTCTTCTAA (SEQ ID No. 12) S2: GTGGGTGTAGGATGTGTTTAGAAGAGG(SEQIDNo, 13) S3: TACACCCACTCCCAGAATCTGTCTC (SEQ ID No, 14) 自4種基因片段中各取1/z l(50pmole//z 1)，並利用BM Co.之高準確度Taq系統進行PCR。 於1 %之瓊脂糖凝膠中確認約50bps (STP基因）之基因 片段。此基因可編碼出ΤΡ0之羧基端（第1圖）的1 7個胺基酸 (胺基酸位置為第3 3 7至3 5 3 )。 利用pGEM-EPO作為模板以及引子EP11及EP2進行PCR， 可只得到EP0基因。同時將此EP0基因及STP基因作為模板 且利用引子EP1 1及L2以BM Co.之高準確度Taq系統進行 PCR，而得到所欲之融合基因，ESTP基因，約6 3 0 bps。將 此基因選殖至pGEM-T (—種選殖載體），然後進行定序 (pGEM—ESTP，第 3b圖）。 (2 )建構表現載體 以Invitrogen Co.之pcDNA3.1載體作為表現載體。 以限制酶Hindi I I及BamHI處理該PCDNA3· 1載體，使其 變成線形載體，以及亦以相同之限制酶H i n d I I I及B a m Η I分
92166. 第11頁 1237056 五、發明說明（7) 別處理pGEM-ELTP及pGEM-ESTP。使用Qiagen萃取套組於瓊 脂糖凝膠中單離出經切割之pcDNA3. 1、ELTP以及ESTP基 因。接合各基因後，將接合產物引入E· col i NM 5 2 2中。 自菌落（於LB-氨比西林培養皿中培養經轉形之E. col i — 整夜後所形成之菌落）中單離出質體，並以限制酶H i nd I I I 及BamH I處理。然後以1 %之瓊脂糖凝膠電泳篩選出具有 ELTP或ESTP基因之菌落。分別將這些質體指定為 pcDNA-ELTP及 pcDNA-ESTP (第 3a及 3b圖）。 (3 ) C Η 0細胞之轉形作用及表現 於6 0 m in之細胞培養皿中培養C Η 0細胞（D G 4 4 )至細胞流 動率為4 0至80% (1至4x 10¾田胞/60m m之培養皿）。均勻混 合3// 1之超感染試劑（BM Co·)以及97 // 1之細胞培養基 (α -MEM培養基、無血清、無抗生素），加入pcdnA —ELTp ( = 〇· 1// g//z卜約2// g)以及具有dhfr基因之載體 pLTIidhfr26 (ATCC37295，0.2// g)。使該混合物於室溫下 反應5至1 0分鐘’然後加入如上述製備之細胞。一天後， 以含有G418之培養基（G418之含量為500// g/ml，無培養基 之a - Μ E Μ、1 0 % F B S )更新原有的培養基。7至1 〇天後以含 有5 0 0// g/ml G418之G418培養基更新原有的培養基，於培 養期間徹底地殺死不含G 4 1 8抗性基因以及反面對照組之細 胞。於G 4 1 8培養基上選擇出的細胞經充分地培養後，使用 3河（：〇.之£?0£118八套組確認所表現之£1^?蛋白質。將相 同之程序施用至ESTP表現載體，然後以相同方法確認所表 現之ESTP融合蛋白質。
第12頁 92]66蜈動_ l237〇56 . ---------------_______ 五、發明說明（8)
(4)純化經表現之eltP及ESTP 離 如下述，利用抗-EPO單株抗體（R&D Co.)製備用於單 及純化之親和性樹脂： 中&取0 · 3克經C N B r活化之Sepharose 4B，使其於ImM HC1 再~ >閑2 0分鐘’將該樹脂移到管柱中並以丨HC 1沖洗。 & ^ 4毫升之偶聯緩衝液（〇· 1M NaHC〇3、〇· 5M NaC卜pH • 3 )冲洗該樹脂，之後立刻與含於4毫升偶聯緩衝液内之 几EP〇單株抗體（5 0 0// g/瓶）混合，以及於室溫下攪拌該 试官使其反應2小時。以封阻緩衝液（〇 . 2M甘胺酸、pH 8 . 0 )更新偶聯緩衝液，於室溫下攪拌該試管使樹脂反應2小 時。然後，依序以6 · 5毫升之偶聯緩衝液、6 . 5毫升之乙酸 缓衝液（0· 1M乙酸、0· 5M NaC卜pH 4)以及6. 5毫升之偶聯 缓衝液沖洗樹脂。利用如此所得之樹脂製備管柱並且依下 述方法進行純化作用。 於無血清培養基中培養細胞一天，利用C e n t r i p r e p (Mi 1 1 ipore MWCO 1 0, 0 0 0 )將培養基濃縮5倍。將此濃縮液 載入預先以PBS平衡（流速為20毫升/小時）後再以PBS沖洗 之管柱。施加洗提緩衝液（0 · 1Μ甘胺酸、ρ η 2 · 8 )至管柱中 且立即以1Μ Tris滴定洗提液至ΡΗ7. 5。當由SDS-PAGE及銀 染色分析時（第4圖），其純度至少為97%。 (5 )由生物分析法測定活性 於生物分析試驗中係以苯肼、ELTP及ESTP處理老鼠的 脾臟細胞，E L T P及E S T P係經表現及適當的純化，並顯示出 其活性比EP0之活性高。此試驗證明了 ELTP及ESTP中經融
