CN101010338A - 成纤维细胞生长因子21的突变蛋白 - Google Patents
成纤维细胞生长因子21的突变蛋白 Download PDFInfo
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Abstract
本发明涉及新的人成纤维细胞生长因子21,当其在酵母中表达时,与野生型人FGF-21相比具有降低的O-糖基化能力。本发明公开了蛋白质和各自的编码核酸种类。本发明还包括用于扩增所述核酸序列以及产生所述突变蛋白的载体和宿主细胞。本发明还公开了治疗2型糖尿病、肥胖症或代谢综合征的方法。
Description
技术领域
本发明涉及鉴定成纤维细胞生长因子21的新突变蛋白,其在酵母中表达时具有减少的O联糖基化。
背景技术
成纤维细胞生长因子是在发育组织和成熟组织中均广泛表达的大的多肽(Baird等人,Cancer Cells,3:239-243,1991),并且在多种生理学功能(包括血管发生、有丝分裂发生、图式形成、细胞分化、代谢调节和组织损伤修复)中发挥重要的作用(McKeehan等人,Prog.Nucleic Acid Res.Mol.Biol.59:135-176,1998)。根据公开的文献,FGF家族现在由至少23个成员组成,即FGF-1到FGF-23(Reuss等人,Cell Tissue Res.313:139-157(2003).)
已经报道了成纤维细胞生长因子(FGF-21)在肝脏中优选表达(Nishimura等人,Biochimica et Biophysica Acta,1492:203-206,2000);(WO01/36640;和WO01/18172),并且被描述为治疗下列疾病:缺血性血管病、创伤愈合、与肺的、支气管的或肺泡的细胞功能丧失相关的疾病以及多种其他障碍。最近,已经表明在延长FGF-21处理(72小时)后,无论胰岛素存在与否,都会刺激小鼠3T3-L1脂肪细胞中葡萄糖的摄取,并且它在ob/ob和db/db小鼠和8周大的ZDF大鼠中以剂量依赖的方式减少餐后和禁食的血糖、甘油三酯、胰高血糖素的水平,因此提供了FGF-21用于治疗糖尿病和肥胖症治疗的基础。
重组DNA技术的发展使得在宿主细胞中产生外源产物如FGF-21突变蛋白成为可能,所述宿主细胞中引入了编码那些产物的外源DNA序列。此技术的优点是产物以高产量、高纯化形式产生,并且具有较低的污染(如病毒污染)危险。这些重组技术已经广泛的用于在原核以及真核宿主细胞中产生重组蛋白质。
但是,由于这些外源DNA序列的表达效率、载体的不稳定和产生重组产物的宿主细胞对重组产物的胞内降解等问题,使得通过这些技术大规模产生重组产物仍是有限的。另外,重组产物通常不同于其天然对应物。例如,在重组产物的糖基化含量方面,在异源真核宿主中产生的重组产物通常不同于它们天然存在的对应物。这涉及到任何糖类结构的存在与不存在,所述糖类结构在产物上的定位以及糖类的性质。更特别地,还表明来自酵母的重组产物通常与它们的天然对应物相比具有额外的非天然O-聚糖(Van den Steen等人,Crit.Reviews in Biochem.and Mole.Biol.33(3):151-208,1998)。
通过提供在酵母中表达时,与野生型FGF-21相比具有减少的O-糖基化量的FGF-21突变蛋白,本发明解决了与来自酵母的重组蛋白质相关的异常O-糖基化的问题。申请人发现,具有减少的O-糖基化的FGF-21突变蛋白可以在工业发酵条件中产生,并且保留了用于治疗患有下列疾病的患者所必需的生物学活性,所述的疾病包括但不限于II型糖尿病、肥胖症和代谢综合征。
发明概述
在第一个实施方案中,本发明提供了人FGF-21的突变蛋白或其生物学活性肽,其包含除Ser或Thr外的任何氨基酸对Ser167的取代,其中氨基酸的编号基于SEQ ID NO:1,并且当在酵母中表达时,所述突变蛋白与野生型人FGF-21相比具有降低的O-糖基化能力。
本发明的第二个实施方案提供了人FGF-21的突变蛋白或者其生物学活性肽,其包含除Ser或Thr外的任何氨基酸对Ser167的取代,以及半胱氨酸对下列两个或多个氨基酸的取代:精氨酸19、酪氨酸20、亮氨酸21、酪氨酸22、苏氨酸23、天冬氨酸24、天冬氨酸25、丙氨酸26、谷氨酰胺27、谷氨酰胺28、丙氨酸31、亮氨酸33、异亮氨酸35、亮氨酸37、缬氨酸41、甘氨酸42、甘氨酸43、谷氨酸50、谷氨酰胺54、亮氨酸58、缬氨酸62、亮氨酸66、甘氨酸67、赖氨酸69、精氨酸72、苯丙氨酸73、谷氨酰胺76、精氨酸77、天冬氨酸79、甘氨酸80、丙氨酸81、亮氨酸82、甘氨酸84、丝氨酸85、脯氨酸90、丙氨酸92、丝氨酸94、苯丙氨酸95、亮氨酸100、天冬氨酸102、酪氨酸104、酪氨酸107、丝氨酸109、谷氨酸110、脯氨酸115、组氨酸117、亮氨酸118、脯氨酸119、天冬酰胺121、赖氨酸122、丝氨酸123、脯氨酸124、组氨酸125、精氨酸126、天冬氨酸127、丙氨酸129、脯氨酸130、甘氨酸132、丙氨酸134、精氨酸135、亮氨酸137、脯氨酸138或亮氨酸139,其中氨基酸的编号基于SEQID NO:1,并且当在酵母中表达时,所述突变蛋白与野生型人FGF-21相比具有降低的O-糖基化能力。
