TW201326198A - 纖維母細胞生長因子２１變體 - Google Patents
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Abstract
本發明係關於人類纖維母細胞生長因子21(FGF21)之藥理學上有效及/或穩定之變體、包含FGF21變體之醫藥組合物及使用該等變體治療II型糖尿病、肥胖症、異常血脂症或代謝症候群或其任一組合之方法。
Description
本發明係關於人類纖維母細胞生長因子21(FGF21)之變體、包含FGF21變體之醫藥組合物及治療II型糖尿病、肥胖症、異常血脂症或代謝症候群或其任何組合之方法。
FGF21係扮演葡萄糖及脂質體內平衡之重要代謝調節劑功能的激素。FGF21藉由上調GLUT1表現而促進脂肪細胞中之葡萄糖吸收，其機制與胰島素之機制不同。在患糖尿病之齧齒動物及猴中，人類FGF21會降低空腹時血清中的葡萄糖濃度，且會降低空腹時血清中三酸甘油酯、胰島素及升糖素之濃度。另外，在飲食誘導型肥胖症之齧齒動物模型中，投與FGF21會以劑量依賴性方式達到累積性體重減輕。因此，FGF21具有治療糖尿病、肥胖症、異常血脂症及代謝症候群之潛在用途。
已在WO 2010/065439、WO 2006/028595及WO 2005/061712中闡述人類FGF21之變體。
與人類野生型FGF21及已知之人類FGF21變體有關之問題係該等分子之藥理效能及/或醫藥穩定性較低。因此，業內仍需要有效及/或穩定之替代性FGF21變體。
本發明提供人類FGF21之替代性變體，其具有優於人類野生型FGF21及業內所揭示之已知之人類FGF21變體之優點。該等優點包括改良之藥理學效能及/或改良之醫藥穩
定性。本發明之某些FGF21變體具有一或多種可用於有效製造及/或調配作為治療蛋白質之有利生理化學特性，包括降低之活體內蛋白水解降解、降低之對氧化之敏感性、降低之高濃度下聚集之傾向、降低之哺乳動物細胞系統中產生期間翻譯後修飾之程度、增加之與某些防腐劑之相容性及/或改良之化學穩定性。另外，本發明之FGF21變體潛在地可用於治療II型糖尿病、肥胖症、異常血脂症或代謝症候群或其任一組合。
本發明提供人類纖維母細胞生長因子21(FGF21)之變體，其中胺基酸序列為HPIPDSSPLLQFGGQVRQRYLYTDDAQQTECHLEIREDGTVGCAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFREX1LLEDGYNVYQSEAHGLX2LHLPGDKSPHRKPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDP X3 X4LV X5PSQLLSPSFLG(SEQ ID NO：13)其中X1為L或D，X2為P或W，X3為L或Y，X4為S或R，且X5為G或E。
本發明提供人類纖維母細胞生長因子21(FGF21)之變體，其中胺基酸序列為HPIPDSSPLLQFGGQVRQRYLYTDDAQQTECHLEIREDGTVGCAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFREDLLEDGYNVYQSEAHGLPLHLPGDKSPHRKPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLRLVEPSQLLSPSFLG(SEQ ID NO：1)。
本發明亦提供包含如本文所述之本發明之人類FGF21變體及至少一種醫藥上可接受之載劑、稀釋劑或賦形劑以及視情況其他治療成份之醫藥組合物。
本發明亦提供治療患者之II型糖尿病、肥胖症、異常血脂症或代謝症候群或其任一組合之方法，其包含向患者投與如本文所述之本發明之人類FGF21變體。
本發明亦提供治療患者之II型糖尿病、肥胖症、異常血脂症或代謝症候群或其任一組合之方法，其包含向患者投與如本文所述之本發明之醫藥組合物。
另外，本發明提供如本文所述之本發明之人類FGF21變體用於療法中。較佳地，本發明提供如本文所述之本發明之人類FGF21變體用於治療II型糖尿病、肥胖症、異常血脂症或代謝症候群或其任一組合。
另外，本發明提供如本文所述之本發明之人類FGF21變體之用途，該變體係用於製造用以治療II型糖尿病、肥胖症、異常血脂症或代謝症候群或其任一組合之藥劑。
全長人類野生型FGF21為208個胺基酸之多肽，其含有27個胺基酸之信號肽。成熟人類野生型FGF21包含不含27個胺基酸之信號肽之全長多肽，從而產生181個胺基酸之多肽(SEQ ID NO：2)。本發明FGF21變體之胺基酸位置之改變係根據成熟人類野生型FGF21之多肽(SEQ ID NO：2)中之胺基酸位置來確定。因此，本文闡述為「A31C」之取代係指在成熟人類野生型FGF21蛋白質之31位處用胺基酸
Cys取代野生型胺基酸Ala。