92166.ptd
1237056 五、發明說明（9) 合的魏基端不會抑制Epo本身之活性。 (6 )藥物動力學實驗 藥物動力學實驗係於老鼠中進行，以決定所製備之 ELTP及ESTP於體内是否確實具有經延長之半生期。對4隻 老鼠經由其靜脈以2 〇單位/老鼠之量投予候選物質。為了 辨識血液中候選物質隨時間消失之濃度，以每3 〇分鐘之間 隔自老昧v*抽取血液樣本以及使用Boehringer Mannheim C ο ·之E I A套組測量濃度。於老鼠之藥物動力學實驗中，該 候述物吳（ELTP及ESTP)顯示出比對照組ΕΡ0還要長之半生 期（第5圖）。 於下列實施例中將更詳細說明本發明。然而，應了解 下列實施例係欲用來說明本發明而不是以任何方法限制本 發明之範疇。 實施例1 :製備基因 分別使用50 pm〇ie之引子EP1及EP2 (與EPO cDNA之末 端互補，得自人類胎兒肝臟cDNA資料庫，Invi trogen Co·)進行習知之 PCR (Bioneer Co·之 PCR PreMix Kit)而 獲得EPO cDNA。依照相同方法，使用引子T1及T2 (與TPO cDNA之末端互補）獲得TPO cDNA。利用BM Co·之高準確度 Taq系統於條件為以52°C 40秒進行退火、72它 55秒以及 94°C 20秒下進行30次循環之PCR，而得EPO cDNA與TPO cDNA。分別將藉由PCR得到的EPO cDNA與TPO cDNA選殖至 pGEM-T (Promega Co·，為一種選殖之載體）中。亦即，自 1%瓊脂糖凝膠洗提PCR產物然後連接至pGEM-T，再將
92166.1032048 第14頁 1237056 五、發明說明αο) pGEM-T引入E_ col i NM5 2 2中。於LB-氨比西林培養皿（含 有X-gal/IPTG)中培養經轉形之ε· coli—整夜。自白色菌 落中純化出質體，並以限制酶Sac I及Sac I I處理，以分 別篩選出含有cDNAs之菌落。將此階段所得之載體指定為 pGEM-EPO及pGEM-TP0，然後進行定序，且將其作為後續程 序之模板。 本發明中所使用之TP0的LTP、CTP基因係利用BM Co. 之高準確度Taq系統及以pGEM-TPO為模板、ELI及T2為弓|子 (50 pmole)於條件為52。〇 40秒進行退火、72°C 45秒以及 9 4°C 2 0秒下進行30次循環之PCR而得。該引子ELI自限制 酶StuI之位置含有一基因，該基因係位於近epo之3,末端 之處’相對於3’末端，同時亦位於LTP基因之5,端部分。 於1 %之瓊脂糖凝膠中確認約534 bps之基因片段（LTP基 因）。此基因可編碼出Tp〇之羧基端（第1圖）的178個胺基酸 (胺基酸位置為第1 7 6至3 5 3 )。 另一方面，以pGEM-EPO為模板、EP1及EP2為引子，進 行PCR可只得到EP0基因。 將以此方法獲得之EP0及LTP基因分別用限制酶StuI處 理。然後’利用Qiagen萃取套組純化EP0及LTP基因之片段 以及以接合酶連接EP0及LTP基因之片段。以EP0及LTP基因 之連接產物作為模板、EP11及L2為引子（50 pmole)於條件 為5 5°C 4 0秒進行退火、7 2°c 6 0秒以及9 4°C 2 0秒下利用 B M C 〇 ·之咼準確度τ a q系統進行3 0次循環之P C R步驟，而得 到具有約1 1 1 3 bps所欲之ELTP融合基因片段。將此基因以
92166£§2047 第 15 頁 1237056 五、發明說明（Π) 如上述之相同方法選殖至選殖載體pGEM_t ,然後進行定 序（pGEM-ELTP，第 3a圖）。 藉由合成及自黏式（sel f-pr iming)pcR步驟得到STp基 因。合成之D N A片段為E S1、S 2、S 3以及L 2。自4種D N A片段 中各取1// 1 (50pmole/// 1 )，於條件為5yc 4〇秒進行退 火、72°C 4 0秒以及94t： 20秒下利用βΜ co·之高準確度 T a q系統進行1 5次循環之p C R。於1 %之瓊脂糖凝膠中確認約 5 0bps (STP基因）之基因片段。此基因可編碼出τρο之羧基 端（第1圖）的1 7個胺基酸（胺基酸位置為第3 3 7至3 5 3 )。 使用p G E Μ - E P 0作為模板、引子e P 1 1及E P 2以如上述之 相同方法進行PCR，可只得到ΕΡΟ基因。將此ΕΡΟ基因及STP 基因作為模板且利用引子Ε Ρ 1 1及L 2以BM Co.之高準確度 Taq系統於條件為58t： 40秒進行退火、72°C 50秒以及94 °C 2 0秒下進行總共為3 0次循環之PCR，而得到所欲之融合 基因，ESTP基因，約630 bps。將此基因選殖至選殖載體 pGEM-T (—種選殖載體），然後進行定序（PGEM-ESTP，第 3b圖）。