本发明的第三个实施方案提供了人FGF-21的突变蛋白或者其生物学活性肽,其包含除Ser或Thr外的任何氨基酸对Ser167的取代,以及带电的和/或极性却不带电的氨基酸对下列一个或多个位置上氨基酸的取代:甘氨酸42、谷氨酰胺54、精氨酸77、丙氨酸81、亮氨酸86、苯丙氨酸88、赖氨酸122、组氨酸125、精氨酸126、脯氨酸130、精氨酸131、亮氨酸139、丙氨酸145、亮氨酸146、异亮氨酸152;丙氨酸154;谷氨酰胺156、甘氨酸161、丝氨酸163、甘氨酸170或丝氨酸172,其中氨基酸的编号基于SEQ ID NO:1,并且当在酵母中表达时,所述突变蛋白与野生型人FGF-21相比具有降低的O-糖基化能力。
其他实施方案涉及了第一、第二和第三个实施方案的突变蛋白的编码多核苷酸;含有所述多核苷酸的载体以及携带所述载体的宿主细胞。另一些实施方案涉及产生多肽、产生能够制备所述多肽的细胞和产生含有所述多肽的编码DNA的载体的方法。
另一实施方案涉及治疗患有一种或多种下列病症的患者的方法:肥胖症、II型糖尿病、抗胰岛素性、高胰岛素血症、葡萄糖不耐受、高血糖症或代谢综合征,所述方法包括向所述需要此类治疗的患者施用治疗有效量的第一、第二或第三个实施方案的人FGF-21突变蛋白。
发明详述
为了本发明的目的,按照在本文的公开和权利要求的,下列术语定义如下。
人FGF-21是含有27个氨基酸前导序列的208个氨基酸的多肽。人FGF-21与小鼠FGF-21具有~79%的氨基酸同一性,并且与大鼠FGF-21具有~80%的氨基酸同一性。人FGF-21是本发明突变蛋白的优选多肽模板,但是认为本领域的技术人员可以容易的根据可选的哺乳动物FGF-21多肽序列制备突变蛋白。
从下列显示的成熟人181个氨基酸的FGF-21多肽(SEQ ID NO:1),可以确定本发明突变蛋白的氨基酸位置:
1 10 20
His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val Arg Gln Arg Tyr
30 40
Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr
50 60
Val Gly Gly Ala Ala Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro
70 80
Gly Val Ile Gln Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly
90 100
Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu Leu Leu Leu
110 120
Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly Leu Pro Leu His Leu Pro Gly
130 140
Asn Lys Ser Pro His Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro
150 160
Gly Leu Pro Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val
170 180
Gly Ser Ser Asp Pro Leu Ser Met Val Gly Pro Ser Gln Gly Arg Ser Pro Ser Tyr Ala
Ser
成熟人181个氨基酸的FGF-21多肽的相应编码DNA序列是(SEQ IDNO:2):
CACCCCATCCCTGACTCCAGTCCTCTCCTGCAATTCGGGGGCCAAGTCCGGCA
GCGGTACCTCTACACAGATGATGCCCAGCAGACAGAAGCCCACCTGGAGATC
AGGGAGGATGGGACGGTGGGGGGCGCTGCTGACCAGAGCCCCGAAAGTCTC
CTGCAGCTGAAAGCCTTGAAGCCGGGAGTTATTCAAATCTTGGGAGTCAAGA
CATCCAGGTTCCTGTGCCAGCGGCCAGATGGGGCCCTGTATGGATCGCTCCAC