重要的是，應注意，特定變體中一個胺基酸殘基之取代可影響整個變體之特性，且整體效應可對藥理學效能及/或醫藥穩定性有益或有害。例如，一個胺基酸取代(P115W)增加FGF21變體之效能，然而據信，P115W亦促成造成聚集之自相互作用(參見實例5)。因此，該整體效應對變體有害，且因此，本發明之較佳之FGF21變體不包括取代P115W。
本發明之某些人類FGF21變體係有效之生物活性蛋白質，如實例2及3中之SEQ ID NO：1所證明。本發明之較佳之FGF21變體含有不僅共同改良藥理學效能且亦與其他胺基酸改變相容之胺基酸取代，該等其他胺基酸改變又提供改良之生理化學性質及增加之活體內穩定性。本發明之較佳之FGF21變體中改良效能之胺基酸取代之群包括D127K、S167R、G174L、R175L及A180L(參見實例2及3)。
人類野生型FGF21之濃縮蛋白質溶液於醫藥防腐劑(例如間-甲苯酚)之暴露增加蛋白質形成聚集體之傾向。藉由引入額外二硫鍵而得之結構穩定性改良人類野生型FGF21之防腐劑相容性以及熱穩定性。本發明之FGF21變體納入顯著改良熱穩定性及防腐劑相容性而不減損生物活性之胺基酸取代A31C及G43C。先前已闡述亦包括A31C/G43C取代之高效能FGF21變體。雖然彼等所報導變體顯示相對於野生型FGF21顯著改良之防腐劑相容性，但其仍傾向於聚集。已知聚集會增加免疫原性之風險，藉此降低該等變體
作為治療蛋白質之可接受性。
為最小化此有害的聚集，本發明之較佳變體包括胺基酸取代L98D，該胺基酸取代L98D使得於高濃度下形成顯著較少之高分子量聚集體(參見實例5)。有利地，胺基酸取代L98D並不降低變體之效能。
用於製造本發明FGF21變體之較佳商業表現系統係哺乳動物CHO-K1細胞系。然而，哺乳動物細胞系CHO-K1及HEK293可藉由179位處酪胺酸側鏈之硫酸化而致使成熟人類野生型FGF21進行翻譯後修飾。179位及180位(若存在)處酪胺酸殘基之硫酸化降低效能，且係不合意之產物異質性之來源。因此，當自CHO-K1或HEK293細胞系表現在179位及/或180位處具有Tyr之FGF21蛋白質時，一定比例之經表現蛋白質可在179位處經硫酸化，其他蛋白質可在180位處經硫酸化，而其他蛋白質可在兩個位置處經硫酸化，且一些蛋白質在兩個位置處均未經硫酸化。此產生具有降低之效能之異質且不可預測之蛋白質群體。
本發明之FGF21變體藉由在變體中納入胺基酸取代Y179F而解決此有害硫酸化。Y179F消除了由在CHO-K1及HEK293細胞中產生而造成之硫酸化(參見實例4)。另外，胺基酸取代Y179F與本發明其他有利之胺基酸取代相容，且經測定為關於效能之中性改變。
人類野生型FGF21在活體內易受蛋白水解降解之影響。在靜脈內或皮下注射之後自小鼠或食蟹猴之血清回收之主要蛋白水解片段係於171位處終止之片段。先前，橫跨殘
基1至171之FGF21片段之效能已在活體外效能分析中經測定為約1/100。消除此蛋白水解解離位點藉由增加於活性藥物之暴露而改良藥物功效。已顯示胺基酸取代G170E顯著減慢在小鼠中之解離(未顯示數據)，且在食蟹猴中於24小時之後實際上消除了171位處之蛋白水解(參見實例6)。G170E取代並不影響效能且與期望之生理化學穩定性特徵相容。因此，將胺基酸取代G170E納入本發明之較佳之FGF21變體中。
人類野生型FGF21易受CHO-K1製造中產生之羧肽酶之影響，且胺基酸取代S181G減慢此處理，藉此降低所表現蛋白質之長度之異質性(即，由細胞系表現之成熟蛋白質中胺基酸殘基之數目之異質性)。儘管胺基酸取代S181G並不消除哺乳動物細胞表現中之C-末端蛋白水解，但在本發明FGF21變體中發現之其他期望胺基酸取代之背景下，其相當有效地減慢蛋白水解同時維持期望效能。鑒於此有利特性，將胺基酸取代S181G納入本發明FGF21變體中。
本發明亦涵蓋編碼上述變體之多核苷酸，該等多核苷酸可呈RNA形式或呈DNA形式，該DNA包括cDNA及合成DNA。DNA可為雙鏈或單鏈形式。編碼本發明變體之編碼序列可因遺傳密碼之冗餘度或簡併度而變化。
編碼本發明變體之多核苷酸可包括以下：僅變體之編碼序列；變體之編碼序列及其他編碼序列，例如功能多肽或前導序列或分泌序列或前蛋白序列；變體之編碼序列及非編碼序列，例如內含子或變體之編碼序列之非編碼序列5'
及/或3'。因此，術語「編碼變體之多核苷酸」涵蓋可不僅包括變體之編碼序列且亦包括包含其他編碼及/或非編碼序列之多核苷酸之多核苷酸。
本發明之多核苷酸將在將序列可操作地連接至表現控制序列之後在宿主細胞中表現。表現載體通常可作為游離基因體或作為宿主染色體DNA之組成部分而在宿主有機體中複製。通常，表現載體會含有選擇標記(例如四環素(tetracycline)、新黴素(neomycin)及二氫葉酸還原酶)，以使得可選擇彼等經期望DNA序列轉化之細胞。
本發明之FGF21變體可易於在以下細胞中產生：哺乳動物細胞，例如CHO、NS0、HEK293或COS細胞；細菌細胞，例如大腸桿菌(E.coli)、枯草桿菌(Bacillus subtilis)或螢光假單胞菌(Pseudomonas fluorescence)；或真菌或酵母細胞。使用業內眾所周知之技術來培養宿主細胞。較佳之哺乳動物宿主細胞係含有麩醯胺酸合成酶(GS)表現系統之CHOK1SV細胞系(參見US 5,122,464)。