實施例2 :建構表現載體pcDNA-ELTP及pcDNA-ESTP 以Invi trogen Co.之pcDNA3. 1載體作為表現載體。分 別將ELTP及ESTP基因選殖至於末端含有Hindi I I及BamHI識 別位置之pGEM-T載體中。分別以限制酶Hindi I I及Bam HI處 理 pcDNA3.1、pGEM-ELTP及 pGEM-ESTP。利用 Qiagen萃取套 組於瓊脂糖凝膠中單離直線化的pcDNA3. 1、ELTP及ESTP基 因。使pcDNA3. 1與ELTP或ESTP接合後，將接合產物引入Ε·
92]66'ϋ&32048 第 ΐβ 頁 1237056 五、發明說明（12) col i NM 5 2 2中。自菌落（於LB -氨比西林培養孤中培養經轉 形之E· col i—整夜後所形成之菌落）中單離出質體，並以 限制酶H i nd I I I及Bam Η I處理。然後以1 %之瓊脂糖凝膠電泳 篩選出具有ELTP或ESTP基因之菌落。分別將這些質體指定 為 pcDNA-ELTP及 pcDNA —ESTP (第 3圖）。 實施例3 : CH0細胞之轉形作用及表現 於6〇111111之細胞培養皿中培養（：110細胞（〇644)至細胞流 動率為40至80%(1至4\10%田胞/6〇11]111之培養皿）。均勻混 合3" 1之超感染試劑（BM Co·)以及97 // 1之細胞培養基 (a -ME Μ培養基、無血清、無抗生素），加入質體 pcDNA-ELTP ( = 0.1// g/" 1，約 2" g)以及具有 dhfr基因之 載體 pLTRdhfr26 (ATCC3 72 9 5，〇· 2// g)。使該混合物於室 溫下反應5至1 0分鐘，然後加入如上述製備之細胞。一天 後，以含有G418之培養基（G418之含量為5 0 0// g/m卜無培 養基之α -MEM、10% FBS)更新原有的培養基。7至10天後 以含有500" g/ml G418之G418培養基更新原有的培養基， 於培養期間徹底地殺死不含G 41 8抗性基因以及反面對照組 之細胞。於G 4 1 8培養基上選擇出的細胞經充分地培養後， 使用6诞（：〇.之£?0£1^18八套組確認所表現之£1^?蛋白質。 將相同之程序施用至pcDNA-ESTP。 實施例4 ··純化經表現之融合蛋白質 如下述’利用抗-EP0單株抗體（R&D Systems Co.)製 備用於單離及純化之親和性樹脂： 取0· 3克經CNBr活化之Sepharose 4B，使其於ImM HC1
第17頁 92166β^2049 1237056 五、發明說明（13) -
中膨 >閏20分鐘’將該樹脂移到管柱中並以lmM 丨沖洗。 再以4毫升之偶聯緩衝液（0_1M NaHC03、〇·5Μ NaC1/pH 8. 3)沖洗該樹脂’之後立刻與含於試管中4毫升偶聯缓衝 液内之抗-ΕΡ0單株抗體（ 5 0 0# g/瓶）混合，以及於室溫下 授摔該試管使其反應2小時。以封阻緩衝液（〇 2M甘$ 酸、pH 8. 0)更新偶聯缓衝液，於室溫下攪拌該試管使樹 脂反應2小時。然後，依序以6. 5毫升之偶聯緩衝液、6. 5 ，升之乙酸緩衝液（〇· 1M乙酸、〇· 5M NaC卜pH 4)以及6., 毫升之偶聯缓衝液沖洗樹脂。利用如此所得之樹脂製備管 才主並且依下述方法進行純化作用。 於無血清培養基中培養細胞一天，利用centriprep (」^11丨？〇1^»^(：0 1 0,0 0 0 )將培養基濃縮至約5倍。將此濃 縮液載入預先以PBS平衡（流速為20毫升/小時）之管柱後， 再以P B S沖洗。施加洗提缓衝液（〇 · 1 μ甘胺酸、pjj 2 8)至 嘗才主中且立即以1 Μ T r i s滴定洗提液至p jj 7 · 5。第4圖顯示 、經純化的ELTP及ESTP之SDS-PAGE結果。當由SDS —PAGE及銀 染色分析時，其純度至少為9 7%。 實施例5 :由生物分析法測定活性 將劑置為60¾克/公斤之本讲注入老鼠體内，一天一 人共注射兩天。3天後’分離出變大的脾臟並以均質器研 磨而得脾臟細胞。將脾臟細胞稀釋成β χ丨〇 |田胞/毫升以 及將1 0 0// 1之細胞溶液引入9 6孔盤之各個孔井中。於各個 孔井中加入0至5 0 0mU/ml純正的ΕΡΟ以及丨〇〇11111/1111經表現之 融合蛋白質，然後將9 6孔盤置於C 0 2、3 7°C之培養器中停