TTTGACCCTGAGGCCTGCAGCTTCCGGGAGCTGCTTCTTGAGGACGGATACAA
TGTTTACCAGTCCGAAGCCCACGGCCTCCCGCTGCACCTGCCAGGGAACAAG
TCCCCACACCGGGACCCTGCACCCCGAGGACCAGCTCGCTTCCTGCCACTACC
AGGCCTGCCCCCCGCACTCCCGGAGCCACCCGGAATCCTGGCCCCCCAGCCC
CCCGATGTGGGCTCCTCGGACCCTCTGAGCATGGTGGGACCTTCCCAGGGCCG
AAGCCCCAGCTACGCTTCC
使用三字母密码识别氨基酸或可选的用标准单字母密码指示氨基酸。通过原始氨基酸的三字母密码,之后是是氨基酸数,之后是取代氨基酸的三字母密码来指示突变。每个突变蛋白的数字指示基于成熟野生型人FGF-21的181个氨基酸的序列。例如,将用非极性/疏水氨基酸——丙氨酸(Ala)对第167位的丝氨酸(即Ser167)的取代指示为Ser167Ala或S167A。在类似的形式中,将用含硫氨基酸——半胱氨酸(Cys)对第118位的亮氨酸和第134位的丙氨酸(Leu118,Ala134)的双取代指示为Leu118Cys/Ala134Cys或者L118C/A134C。
术语“氨基酸”在本文中以其最广泛的含义使用,并且包括天然存在的氨基酸以及非天然存在的氨基酸,包括氨基酸类似物以及衍生物。后者包括含有氨基酸一部分的分子。考虑到这种广泛的定义,本领域的技术人员将认识到在本文提及的氨基酸包括例如天然存在的蛋白质性(proteogenic)的L-氨基酸;D-氨基酸;化学修饰的氨基酸如氨基酸类似物和衍生物;天然存在的非蛋白质性的氨基酸如正亮氨酸、β-丙氨酸、鸟氨酸等;以及具有本领域已知的为氨基酸特性的性质的化学合成化合物。
将人FGF-21突变蛋白定义为包括野生型成熟蛋白质的至少一个氨基酸被另一个氨基酸取代的人FGF-21。在本文引用作为参考的美国专利申请60/528,582中描述了FGF21突变蛋白的实例。一般而言,突变蛋白具有野生型蛋白质的一些修饰性质、结构或功能。例如,突变蛋白可以在浓缩的溶液中具有增强的或改进的物理稳定性(例如,疏水介导的聚集较少),同时保持了有利的生物活性谱。突变蛋白与药物防腐剂(例如m-甲酚、苯酚、苯甲醇)的相容性增加,使得能够制备在储存期间保持了此蛋白质物理化学性质和生物学活性的可保存的药物制剂。当在酵母中表达时突变蛋白可以具有减少的O-糖基化。此类O-糖基化可以在蛋白质上引入新的免疫决定簇,并且因此当它施用给人时是抗原性的;可以改变蛋白质的药物动力学性质;和/或可以影响蛋白质的生物学活性。因此,具有比野生型FGF-21的O-糖基化减少的酵母产生的突变蛋白有更低的免疫原性并且具有有利的药物动力学模式,同时保持了生物学潜能。如本文所用,这些术语不是限制的,所给突变蛋白具有一个或多个野生型蛋白质的修饰性质是完全可能的。
“治疗有效量”是给予患者治疗益处所需的活性剂的最小量。例如,对患有或倾向于患有II型糖尿病、肥胖症或代谢综合征的患者,或预防这些疾病的“治疗有效量”是这种量,即它诱导、改善或另外引起与前述疾病相关的病理症状、疾病进展、生理学情况的改进,或抵抗患有前述疾病。对于本发明的目的,“对象”或“患者”优选是人。
II型糖尿病的特征在于无论胰岛素的可用性而过量产生葡萄糖,并且作为不充分葡萄糖清除的结果--循环葡萄糖水平过高。
可以将葡萄糖不耐受定义为对葡萄糖的异常敏感。
将高血糖症定义为血液中糖(葡萄糖)过量。
当你的血液葡萄糖水平下降到很低,以致不足以为你身体活动提供充足的能量时,发生低血糖。
将高胰岛素血症定义为血液中高于正常水平的胰岛素。
将抗胰岛素性定义为正常量的胰岛素产生了低于正常的生物应答的状态。
在人类对象方面,可以将肥胖症定义为体重超出了给定人群理想体重的百分之二十(R.H.Williams,Textbook of Endocrinology,1974,904-916页)。
可以将代谢综合征定义为至少三种下列征兆的集合:腹部肥胖-在大多数人中,40英寸腰围或更大;高血糖-在禁食后至少为110毫克每分升(mg/dl);高甘油三酯-血流中至少为150mg/dL;低HDL-低于40mg/dl;以及,血压为130/85或更高。