可藉由眾所周知之方法將含有目標多核苷酸序列(例如FGF21之變體及表現控制序列)之載體轉移至宿主細胞中，該等方法端視細胞宿主之類型而變化。例如，氯化鈣轉染通常用於原核細胞，而磷酸鈣處理或電穿孔可用於其他細胞宿主。
可採用各種蛋白質純化方法，且該等方法已為業內所知，且闡述於(例如)Deutscher,Methods in Enzymology 182：83-89(1990)及Scopes，Protein Purification：Principles and Practice，第3版，Springer，NY(1994)中。
可藉由業內已知任一可達成通常意欲治療II型糖尿病、肥胖症、異常血脂症或代謝症候群或其任一組合之目的的方式來投與本發明FGF21變體之醫藥組合物。較佳之投與途徑為非經腸的。投與劑量應取決於接受者之年齡、健康及體重；併行治療(若有)之種類；治療頻率及期望效應之特性。典型劑量水平可使用標準臨床技術來最佳化，且將取決於投與模式及患者之病況，且可由業內一般技術人員來確定。
根據已知方法調配本發明之FGF21變體來製備醫藥上可用之組合物。令人滿意的調配物係可用適當稀釋劑進行復水之穩定性凍乾產物或含有可選擇之醫藥上可接受之載劑、防腐劑、賦形劑或穩定劑之高純度水溶液[Remington，The Science and Practice of Pharmacy，第19版，Gennaro編輯，Mack Publishing公司，Easton，PA 1995]。
本發明FGF21變體可與醫藥上可接受之緩衝液合併，並調節pH以提供可接受之穩定性及可接受之pH以供投與。另外，本發明FGF21變體可置於容器(例如小瓶、藥盒、筆式遞送裝置(pen delivery device)、注射器、靜脈內投與管或靜脈內投與袋)中，其中容器為單位劑量容器。
術語「異常血脂症」意指脂蛋白代謝之病症，包括脂蛋白產生過剩或不足。異常血脂症可表現為血液中總膽固醇、低密度脂蛋白(LDL)膽固醇及三酸甘油酯濃度之升高及/或高密度脂蛋白(HDL)膽固醇濃度之降低。
術語「代謝症候群」係藉由身體中之一群代謝風險因子來描述。其包括：腹部脂肪-在大多數人中，40英吋腰或更大；空腹後高血糖-至少110毫克/分升(mg/dl)；血流中高三酸甘油酯-至少150 mg/dL；低HDL-低於40 mg/dl；及/或130/85或更高之血壓。
術語「肥胖症」定義為存在過量之與瘦體重成比例之皮下脂肪之病況(Stedman's Medical Dictionary，第28版，2006，Lippincott Williams & Wilkins)。
「患者」為哺乳動物，較佳人類。
術語「治療(treating)」(或「treat」或「treatment」)意指減慢、降低或逆轉症狀、病症、病況或疾病之進展或嚴重程度。
術語「治療有效量」係指在單一或多重劑量投與患者後提供期望治療之本發明FGF21變體之量或劑量。
術語「II型糖尿病」之特徵在於儘管可利用胰島素但葡萄糖產生過量，且循環葡萄糖含量由於不充分之葡萄糖清除而依然過高。
可基本上如下所述來實施以下實例。
使用中國倉鼠卵巢(CHOK1SV)細胞在哺乳動物細胞表現系統中產生本發明之FGF21變體。將編碼FGF21變體之基因亞選殖至含麩醯胺酸合成酶(GS)之表現質粒骨架(基於pEE12.4之質粒)中。將編碼FGF21變體之cDNA序列與較佳
之信號肽序列之編碼序列框架內融合以增加期望產物至組織培養基中之分泌。較佳之信號肽序列為如胺基酸序列SEQ ID NO：3、SEQ ID NO：4、SEQ ID NO：5或SEQ ID NO：6中所展示之多肽。
藉由病毒巨細胞病毒(CMV)啟動子來驅動表現。使用電穿孔及適量之重組表現質粒來穩定地轉染CHOK1SV細胞，且將經轉染細胞以充足細胞密度維持於懸浮培養物中。藉由在含甲硫胺酸磺醯亞胺(MSX)之無血清培養基中生長並在35℃至37℃及5%至7% CO2下培育進行經轉染細胞之選擇。
藉由使用流式細胞儀來生成以選殖方式獲得之細胞系。FGF21變體在哺乳動物細胞中之表現通常產生天然N末端序列HPIP，即在N末端處無甲硫胺酸殘基，例如由SEQ ID NO：1之胺基酸序列所展示之FGF21變體。
藉由以下方法來純化自CHO細胞分泌至培養基中之FGF21變體：將澄清的細胞培養基加熱至50℃至60℃，並保持多達兩個小時，冷卻，用清潔劑(Triton X-100)處理以達成病毒滅活，並施加至Capto MMC(GE Healthcare)混合模式層析管柱。使用pH 8緩衝液自管柱溶析FGF21變體，且用50 mM檸檬酸、150 mM NaCl溶液將隨後之產物池調節至3.2至3.5之pH範圍，並保持一小時以達成病毒滅活。藉由添加Tris緩衝液將溶液調節至介於pH 6.7至7.3之間，並使用苯基瓊脂糖高性能樹脂(GE Healthcare)藉由疏水交換層析來進一步純化FGF21變體。用遞減梯度之pH 7下之