1237056 五、發明說明（14) 留2 2小時。於各個孔井中加 (2Π" rw η 五认*门 入50" 1二甲基3[Η]-胸腺核苷 IZU/z Li/ml)’再於相同之拉益 徊了丨北山 ^ ^ ^ 口養杰中反應2小時，以及使各 個孔井中之反應溶液吸收至 ^ 以座w、士斗、名 王坡㈤濾紙（Nunc 1 - 7 3 1 6 4 )上。 生里風水〉月洗该滤紙3次，铁括 之访g ^人曰 然後以冷計數器測量各濾紙 之放射性含量。此試驗顯示屮 活报你“ ，出融合蛋白質（ELTP及ESTP)之 /古性與純正的EP〇之活性相去 融人石^ 田或同於純正的ΕΡ0，並證明了 一。至ΕΡ0的羧基端不會抑制Ep〇本身之活性。 貝施例6 :藥物動力學實驗 藥物動力學實驗係於老鼠中進行，以決定所製備之候 例，貝於體内是否確實具有經延長之半生期。將依照實施 、、之步驟純化出的融合蛋白質以2 〇單位/老鼠之量經由靜 萨左射投予至4隻老鼠體内。為了辨識血液中候選物質隨 :間消失之濃度，以每3 0分鐘之間隔自老鼠抽取血液樣本 t及使用Boehringer Mannheim Co.之El A套組測量濃度。 、、、°果顯示於第5圖。由第5圖可知，ELTP及ESTP分別顯示出 其半生期約為對照組ΕΡ0之半生期的3倍（epo之半生期為22 刀鐘’ ELTP之半生期為60分鐘以及ESTP之半生期為57分 鐘）。 [產業上應用性] 根據本發明之融合蛋白質，其於體内具有高度經延長 之EP〇半生期，這是因為存在於融合蛋白質中之胺基酸使 糖鏈增加且不會影響ΕΡ0固有的活性。再者，由於該融合 蛋白質（LTP或STP)係已存在於體内之胜肽，所以當此融合 蛋白質施用至人體時不會造成任何抗原力的問題。
92166伽翻li 第19頁 1237056 五、發明說明（15) 序列表 <110> Cheil Jedang Corporation <120> Fusion protein having the enhanced in vivo activity of erythropoietin
<130> PC01-0368-JIJD <160〉 14 <170> Kopatentln 1.71 <210> 1 <211> 178
<212> PRT <213> Homo sapiens <220>
<221> PEPTIDE <222> (1)·.(178) <223> Amino acids in the positions of 176 to 353 of human thrombopoiet in(TPO) <400> 1
Ala Pro Pro Thr Thr Ala Val Pro Ser Arg Thr Ser Leu Val Leu Thr 1 5 10 15
Leu Asn Glu Leu Pro Asn Arg Thr Ser Gly Leu Leu Glu Thr Asn Phe 20 25 30
Thr Ala Ser Ala Arg Thr Thr Gly Ser Gly Leu Leu Lys Trp Gin Gin 35 40 45
Gly Phe Arg Ala Lys lie Pro Gly Leu Leu Asn Gin Thr Ser Arg Ser 50 55 60
Leu Asp Gin lie Pro Gly Tyr Leu Asn Arg lie His Glu Leu Leu Asn 65 70 75 80
Gly Thr Arg Gly Leu Phe Pro Gly Pro Ser Arg Arg Thr Leu Gly Ala 第20頁 92166._3 謂 32、 1237056 五、發明說明（16) 85 90 95
Pro Asp lie Ser Ser Giy Thr Ser Asp Thr Gly Ser Leu Pro Pro Asn 100 105 110
Leu Gin Pro Gly Tyr Ser Pro Ser Pro Thr His Pro Pro Thr Gly Gin 115 120 125
Tyr Thr Leu Phe Pro Leu Pro Pro Thr Leu Pro Thr Pro Val Val Gin 130 135 140
Leu His Pro Leu Leu Pro Asp Pro Ser Ala Pro Thr Pro Thr Pro Thr 145 150 155 160
Ser Pro Leu Leu Asn Thr Ser Tyr Thr His Ser Gin Asn Leu Ser Gin 165 170 175
Glu Gly <210> 2 <211〉 17
<212> PRT <213> Homo sap i ens <220〉 <221〉 PEPTIDE <222> (1)..(17) <223〉 Amino acids in the positions of 337 to 353 of human thrompoietin(TPO) <400〉 2
Pro Leu Leu Asn Thr Ser Tyr Thr His Ser Gin Asn Leu Ser Gin Glu 1 5 10 15
Gly <210> 3 <211〉 370
Hll 第21頁 92166.PJ32Q53 1237056 五、發明說明（17)