本发明提供了糖基化突变蛋白,其中与天然FGF-21相比,它改变了糖基化位点的数量和/或类型。一个此类实施方案包括含有更少量O联糖基化位点的FGF-21突变蛋白。不存在鉴定O联糖基化位点的共有氨基酸序列,这使此类鉴定成为困难的工作。通常,O联糖基化发生在丝氨酸或苏氨酸残基的侧链。一旦鉴定了O联糖基化位点,排除此序列的氨基酸取代可以移除存在的O-联糖类链。本发明中鉴定的O联糖基化位点包括Ser163、Ser 164、Ser 167、Ser 172和Ser 176。O-糖基化的首要位点是Ser167。申请人已经公开了排除Ser167位点,使得酵母表达的突变蛋白的O-糖基化显著减少。尽管Ser167是移除O-糖基化的突变优选位点,在人FGF-21中对O-糖基化的其他位点(Ser163、Ser164、Ser172和Ser176)的突变也在本发明的范围内。
因此,在第一个实施方案中,本发明提供了人FGF-21的突变蛋白或其生物学活性肽,其包含除Ser或Thr外的任何氨基酸对Ser167的取代,其中氨基酸的编号基于SEQ ID NO:1,并且其中当在酵母中表达时,所述突变蛋白与野生型人FGF-21相比具有降低的O-糖基化能力。第一个实施方案优选的突变蛋白是Ser167Ala、Ser167Glu、Ser167Asp、Ser167Asn、Ser167Gln、Ser167Gly、Ser167Val、Ser167His、Ser167Lys和Ser167Tyr。
本发明的第二个实施方案提供了人FGF-21的突变蛋白或者其生物学活性肽,其包含除Ser或Thr外的任何氨基酸对Ser167的取代以及半胱氨酸对下列两个或多个的取代:精氨酸19、酪氨酸20、亮氨酸21、酪氨酸22、苏氨酸23、天冬氨酸24、天冬氨酸25、丙氨酸26、谷氨酰胺27、谷氨酰胺28、丙氨酸31、亮氨酸33、异亮氨酸35、亮氨酸37、缬氨酸41、甘氨酸42、甘氨酸43、谷氨酸50、谷氨酰胺54、亮氨酸58、缬氨酸62、亮氨酸66、甘氨酸67、赖氨酸69、精氨酸72、苯丙氨酸73、谷氨酰胺76、精氨酸77、天冬氨酸79、甘氨酸80、丙氨酸81、亮氨酸82、甘氨酸84、丝氨酸85、脯氨酸90、丙氨酸92、丝氨酸94、苯丙氨酸95、亮氨酸100、天冬氨酸102、酪氨酸104、酪氨酸107、丝氨酸109、谷氨酸110、脯氨酸115、组氨酸117、亮氨酸118、脯氨酸119、天冬酰胺121、赖氨酸122、丝氨酸123、脯氨酸124、组氨酸125、精氨酸126、天冬氨酸127、丙氨酸129、脯氨酸130、甘氨酸132、丙氨酸134、精氨酸135、亮氨酸137、脯氨酸138或亮氨酸139,其中氨基酸的编号基于SEQ ID NO:1,并且当在酵母中表达时,所述突变蛋白与野生型人FGF-21相比具有降低的O-糖基化能力。优选的,短语“两个或多个”意思是半胱氨酸对2、3、4、5、6、7、8、9、10、11、12、13或14个位于上面所示位置的氨基酸残基的取代。更优选的,其意思是半胱氨酸对2或4个位于上面所示位置的氨基酸残基的取代。
本领域的技术人员还将认识到天然的半胱氨酸——半胱氨酸75和半胱氨酸93也可以用作引入新的可以给予改良性质的二硫键的位置。特别希望的是在丝氨酸85或苯丙氨酸73上引入半胱氨酸取代,以及分别在半胱氨酸93或半胱氨酸75上的伴随改变,其中后一个位点用任何其他氨基酸取代。
在美国专利申请60/528,582中描述了除了在Cys75-Cys93上天然存在的二硫键之外,还具有改造的二硫键的FGF21突变蛋白。第二个实施方案最优选的突变蛋白是Leu118Cys-Ala134Cys-Ser167Ala;Leu21Cys-Leu33Cys-Ser167Ala;Ala26Cys-Lys122Cys-Ser167Ala;或Leu21Cys-Leu33Cys/Leu118Cys-Ala134Cys-Ser167Ala。
本发明的第三个实施方案提供了人FGF-21的突变蛋白或者其生物学活性肽,其包含除Ser或Thr外的任何氨基酸对Ser167的取代,以及带电的和/或极性却不带电的氨基酸对下列位置的一个或多个氨基酸的取代:甘氨酸42、谷氨酰胺54、精氨酸77、丙氨酸81、亮氨酸86、苯丙氨酸88、赖氨酸122、组氨酸125、精氨酸126、脯氨酸130、精氨酸131、亮氨酸139、丙氨酸145、亮氨酸146、异亮氨酸152、丙氨酸154、谷氨酰胺156、甘氨酸161、丝氨酸163、甘氨酸170或丝氨酸172,其中氨基酸的编号基于SEQ ID NO:1,并且当在酵母中表达时,所述突变蛋白与野生型人FGF-21相比具有降低的O-糖基化能力。
将带电的氨基酸定义为带正电或带负电的氨基酸。将带正电的氨基酸定义为包括组氨酸、赖氨酸、精氨酸,以及其非天然存在的类似物(例如γ-氨基丁酸、鸟氨酸等)。将带负电的氨基酸定义为包括天冬氨酸、谷氨酸、以及其非天然存在的类似物(例如氨基己二酸)。将极性但是不带电的氨基酸定义为包括丝氨酸、苏氨酸、天冬酰胺、谷氨酰胺,以及其非天然存在的类似物。第三个实施方案优选的突变蛋白是Gln54Glu-Ser167Ala、Leu139Glu-Ser167Ala、Ala145Glu-Ser167Ala、Leu146Glu-Ser167Ala、Ile152Glu-Ser167Ala、Gln156Glu-Ser167Ala、Ser163Glu-Ser167Ala和Ile152Glu-Ser163Glu-Ser167Ala。