硫酸鈉溶析疏水相互作用管柱。將經HIC純化之FGF21變體之緩衝液更換為含NaCl之pH 8下之Tris緩衝液，並藉由Source 30Q樹脂(GE Healthcare)上之陰離子交換層析進一步加以純化。用遞增濃度之pH 8下之氯化鈉溶析陰離子交換管柱。使經純化FGF21變體通過Planova 20N(Asahi Kasei Medical)病毒滯留過濾器，隨後使用再生纖維素膜(Millipore)上之切向流超濾而濃縮/透析至10 mM檸檬酸鹽、150 mM NaCl pH 7中。
藉由CMV驅動之含有野生型小鼠βKlotho之編碼序列及殺稻瘟菌素(blasticidin)抗性標記之哺乳動物表現載體之逆轉錄病毒轉導自3T3-L1纖維母細胞生成3T3-L1-βKlotho纖維母細胞。在15 μM殺稻瘟菌素之存在下生長14天之後選擇抗殺稻瘟菌素細胞，並利用抗βKlotho抗體藉由免疫墨點來驗證βKlotho蛋白質之表現。將3T3-L1-βKlotho纖維母細胞維持於含10%牛血清及15 μM殺稻瘟菌素之達爾伯克改良伊格爾培養基(Dulbecco's Modified Eagle Medium，DMEM)中直至平鋪以供實驗使用為止。
對於葡萄糖吸收而言，以20,000個細胞/孔將3T3-L1-βKlotho纖維母細胞平鋪於96孔板中，並在含10%牛血清之DMEM中培育48小時。將細胞在有或沒有目標FGF21變體之情況下在含0.1%牛血清白蛋白(BSA)之DMEM中培育3小時，隨後在有或沒有目標FGF21變體之情況下在含有100
μM 2-去氧-D-(14C)葡萄糖之克-林二氏磷酸鹽(Krebs-Ringer phosphate，KRP)緩衝液(15 mM Hepes,pH 7.4,118 mM NaCl,4.8 mM KCl,1.2 mM MgSO4,1.3 mM CaCl2,1.2 mM KH2PO4,0.1% BSA)中培育1小時。藉由在含1 mM 2-去氧-D-(14C)葡萄糖之克-林二氏碳酸氫鹽/Hepes(KRBH)緩衝液中培育所選孔來測定非特異性結合。藉由向細胞中添加20 μM細胞遲緩素B來終止反應，且使用液體閃爍計數器來量測葡萄糖吸收。
在3T3-L1-βKlotho纖維母細胞葡萄糖吸收分析中SEQ ID NO：1之FGF21變體之活體外效能為0.026 nM。當與已知之SEQ ID NO：11之FGF21變體(揭示於WO 2006/028595中)相比時，SEQ ID NO：1之FGF21變體為有效之FGF21變體。在3T3-L1-βKlotho纖維母細胞葡萄糖吸收分析中SEQ ID NO：11之FGF21變體之活體外效能為0.49 nM。
293-βKlotho-SRE luc報導細胞之構築：在達爾伯克改良伊格爾培養基之含10%胎牛血清(FBS)之生長培養基中在37℃、5% CO2下培養HEK-293(人類胚胎腎細胞)。用含有CMV啟動子驅動之人類βKlotho表現盒之質粒及含有血清響應元件(SRE)驅動之螢光素酶表現盒之質粒共轉染細胞。βKlotho表現質粒亦含有SV40啟動子驅動之新黴素磷酸轉移酶表現盒以賦予對胺基糖苷類抗生素G418之抗性。用600 μg/mL之G418選擇經轉染HEK-293
細胞，以選出經轉染質粒已經整合至基因組中之細胞。藉由稀釋來選殖所選細胞，並在添加FGF21後24小時針對螢光素酶產物之增加加以測試。將展示螢光素酶之最大之FGF21依賴性增加之純系選為用於量測相對FGF21變體活性之細胞系。
293-βKlotho-SRE luc FGF21活性分析：沖洗293-βKlotho-SRE luc細胞並將其置於CD 293懸浮培養基(Invitrogen)中。細胞在懸浮物中在37℃、6% CO2、125 rpm下過夜生長。將細胞計數，藉由離心沈澱，並將其再懸浮於含0.1% BSA之CD 293培養基中。將細胞以25,000個細胞/孔置於白色96孔板中。對於每一FGF21變體，在CD 293/0.1% BSA中製備4倍連續稀釋液，從而生成8份稀釋液，其中終濃度為100 nM至0.006 nM。將稀釋液以一式三份添加至細胞中，並在37℃、5% CO2下培育16-20小時。藉由添加等體積之OneGloTM螢光素酶受質(Promega)並量測相對螢光來測定螢光素酶含量。使用四參數邏輯斯諦模型(XL擬合第5.1版)分析數據以擬合曲線並確定EC50。
在人類293細胞-βKlotho-SRE luc分析中SEQ ID NO：1之FGF21變體之活體外效能為0.25 nM。當與已知之SEQ ID NO：11之FGF21變體(揭示於WO 2006/028595中)相比時，SEQ ID NO：1之FGF21變體為有效之FGF21變體。在人類293細胞-βKlotho-SRE luc分析中SEQ ID NO：11之FGF21變體之活體外效能為22.39 nM。
在CHOK1SV細胞中之哺乳動物蛋白質表現期間人類野生型FGF21在179位處易受酪胺酸硫酸化之影響(未顯示數據)。此硫酸化導致產物異質性，意味著可產生不同形式之蛋白質(即，經及未經硫酸化)。產物同質性係生物醫藥產物之期望屬性。在治療蛋白質之產生期間發生之翻譯後修飾係不合意的，此乃因該等修飾可致使活性或其他生物醫藥性質不同。