<212> PRT <213> Artificial Sequence <220〉 <223> Fusion protein(ELTP) of erythropoietin(EPO) and carboxy terminal peptide(LTP) of human thrombopoietin <400> 3
Met Gly Val His Glu Cys Pro Ala Trp Leu Trp Leu Leu Leu Ser Leu 15 10 15
Leu Ser Leu Pro Leu Gly Leu Pro Val Leu Gly Ala Pro Pro Arg Leu 20 25 30 lie Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu Leu Glu Ala Lys Glu 35 40 45
Ala Glu Asn lie Thr Thr Gly Cys Ala Glu His Cys Ser Leu Asn Glu 50 55 60
Asn lie Thr Val Pro Asp Thr Lys Val Asn Phe Tyr Ala Trp Lys Arg 65 70 75 80
Met Glu Val Gly Gin Gin Ala Val Glu Val Trp Gin Gly Leu Ala Leu 85 90 95
Leu Ser Glu Ala Val Leu Arg Gly Gin Ala Leu Leu Val Asn Ser Ser 100 105 110
Gin Pro Trp Glu Pro Leu Gin Leu His Val Asp Lys Ala Val Ser Gly 115 120 125
Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu Gly Ala Gin Lys Glu 130 135 140
Ala lie Ser Pro Pro Asp Ala Ala Ser Ala Ala Pro Leu Arg Thr lie 145 150 155 160
Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val Tyr Ser Asn Phe Leu 165 170 175 mi 92166 ¢32054 第22頁 1237056 五、發明說明（18)
Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala Cys Arg Thr Gly Asp 180 185 190
Ala Pro Pro Thr Thr Ala Val Pro Ser Arg Thr Ser Leu Val Leu Thr 195 200 205
Leu Asn Glu Leu Pro Asn Arg Thr Ser Gly Leu Leu Glu Thr Asn Phe 210 215 220
Thr Ala Ser Ala Arg Thr Thr Gly Ser Gly Leu Leu Lys Trp Gin Gin 225 230 235 240
Gly Phe Arg Ala Lys lie Pro Gly Leu Leu Asn Gin Thr Ser Arg Ser 245 250 255
Leu Asp Gin lie Pro Gly Tyr Leu Asn Arg lie His Glu Leu Leu Asn 260 265 270
Gly Thr Arg Gly Leu Phe Pro Gly Pro Ser Arg Arg Thr Leu Gly Ala 275 280 285
Pro Asp lie Ser Ser Gly Thr Ser Asp Thr Gly Ser Leu Pro Pro Asn 290 295 300
Leu Gin Pro Gly Tyr Ser Pro Ser Pro Thr His Pro Pro Thr Gly Gin 305 310 315 320
Tyr Thr Leu Phe Pro Leu Pro Pro Thr Leu Pro Thr Pro Val Val Gin 325 330 335
Leu His Pro Leu Leu Pro Asp Pro Ser Ala Pro Thr Pro Thr Pro Thr 340 345 350
Ser Pro Leu Leu Asn Thr Ser Tyr Thr His Ser Gin Asn Leu Ser Gin 355 360 365
Glu Gly 370 <210〉 4 <211〉 209
<212〉 PRT