本发明其他实施方案提供了人FGF-21的突变蛋白或其生物学活性肽,其包括本发明的第一个实施方案、本发明的第二个实施方案和本发明的第三个实施方案的组合,其中与野生型人FGF-21相比,当在酵母中表达时,所述的突变蛋白具有降低的O-糖基化能力。
尽管本发明的实施方案涉及当在酵母中表达时,具有比野生型人FGF-21的O-糖基化能力降低的FGF-21突变蛋白,维持与野生型FGF-21相当的突变蛋白的生物学潜能也是需要考虑的重要因素。因此,将本发明突变蛋白的生物学潜能定义为突变蛋白影响葡萄糖摄取的能力(如在体外3T3-L1细胞测定中所测量的)(实施例2)和/或降低血浆葡萄糖水平和血浆甘油三酯的能力(如在体外ob/ob小鼠测定中所测量的)(实施例3)。
可以通过本领域任何已知的方法产生和/或分离根据本发明施用的FGF-21突变蛋白。产生此突变蛋白最优选的方法是通过重组DNA方法并且是本领域的技术人员众所周知的。在此引用作为参考的Current Protocolsin Molecular Biology(John Wiley & Sons,Inc.)中描述了此类方法。
另外,优选的实施方案包括衍生自本文所描述突变蛋白的生物学活性肽,并且此肽会包含至少一种所描述的取代,会比相应的非突变肽表现出降低的O-糖基化能力,并且会具有生物学活性。通过肽影响葡萄糖摄取(如在体外3T3-L1细胞测定中所测量的)(实施例2)、和/或降低血浆葡萄糖水平以及血浆甘油三酯(如在体外ob/ob小鼠测定中所测量的)(实施例3)的能力来定义此生物学活性。可以通过本领域技术人员已知的任何方法产生该肽,所述方法的实例包括但不限于酶促消化、化学合成或重组DNA方法。
在本领域中已经确定的是某些成纤维细胞生长因子的肽片段具有生物学活性。参见例如,Baird等人,Proc.Natl.Acad.Sci(USA)85:2324-2328(1988),和J.Cell.Phys.Suppl.5:101-106(1987)。例如,已知二肽基肽酶IV(DPP-IV)是参与灭活神经肽、内分泌肽和细胞因子的丝氨酸类型蛋白酶(Damme等人Chem.Immunol.72:42-56,(1999))。FGF-21的N端(HisProIlePro)包含两个可能为DPP-IV底物的二肽,得到在N端截短多达4个氨基酸的FGF-21的片段。出乎意料的,已经证明了此野生型FGF-21片段保留了生物学活性(表1),因此,在N端截短多达4个氨基酸的本发明突变蛋白与本发明任何实施方案的氨基酸取代相组合。此外,申请人已经发现从N端截短5个或更多个的氨基酸没有影响生物学活性。
本发明还包括可以是RNA形式或DNA形式的上述突变蛋白的编码多核苷酸,其中DNA包括cDNA、基因组DNA和合成DNA。DNA可以是双链或单链的。编码本发明突变蛋白的编码序列可以由于遗传密码的丰余或简并而不同。
本发明突变蛋白的编码多核苷酸可以包括下列:突变蛋白的唯一编码序列、突变蛋白的编码序列和其他编码序列如功能性多肽或前导序列或分泌序列或蛋白原序列;突变蛋白的编码序列和非编码序列,如内含子或突变蛋白编码序列的5’和/或3’非编码序列。因此,术语“编码突变蛋白的多核苷酸”涉及不仅包括突变蛋白的编码序列,而且包括含有其他编码序列和/或非编码序列的多核苷酸的多核苷酸。
本发明还涉及所述多核苷酸的变体,该变体编码包含有所示取代的多肽的片段、类似物和衍生物。该多核苷酸变体可以是天然存在的人FGF-21序列的等位基因变体、非天然存在的变体或如上所述截短的变体。因此,本发明还包括编码上述突变蛋白的多核苷酸以及此多核苷酸的变体,该变体编码比相应的非突变片段、衍生物或类似物表现出降低的O-糖基化能力的公开突变蛋白的片段、衍生物或类似物。此类核苷酸变体包括缺失变体、取代变体、截短的变体以及添加或插入变体,只要存在至少一个第一、第二或第三个实施方案所示的氨基酸取代。
在序列有效连接到表达调控序列上后,本发明的多核苷酸将在宿主细胞中表达。这些表达载体在宿主生物中一般作为附加体或作为宿主染色体DNA的整合部分复制。通常,表达载体会包含选择性标记,例如四环素、新霉素和二氢叶酸还原酶,以用于检测那些转化了所需DNA序列的细胞。优选的,宿主细胞是真菌或酵母细胞。