為評價FGF21變體是否經硫酸化，將1 μL等份試樣與99 μL 0.1%三氟乙酸(TFA)混合。藉由液相層析-質譜法(LC-MS)使用以下條件來分析試樣：流動相A為0.1% TFA/10%乙腈，流動相B為存於乙腈中之0.1% TFA，管柱為C8管柱，3.5 μm 2.1×150 mm，具有2.1×12.5 mm C8保護管柱，注入體積為12-20 μL，此取決於試樣濃度以便注入大約1 μg之蛋白質。
將Waters Micromass LCT PremierTM質譜儀設置為如下：質量範圍介於400 amu至1990 amu之間，極性ES+，毛細管2000，試樣錐電壓為40 V，孔1電壓為30 V，源溫度為105℃，錐氣流為50 L/小時，去溶劑化溫度為300℃，且去
溶劑化氣流為600 L/小時。
自表2可看出，預期質量(未經硫酸化)與SEQ ID NO：1之FGF21變體之所觀察質量大致相同，此表明未在SEQ ID NO：1之FGF21變體中檢測到硫酸化。因此，胺基酸取代Y179F防止在SEQ ID NO：1之FGF21變體中之179位處發生硫酸化。此結果提供更同質之產物，從而使其更可接受作為治療蛋白質產物。
為量測蛋白質自締合及聚集之量，使用分析型尺寸排除層析(SEC)方法來量測高分子量聚集體之百分比(HMW%)。藉由SEC來表徵蛋白質之初始儲備溶液以測定HMW之起始含量(表3)。
每一蛋白質之試樣係藉由在4℃下以2 mg/mL之濃度過夜
透析(使用分子量截止值為10,000道爾頓(dalton)之透析盒)至試樣緩衝液(闡述於表4-6中)中來製備。在透析之後，無菌過濾(0.22 μm膜)試樣，並藉由280 nm下之吸光度加以量化。接下來，使用10,000 MW截止離心過濾器在4℃下以3000 rpm將試樣濃縮至60 mg/mL之目標濃度。在濃縮試樣之後，藉由280 nm下之吸光度來量化蛋白質之濃度，並使用SEC分析來測定HMW%。
SEC方法利用尺寸為30 cm×0.78 cm之TosoHaas型TSK-GEL® G2000SWXL管柱。流動相為0.1 M磷酸鈉，pH 7.4，流速為0.5 mL/分鐘。將低濃度試樣作為10 μL注入劑施加，並在214 nm之吸收波長下監測，而將濃縮試樣作為1 μL注入劑施加，並在280 nm下監測。
表4報告SEQ ID NO：8及SEQ ID NO：9之FGF21變體之濃縮溶液中之蛋白質濃度及HMW%。保持單體形式(非聚集形式)之每一變體之百分比未列於表中，但等於100%減去所報告之HMW%。SEQ ID NO：8之FGF21變體與SEQ ID NO：9之FGF21變體僅在胺基酸115位處不同，其中SEQ ID NO：8之FGF21變體在115處含效能增強殘基色胺酸(P115W)，且SEQ ID NO：9之FGF21變體含野生型殘基脯胺酸(P115P)。在各種調配物緩衝液條件下，SEQ ID NO：8之FGF21變體(P115W)之HMW%顯著高於SEQ ID NO：9之FGF21變體，此證明115位處具有色胺酸對促進聚集及自締合之因果作用。同樣，SEQ ID NO：10之FGF21變體(其在115位處含有色胺酸殘基)與SEQ ID NO：1之FGF21變體(其
在115位處含有胺基酸脯胺酸)相比具有實質上升高之HMW%(表5)。
在SEC分析中以HMW%形式量測物理穩定性及與0.9%
之防腐劑含量濃度下之苯甲醇之相容性，在存在或不存在0.02%吐溫(Tween)-80下，在含150 mM NaCl之pH 7.0下之緩衝液10 mM組胺酸中監測。以30 mg/mL製備試樣並在4℃、25℃及40℃下培育4週。藉由SEC方法針對HMW%分析剛剛調配之FGF21變體(即零時)及彼等經培育4週者。表6匯總分析結果，其比較零時試樣(「初始」)及彼等在40℃下培育4週者。
SEQ ID NO：1之FGF21變體含有胺基酸取代L98D。SEQ ID NO：9之FGF21變體不含胺基酸取代L98D，而是在98位處含有野生型胺基酸白胺酸。當在調配條件下以30 mg/mL調配每一蛋白質時觀察到胺基酸取代L98D之益處(表6)。在所有測試條件下，將FGF21變體在40℃下加壓(stress)4週致使SEQ ID NO：9之FGF21變體之HMW%實質上高於SEQ ID NO：1之FGF21變體。另外，0.9%苯甲醇(一種用於
多用醫藥製劑中之常見防腐劑)之添加對於SEQ ID NO：9之FGF21變體而言加劇了HMW%之增加，但對於SEQ ID NO：1之FGF21變體並未如此。在分析初始試樣製劑時亦觀察到此與苯甲醇之不相容性，其中在存在0.9%苯甲醇下之HMW%為11%，與之相比，在不存在苯甲醇下僅為0.97%。SEQ ID NO：9之FGF21變體及SEQ ID NO：1之FGF21變體均不含P115W殘基，因此，該等條件下較差之物理穩定性不可歸因於P115W殘基。在實施胺基酸取代L98D之後，觀察到存在0.9%苯甲醇下之增強之物理穩定性。