IH1I 第23頁 92166.P032O55 Ϊ237056 五、發明說明（19) <2l3> Artificial Sequence <220 <223> Fusion protein(ESTP) of erythropoietin(EPO) and carboxy terminal peptide(STP) of human thrombopoietin <400> 4
Met Gly Val His Glu Cys Pro Ala Trp Leu Trp Leu Leu Leu Ser Leu 15 10 15
Leu Ser Leu Pro Leu Gly Leu Pro Val Leu Gly Ala Pro Pro Arg Leu 20 25 30 lie Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu Leu Glu Ala Lys Glu 35 40 45
Ala Glu Asn lie Thr Thr Gly Cys Ala Glu His Cys Ser Leu Asn Glu 50 55 60
Asn lie Thr Val Pro Asp Thr Lys Val Asn Phe Tyr Ala Trp Lys Arg 65 70 75 80
Met Glu Val Gly Gin Gin Ala Val Glu Val Trp Gin Gly Leu Ala Leu 85 90 95
Leu Ser Glu Ala Val Leu Arg Gly Gin Ala Leu Leu Val Asn Ser Ser 100 105 110
Gin Pro Trp Glu Pro Leu Gin Leu His Val Asp Lys Ala Val Ser Gly 115 120 125
Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu Gly Ala Gin Lys Glu 130 135 140
Ala lie Ser Pro Pro Asp Ala Ala Ser Ala Ala Pro Leu Arg Thr lie 145 150 155 160
Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val Tyr Ser Asn Phe Leu 165 170 175
Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala Cys Arg Thr Gly Asp 180 185 190 11Ι·Ι 第24頁 92166.pt032〇56 1237056 五、發明說明 (20) Pro Leu Leu Asn Thr Ser Tyr Thr His Ser Gin Asn Leu Ser Gin Glu 195 200 205 Gly <210〉 5 <211〉 29 <212> DNA <2X3> Artificial Sequence <220> <223〉 Primer EP1 having the nucleotide sequence complementary to the terminal sequence of ΕΡ0 cDNA <400> 5 atgggggcac gaatgtcctg cctggctgg 29 <210> 6 <211〉 20 <212> DNA <213> Artificial Sequence <220〉 <223〉 Primer EP2 having the nucleotide sequence complementary to the terminal sequence of ΕΡ0 cDNA <400〉 6 gtcccctgtc ctgcaggcct 20 <210> 7 <211〉 24 <212> DNA < 2 1 3 > Artific i a 1 Sequence 1111111 第25頁 92166t?32057 1237056 五、發明說明（21) < 2 2 0 > <223〉 Primer T1 having the nucleotide sequence complementary to the terminal sequence of IPO cDNA <400> 7 atggatctga ctgaattgct cctc 24 <210 8 <211〉 27 <212> DNA <213〉 Artificial Sequence <220> <223> Primer T2 having the nucleotide sequence complementary to the terminal sequence of TPO cDNA <400> 8 ttacccttcc tgagacagat tctggga 27 <210〉 9 <211> 43 <212〉 DNA <213> Artificial Sequence <220> <223〉 Primer ELI for