用于表达本发明突变蛋白的酵母细胞包括巴斯德毕赤酵母(Pichiapastoris)、酿酒酵母(Saccharomyces cerevisiae)、粟酒裂殖酵母(Schizosaccharomyces pombe)和Pichia angust。酵母宿主细胞包含具有表达调控序列如启动子(包括3-磷酸甘油酸激酶或其他糖酵解酶)的合适载体以及所需的复制起点、终止序列等。本发明优选的酵母宿主是其中表达载体整合到宿主染色体DNA中的巴斯德毕赤酵母。黑曲霉(Aspergillusniger)、里氏木霉(Trichoderma reesei)以及裂褶菌(Schizophyllum commune)是真菌宿主的实例,尽管其他也可以作为选择使用。
通过众所周知的方法(该方法根据细胞宿主类型不同而不同),可以将包含目标多核苷酸序列(例如FGF-21的突变蛋白和表达调控序列)的载体转移到宿主细胞中。例如,通常对原核细胞使用氯化钙转染,而对其他细胞宿主使用磷酸钙处理或电穿孔。
可以使用多种蛋白质纯化方法,并且此类方法在本领域中是已知的,在例如Deutscher,Methods in Enzymology 182:83-9(1990)和Scopes,Protein Purification:Principles and Practice,Springer-Verlag,NY(1982)中进行了描述。例如,所选择的纯化步骤会取决于用于FGF-21突变蛋白的产生方法的性质。
包含FGF-21突变蛋白的组合物应当以与良好的医学实践相一致的形式配制并且给药,要考虑的是患者的临床情况、FGF-21突变蛋白组合物递送的位置、施用方式、施用日程安排以及技术人员已知的其他因素。因此,用于本文目的的FGF-21的“治疗有效量”是通过此类考虑来确定的。
通过能达到治疗II型糖尿病、肥胖症或代谢综合征通常所需目的的本领域已知的任何方法来施用本发明FGF-21突变蛋白的药物组合物。优选的施用途径是肠胃外,在本文定义的是指包括静脉内的、肌内的、腹膜内的、胸骨内的(intrastemal)、皮下的以及关节内注射和输注的施用方式。施用剂量将取决于受体的年龄、健康状况和体重、并行治疗的类型,治疗频率(若有的话)和所需作用的性质。在本发明范围内的组合物包括其中FGF-21突变蛋白以能够有效实现治疗II型糖尿病、肥胖症或代谢综合征的所需医学效果的量存在的所有组合物。尽管在一个患者和另一个患者之间个体需要可以不同,所有成分有效量的最佳范围的确定是在普通临床医生能力范围内的。
可以根据制备可药用组合物的已知方法配制本发明的FGF-21突变蛋白。所需的制剂可以是一种用适当高纯度稀释剂或水溶液重组的稳定冻干产物与可选的可药用载体、防腐剂、赋形剂或稳定剂[Remington’sPharmaceutical Sciences第16版(1980)]。本发明的突变蛋白可以与可药用缓冲液组合,并且将pH调节到能提供可接受的稳定性和可施用的pH。此外,本发明的突变蛋白可置于选自下列的容器中:小瓶、药筒、钢笔递送装置、注射器、静脉内施用管和静脉内施用袋,其中该容器是单位剂量的容器。
对于肠胃外施用,通常以单位剂量注射形式(溶液、悬液或乳液),通过将一种或多种在所需程度的纯度下的FGF-21突变蛋白与可药用的载体(即在使用的剂量和浓度下对受体无毒并且与制剂的其他成分相容的载体)混合,来配制FGF-21突变蛋白。优选的,可以添加一种或多种可药用的抗微生物剂。苯酚、m-甲酚和苯甲醇是优选的可药用抗微生物剂。
可选的,可以添加一种或多种可药用盐来调节离子强度或张力。可以添加一种或多种赋形剂来另外调节制剂的等渗性。甘油、氯化钠和甘露醇是等渗性调节赋形剂的实例。
本领域的技术人员可以容易的优化包含FGF-21突变蛋白的治疗性组合物的药学有效剂量和施用方案,其通过好的医学实践和个体患者的临床条件确定。施用适当剂量的FGF-21突变蛋白可以使得通过更快和更有效的葡萄糖利用来降低血液葡萄糖水平以及增加能量支出,因此可用于治疗2型糖尿病、肥胖症和代谢综合征。
此外,与施用了胰岛素的大鼠相比,在瘦的ZDF大鼠中FGF-21不会诱导低血糖(WO03/011213)。这一数据表示出FGF-21以不依赖于胰岛素的方式影响血浆葡萄糖水平,这表明本发明的FGF-21突变蛋白还可以用于治疗I型糖尿病。
在本发明另一方面中,本文描述的人FGF-21突变蛋白或其生物学活性肽用作药物。
在本发明的另一方面中,有效量的在本文描述的FGF-21突变蛋白或其生物学活性肽用于制备治疗或预防选自下列一种或多种病症的药物,所述的病症选自II型糖尿病、肥胖症或代谢综合征。
现对本发明进行了详细的描述,通过参考下列实施例,将更加明确的了解同样的事物。在此涉及的实施例仅用于说明目的而不不意在限制本发明。
本文涉及的所有专利和出版物引为参考。
实施例1
酵母中FGF-21突变蛋白的表达和纯化