該等數據表明，尤其在某些防腐劑(例如苯甲醇)存在下，某些取代會因聚集成高分子量物質而影響整體蛋白質之穩定性。對於治療蛋白質而言，將該等HMW聚集體最小化是較佳的。此可藉由FGF21變體蛋白質中之某些取代(例如SEQ ID NO：1中展示之FGF21變體中之L98D)來達成。其他取代(例如P115W)具有有害效應，例如會增加變體中聚集的程度。
經皮下投與雄性食蟹猴(n=2隻/組)SEQ ID NO：1之FGF21變體之單一2 mg/kg注射劑。隨時間取得血清(在0.25小時至12小時之後抽取)，以便經由質譜法量化活性化合物之含量而評估24小時在活體內蛋白水解作用。
實施液相層析質譜法(LC/MS)分析。用共價結合至磁性
珠粒之抗FGF21單株抗體免疫沈澱每一血清試樣之100 μL等份試樣。將經免疫沈澱試樣分成單獨等份試樣，從而使得能夠檢測完整蛋白質及經胰蛋白酶消化之蛋白質。將完整蛋白質注入含經C5塗覆之3 μm粒子且內徑為100×0.32 mm之Discovery® Bio大孔管柱上。將經胰蛋白酶消化之試樣注入含經C18塗覆之3 μm粒子且內徑為100×0.32 mm之Discovery Bio大孔管柱上。所有注入之層析條件係使用由流動相A(0.1/100，甲酸：水)及流動相B(0.1/100，甲酸：乙腈)組成之二元梯度。將來自LC之流出液直接連接至Micromass Synapt® Q-Tof質譜儀以便以正離子模式進行質譜檢測。使用Masslynx(v 4.1)及MaxEntl反褶積軟體收集來自Q-Tof質譜儀之數據。
已發現FGF21蛋白質之171位處之解離會使蛋白質之生物活性降低超過100倍。因此，意欲降低此位點處之蛋白水解作用以增強全活性藥物之暴露。當以上述LC/MS方法分析時，SEQ ID NO：1之FGF21變體在24小時評價期間未顯示出可檢測到蛋白水解產物。該等數據證明，當與SEQ ID NO：7之FGF21變體(其不含G170E之胺基酸取代)相比時，SEQ ID NO：1之FGF21變體中之G170E取代會使雄性食蟹猴在24小時期間之活體內蛋白水解降解作用減少。
雄性ob/ob(B6.V-Lep ob /Lep +/OlaHsd)小鼠及年齡匹配之ob/m瘦對照(B6.V-Lep +/OlaHsd)在到達時為7-8週齡，且在
治療起始時為10-11週齡。到達後，以3隻/籠子圈養所有小鼠，且在治療開始之前使其適應3週。餵養小鼠Purina齧齒動物飼料5015，並隨意給予水。以12小時之光照/黑暗循環圈養小鼠，其中將環境溫度設於70℉下。治療開始之前一天，使小鼠禁食2小時且藉由尾部放血將血液試樣收集至含有肝素之毛細管中。用Ascensia Contour血糖計量測血糖含量，且使用Meso Scale小鼠/大鼠胰島素分析套組(Meso Scale Discovery,Gaithersburg,MD)來量化血漿胰島素含量。在治療起始當天(第0天)，基於前一天之體重、血糖及血漿胰島素將小鼠分類。用無菌鹽水(0.9% NaCl)稀釋FGF21變體，且經由微型滲透Alzet幫浦(mini-osmotic Alzet pump)將其經皮下投與。在第5天，在光照循環開始之後大約2小時，量測餐後血糖及血漿胰島素含量。在第5天使所有小鼠禁食過夜，且在第6天實施口服耐糖試驗(OGTT)。藉由剪尾巴將小鼠血液放至含有肝素之毛細管中，隨後口服投與葡萄糖(2 g/kg)。在口服葡萄糖投與之後30分鐘、60分鐘及120分鐘時，收集其他血液試樣。用來自Cayman Chemicals之葡萄糖分析套組量測血漿葡萄糖。在第6天，對標準化之葡萄糖AUC值實施四參數邏輯斯諦回歸模型擬合。
在第5天，經媒劑治療之小鼠血糖過高，其中所量測平均血糖含量在240.4±15.0 mg/dl(平均值±SEM)，而ob/m瘦對照小鼠之血糖含量為150.6±6.0 mg/dl(平均值±SEM)。SEQ ID NO：1之FGF21變體及SEQ ID NO：11之FGF21變體
二者均以劑量依賴性方式使血糖降低至與ob/m瘦對照相當之含量。SEQ ID NO：1之FGF21變體之ED50為0.7 μg/kg/hr，而SEQ ID NO：11之FGF21變體之ED50為3.1 μg/kg/hr。SEQ ID NO：1之FGF21變體在降低ob/ob小鼠中之血糖方面之效能係SEQ ID NO：11之FGF21變體之大約4.4倍。因此，當與已知之SEQ ID NO：11之FGF21變體(揭示於WO 2006/028595中)相比時，SEQ ID NO：1之FGF21變體為有效之FGF21變體。
HPIPDSSPLLQFGGQVRQRYLYTDDAQQTECHLEIREDGTVGCAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFREDLLEDGYNVYQSEAHGLPLHLPGDKSPHRKPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLRLVEPSQLLSPSFLG
HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS
MRLAVGALLVCAVLGLCLA
MKICSLTLLSFLLLAAQVLLVEG
MKALVILGFLFLSVAVQG
METDTLLLWVLLLWVPGSTG
HPIPDSSPLLQFGGQVRQRYLYTDDAQQTECHLEIREDGTV
GCAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFREDLLEDGYNVYQSEAHGLPLHLPGDKSPHRKPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLRLVGPSQLLSPSFLG
HPIPDSSPLLQFGGQVRQRYLYTDDAQQTECHLEIREDGTVGCAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLWLHLPGDKSPHRKPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPYSLVEPSQLLSPSFLG
HPIPDSSPLLQFGGQVRQRYLYTDDAQQTECHLEIREDGTVGCAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGDKSPHRKPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPYSLVEPSQLLSPSFLG
HPIPDSSPLLQFGGQVRQRYLYTDDAQQTECHLEIREDGTVGCAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLWLHLPGDKSPHRKPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLRLVEPSQLLSPSFLG
DSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGA
ADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHCPGNKSPHRDPAPRGPCRFLPLPGLPPALPEPPGILAPQPPDVGSSDPLAMVGPSQGRSPSYAS
CACCCTATCCCTGACTCCAGCCCTCTGCTGCAGTTTGGCGGACAGGTCCGGCAGCGGTACCTGTACACCGACGACGCCCAGCAGACCGAGTGCCACCTGGAAATCCGGGAGGACGGCACCGTGGGCTGTGCCGCCGACCAGTCCCCTGAGTCCCTGCTGCAGCTGAAGGCCCTGAAGCCTGGCGTGATCCAGATCCTGGGCGTGAAAACCTCCCGGTTCCTGTGCCAGAGGCCTGATGGCGCCCTGTACGGCTCCCTGCACTTCGACCCTGAGGCCTGCTCCTTCCGGGAGGACCTGCTGGAAGATGGCTACAACGTGTACCAGTCCGAGGCTCACGGCCTGCCTCTGCATCTGCCTGGCGACAAGTCCCCCCACCGGAAGCCTGCTCCTAGGGGCCCTGCCAGATTCCTGCCACTGCCTGGCCTGCCTCCAGCTCTGCCTGAGCCTCCTGGCATCCTGGCCCCTCAGCCTCCAGACGTGGGCTCCTCCGACCCTCTGCGGCTGGTCGAGCCTTCCCAGCTGCTGAGCCCTAGCTTCCTGGGC
HPIPDSSPLLQFGGQVRQRYLYTDDAQQTECHLEIREDGTVGCAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFREX1LLEDGYNVYQSEAHGLX2LHLPGDKSPHRKPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGS
SDP X3 X4LV X5PSQLLSPSFLG
X1為L或D
X2為P或W
X3為L或Y
X4為S或R
X5為G或E
<110> 美國禮來大藥廠
<120> 纖維母細胞生長因子21變體
<130> X-19386
<140> 101134800
<141> 2012-09-21
<150> 61/542,906
<151> 2011-10-04
<160> 13
<170> PatentIn version 3.5
<210> 1
<21> 181
<212> PRT
<213> 人工序列
<220>
<223> 合成構築體
<400> 1
<210> 2
<211> 181
<212> PRT
<213> 智人
<400> 2
<210> 3
<211> 19
<212> PRT
<213> 人工序列
<220>
<223> 合成構築體