PCR to obtain the desired gene LT? <400〉 9 gaggcctgca ggacagggga cgccccaccc accacagctg tcc 43 <210〉 10 <211〉 25 Hill 92166.pg32〇58 第26頁 1237056 五、發明說明（22) <211> DNA <213> Artificial Sequence <220> <223〉 Primer EP11 for PCR to obtain the desired fusion gene ΕΙΊΡ and ESTP <400〉 10 taagcttatg ggggtgcacg aatgt 25 <210〉 11 <211〉 29 <212〉 DNA 、 <213〉 Artificial Sequence <220〉 <223> Primer L2 for PCR to obtain the desired fusion gene ELIP and ESTP <400〉 11 tggattctta cccttcctga gacagattc 29 <210〉 12 <211> 35 <212> DNA <213〉 Artificial Sequence <220 <223> Primer ESI for PCR shuffling to obtain the desired gene STP <400> 12 aggggaggcc tgcaggacag gggaccctct tctaa 35 11111 92166.iD32〇59 第 27 頁 1237056 五、發明說明（23)<210> 13 <211〉 27 <212〉 DNA <213> Artificial Sequence <220〉 <223〉 Primer S2 for PCR shuffling to obtain the desired gene SIP <400> 13 gtgggigtag gatgtgttta gaagagg 27 0>1>2>3> 1X IX IX IX <2<2<2<2 14 25 DNA Artificial Sequence
STP <220> <223〉 Primer S3 for PCR shuffling to obtain the desired gene <400> 14 tacacccact cccagaatct gtctc 25
第28頁 9memz§m 1237056 圖式簡單說明 [圖示之簡單說明] 第la及lb圖顯示ΤΡ0之羧基端胜肽（LTP，STP)的核苷 酸及胺基酸序列； 第2aa及2ab圖顯示ELTP之核苷酸及胺基酸序列，ELTP 係ΕΡ0與胜肽LTP之融合蛋白質，以及第2b圖顯示ESTP之核 苷酸及胺基酸序列，ESTP係ΕΡ0與胜肽STP之融合蛋白質； 第3a及3b圖係以流程圖顯示製備該表現載體 (pcDNA-ELTP及 pcDNA-ESTP)之程序； 第4圖係經表現之ELTP及ESTP之電泳圖；以及 第5圖係顯示ΕΡ0，ELTP及ESTP之藥物動力學結果之圖 表0
92166.ρ^32061 第 29 頁
Claims (1)
- 修正 案號 91116686 六、申請專利範圍 1. 一種融合蛋白質，係由序列編號第3號（SEQ ID No. 3) 之胺基酸序列所組成，其中由序列編號第1號（SEQ I D No. 1 )之胺基酸序列所組成之血小板生成素（TP0)之羧 基端胜肽（CTP)係融合於人類促紅素（ΕΡ0)之羧基端。 2. —種融合蛋白質，係由序列編號第4號（SEQ ID No. 4) 之胺基酸序列所組成，其中由序列編號第2號（SEQ I D Ν ο. 2 )之胺基酸序列所組成之血小板生成素（T P 0 )之魏 基端胜肽（CTP)係融合於人類促紅素（EP0)之羧基端。 3. —種可編碼出如申請專利範圍第1或2項之融合蛋白質 之核苷酸。 4. 一種製備於體内具有經加強之人類EP0活性之融合蛋白 質之方法，包括培養以重組載體轉形之宿主細胞株， 該重組載體含有如申請專利範圍第3項之核苷酸。第30頁 92166修正版.ptc
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| KR10-2001-0074975A KR100467750B1 (ko) | 2001-11-29 | 2001-11-29 | 생체내 에리스로포이에틴 활성이 증진된 융합단백질 |
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| TWI507415B (zh) * | 2012-12-18 | 2015-11-11 | Ubi Pharma Inc | 重組蛋白 |