FGF-21在酵母如巴斯德毕赤酵母、甲醇毕赤酵母(Pichia methanolica)或酿酒酵母中表达。为了在巴斯德毕赤酵母中产生,市售系统(Invitrogen,Carlsbad,CA)利用具有强AOX1(乙醇氧化酶)启动子的载体来促进重组蛋白质的高水平表达。可选的,使用来自GAP基因(甘油醛-3-磷酸脱氢酶)的启动子的载体可用于高水平的组成性表达。多拷贝的毕赤酵母表达载体使人们得到了具有整合到基因组中的多拷贝目标基因的菌株。增加重组毕赤酵母菌株中目标基因的拷贝数可以增加蛋白质表达水平。而另一个酵母表达系统是酿酒酵母。表达载体包含来自GAL1基因的启动子和增强子序列。由于GAL1启动子在半乳糖诱导下的强转录活性,它是使用最广泛的酵母启动子之一。
分析性表征(质谱分析)显示在巴斯德毕赤酵母中表达的FGF-21是截短的(在野生型的N端移除了4个氨基酸)。当在小鼠3T3-L1脂肪细胞测定中测定时(见实施例2),此FGF-21截短的变体以与野生型FGF-21同样的水平刺激葡萄糖摄取(表1)。
实施例2
小鼠3T3-L1脂肪细胞中葡萄糖的摄取
3T3-L1细胞获自American Type Culture Collection(ATCC,Rockville,MD)。细胞在含有10%铁强化胎牛血清(Dulbecco’改良的Eagle’s培养基中)的生长培养基(GM)中培养。为了标准脂肪细胞分化,在细胞达到汇合(指示为0天)两天后,将细胞暴露在含有10%胎牛血清、10μg/ml胰岛素、1μM地塞米松和0.5μM异丁基甲基黄嘌呤的分化培养基(DM)中48小时。之后将细胞维持在含有10%胎牛血清和10μg/ml胰岛素的后分化培养基中。
葡萄糖转运测定——通过0.1mM 2-脱氧-D-[14C]葡萄糖聚集测定己糖的摄取,其如下测量:将在12孔板中的3T3-L1脂肪细胞用加热到37℃并且含有0.2%BSA的KRP缓冲液(136mM NaCl、4.7mM KCl、10mMNaPO4、0.9mM CaCl2、0.9mM MgSO4,pH 7.4)洗涤两次,在室内空气中,37℃下在含有0.2%BSA的Leibovitz’s L-15培养基中孵育2小时,再次用含有0.2%BSA的KRP缓冲液洗涤两次,并且在室内空气中,37℃下在缺乏(只有Me2SO)或存在渥曼青霉素的KRP、0.2%BSA缓冲液中温育30分钟。之后加入胰岛素至终浓度为100nM,进行15分钟,并且对最后4分钟测量2-脱氧-D-[14C]葡萄糖的摄取。所有的值减去在10μM松胞菌素B存在下测量的非特异性摄取。用Pierce bicinchoninic acid测定来确定蛋白质浓度。对于每个实验以三次重复或四次重复来常规测量摄取。
将体外潜能标准化到指示为1.0并且用作阳性对照的野生型FGF-21的体外活性。在表1中将本发明FGF-21突变蛋白的体外潜能与野生型FGF-21的体外潜能对比。如表1中所示,对比保持了多种程度的生物学潜能的本发明突变蛋白与野生型FGF-21。
表1
FGF-21突变蛋白 | 表达系统 | 体外潜能* |
野生型 | 大肠杆菌(E.coli) | 1.0 |
ΔHPIP截短的野生型** | 酵母 | 0.9 |
ΔHPIP L118C、A134C | 酵母 | 0.2 |
ΔHPIP L118C、A134C、S167A | 酵母 | 0.2 |
*潜能是基于大肠杆菌产生的野生型FGF-21活性的相对值。
**在N端截短4个氨基酸
实施例3
ob/ob小鼠模型
使用雄性ob/ob小鼠在肥胖症模型中进行研究,以监控用FGF-21治疗后血浆葡萄糖水平和甘油三酯的水平,将其与载体对照组和胰岛素对照组比较。雄性ob/ob小鼠(7周龄)测试组仅用载体(0.9%NaCl)或FGF-21突变蛋白(0.125mg/kg)皮下注射7天(0.1mL,每天一次)。在第七天,最后一次化合物注射后1小时,通过尾部剪断放血收集血液,并且使用标准方法测量血浆葡萄糖水平。在表2中显示了与载体对照相比较,FGF-21突变蛋白降低血浆葡萄糖水平的能力。表2中的数据显示与载体对照相比,本发明的突变蛋白降低了血浆葡萄糖水平。在表3中显示了与载体对照相比,FGF-21突变蛋白降低甘油三酯水平的能力。
表2
FGF-21突变蛋白 | 血浆葡萄糖水平占对照的% |
野生型 | 62% |
L118C-A134C | 70% |
L118C-A134C-S167A | 62% |
表3
FGF-21突变蛋白 | 甘油三酯水平(mg/dL) |
载体对照 | 210 |
野生型 | 116*** |
L118C-A134C | 137** |
L118C-A134C-S167A | 153* |
p值对比载体对照:*p≤0.05;**p≤0.02;***p≤0.001
实施例4
FGF-21突变蛋白的药物稳定性
在模拟的生理和药物制剂条件下,分析本发明FGF-21突变蛋白的稳定性。为了模拟生理条件,室温(RT)下在靶标蛋白质浓度为10mg/ml、pH7.4时,分析突变蛋白在PBS中的稳定性。如果制备后在室温下蛋白质回收有>90%的回收(通过大小排阻层析和/或反相层析确定),那么认为突变蛋白在PBS中的可溶性/物理稳定性是令人满意的。如在表4和表5中指示的,本发明的突变蛋白达到了此标准。