<400> 3
<210> 4
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<212> PRT
<213> 人工序列
<220>
<223> 合成構築體
<400> 4
<210> 5
<211> 18
<212> PRT
<213> 人工序列
<220>
<223> 合成構築體
<400> 5
<210> 6
<211> 20
<212> PRT
<213> 人工序列
<220>
<223> 合成構築體
<400> 6
<210> 7
<211> 181
<212> PRT
<213> 人工序列
<220>
<223> 合成構築體
<400> 7
<210> 8
<211> 181
<212> PRT
<213> 人工序列
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<223> 合成構築體
<400> 8
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<211> 181
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<220>
<223> 合成構築體
<400> 9
<210> 10
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<220>
<223> 合成構築體
<400> 10
<210> 11
<211> 177
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<400> 11
<210> 12
<211> 543
<212> DNA
<213> 人工序列
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<400> 12
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<211> 181
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<223> 合成構築體
<220>
<221> MISC_FEATURE
<222> (98)..(98)
<223> 98位處之Xaa為Leu或Asp
<220>
<221> MISC_FEATURE
<222> (115)..(115)
<223> 115位處之Xaa為Pro或Trp
<220>
<221> MISC_FEATURE
<222> (166)..(166)
<223> 166位處之Xaa為Leu或Tyr
<220>
<221> MISC_FEATURE
<222> (167)..(167)
<223> 167位處之Xaa為Ser或Arg
<220>
<221> MISC_FEATURE
<222> (170)..(170)
<223> 170位處之Xaa為Gly或Glu
<400> 13
Claims (5)
- 一種人類纖維母細胞生長因子21(FGF21)之變體，其中胺基酸序列為HPIPDSSPLLQFGGQVRQRYLYTDDAQQTECHLEIREDGTVGCAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFREDLLEDGYNVYQSEAHGLPLHLPGDKSPHRKPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLRLVEPSQLLSPSFLG(SEQ ID NO：1)。
- 一種醫藥組合物，其包含如請求項1之變體及至少一種醫藥上可接受之載劑、稀釋劑或賦形劑。
- 一種如請求項1之變體之用途，該變體係用於製造用以治療II型糖尿病、肥胖症、異常血脂症或代謝症候群或其任一組合之藥劑。
- 如請求項1之變體，該變體係用於療法中。
- 如請求項1之變體，該變體係用於治療II型糖尿病、肥胖症、異常血脂症或代謝症候群或其任一組合。
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| WO2010065439A1 (en) * | 2008-12-05 | 2010-06-10 | Eli Lilly And Company | Variants of fibroblast growth factor 21 |
| US20120052069A1 (en) | 2009-05-05 | 2012-03-01 | Amgen Inc | Fgf21 mutants and uses thereof |
| UY32607A (es) | 2009-05-05 | 2010-12-31 | Amgen Inc | Mutantes de fgf21 y usos del mismo |
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