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| US7767643B2 (en) | 2000-12-29 | 2010-08-03 | The Kenneth S. Warren Institute, Inc. | Protection, restoration, and enhancement of erythropoietin-responsive cells, tissues and organs |
| EP1670492A4 (en) * | 2003-09-29 | 2009-07-08 | Warren Pharmaceuticals Inc | FABRIC PROTECTION CYTOKINES FOR THE TREATMENT AND PREVENTION OF SEPTICEMIA AND THE FORMATION OF ADHESIONS |
| US7625564B2 (en) | 2006-01-27 | 2009-12-01 | Novagen Holding Corporation | Recombinant human EPO-Fc fusion proteins with prolonged half-life and enhanced erythropoietic activity in vivo |
| US20150038413A1 (en) | 2006-02-03 | 2015-02-05 | Opko Biologics Ltd. | Long-acting polypeptides and methods of producing and administering same |
| WO2007092252A2 (en) * | 2006-02-03 | 2007-08-16 | Modigene Inc | Long-acting polypeptides and methods of producing same |
| US20140113860A1 (en) | 2006-02-03 | 2014-04-24 | Prolor Biotech Ltd. | Long-acting polypeptides and methods of producing and administering same |
| US9249407B2 (en) | 2006-02-03 | 2016-02-02 | Opko Biologics Ltd. | Long-acting coagulation factors and methods of producing same |
| US8048849B2 (en) | 2006-02-03 | 2011-11-01 | Modigene, Inc. | Long-acting polypeptides and methods of producing same |
| US8946155B2 (en) | 2006-02-03 | 2015-02-03 | Opko Biologics Ltd. | Long-acting polypeptides and methods of producing and administering same |
| US10221228B2 (en) | 2006-02-03 | 2019-03-05 | Opko Biologics Ltd. | Long-acting polypeptides and methods of producing and administering same |
| US10351615B2 (en) | 2006-02-03 | 2019-07-16 | Opko Biologics Ltd. | Methods of treatment with long-acting growth hormone |
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| US20150158926A1 (en) | 2013-10-21 | 2015-06-11 | Opko Biologics, Ltd. | Long-acting polypeptides and methods of producing and administering same |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| TWI507415B (zh) * | 2012-12-18 | 2015-11-11 | Ubi Pharma Inc | 重組蛋白 |
| US10428129B2 (en) | 2012-12-18 | 2019-10-01 | Ubi Pharma Inc. | Recombinant protein |
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