预计本发明突变蛋白的药物制剂可以是可保存的多用途制剂,因此,分析了其与常规防腐剂的相容性。为了测试制剂的相容性,在室温下,向含有在PBS(pH7.4)中大约10mg/ml的突变蛋白的溶液中添加防腐剂m-甲酚(3mg/mL终浓度,通常能够达到在中性pH条件下防腐剂效力的European Pharmacopia B标准的浓度)。通过在室温下,反相层析和大小排阻层析后确定主层析峰的蛋白质回收来最初评估在防腐剂存在时的物理稳定性。此外,对比野生型FGF-21,将DLS(动态光散射)测量的聚集程度用m-甲酚存在2小时后颗粒的平均直径来表示。更大的平均直径对应着增加的蛋白质关联和/或聚集程度。在表4中显示了与野生型FGF-21相比,本发明第一个和第二个实施方案的突变蛋白的防腐剂相容性(作为颗粒的功能平均直径)。在大肠杆菌中表达野生型蛋白质,而在酵母(巴斯德毕赤酵母)中表达突变蛋白。
将在PBS中稳定并且与防腐剂相容的本发明突变蛋白表示为与野生型FGF-21相比,具有增强或改进的药物性质。如在表4中所示,与野生型FGF-21相比,具有增强的药物性质的本发明优选的突变蛋白是L118C-A134C和L118C-A134C-S167A。
表4
FGF-21突变蛋白 | 平均颗粒直径(nm)* |
实验#1 | |
野生型FGF-21 | 1356 |
实验#2 | |
野生型FGF-21 | 813 |
L118C-A134C | 7 |
L118C-A134C-S167A | 7 |
*平均颗粒直径代表在37℃温育2小时后,在靶标浓度10mg/ml、m-甲酚3mg/ml下的蛋白质溶解(solution)。
实施例5
O-糖基化分析
将FGF-21突变蛋白在巴斯德毕赤酵母中表达,并且通过使用40℃下的Zorbax,330-SB C8、4.6×50mm、3.5m颗粒柱(移动相C:10%ACN和90%H2O中的0.1%TFA,D:CAN中的0.1%TFA)的HPLC(Waters 2695)从培养基肉汤中纯化出来。
通过标准LC/MS分析测量纯化的FGF-21突变蛋白的O-糖基化水平。在表5中显示了与人野生型FGF-21相比,代表性突变蛋白O-糖基化的百分数。与野生型FGF-21或突变蛋白L118C-A134C的>60%的O-糖基化水平相比,优选的突变蛋白L118C-A134C-S167A的O-糖基化水平仅为3%,这明确证明了S167突变蛋白显著性减少了O-糖基化水平。
表5
FGF-21突变蛋白 | %O-糖基化 |
野生型 | 62% |
L118C-A134C | 63% |
L118C-A134C-S167A | 3% |
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Claims (26)
1.人FGF-21的突变蛋白或者其生物学活性肽,其包含除Ser或Thr外的任何氨基酸对Ser167的取代,其中氨基酸的编号基于SEQ ID NO:1,并且其中当在酵母中表达时,所述突变蛋白与野生型人FGF-21相比具有降低的O-糖基化能力。
2.权利要求1的突变蛋白,其中所述突变蛋白选自Ser167Ala、Ser167Glu、Ser167Asp、Ser167Asn、Ser167Gln、Ser167Gly、Ser167Val、Ser167His、Ser167Lys和Ser167Tyr。
3.分离的多核苷酸,其包含权利要求1的突变蛋白的编码核苷酸序列。
4.权利要求3的多核苷酸,其中所述核酸是DNA。
5.包含权利要求4的DNA的载体。
6.包含权利要求5的载体的宿主细胞。
7.产生多肽的方法,其包括从权利要求6的宿主细胞中表达所述DNA编码的多肽。
8.用于治疗表现出肥胖症、II型糖尿病、抗胰岛素性、高胰岛素血症、葡萄糖不耐受、高血糖症或代谢综合征中的一种或多种的患者的药物组合物,其包括下列:
(a)治疗有效量的权利要求1的FGF-21突变蛋白;以及
(b)可药用的载体。
9.治疗表现出肥胖症、II型糖尿病、抗胰岛素性、高胰岛素血症、葡萄糖不耐受、高血糖症或代谢综合征中的一种或多种的患者的方法,其包括向需要此类治疗的所述患者施用治疗有效量的权利要求1的FGF-21突变蛋白。
10.人FGF-21的突变蛋白或者其生物学活性肽,其包含除Ser或Thr外的任何氨基酸对Ser167的取代,以及半胱氨酸对下列两个或多个氨基酸的取代:精氨酸19、酪氨酸20、亮氨酸21、酪氨酸22、苏氨酸23、天冬氨酸24、天冬氨酸25、丙氨酸26、谷氨酰胺27、谷氨酰胺28、丙氨酸31、亮氨酸33、异亮氨酸35、亮氨酸37、缬氨酸41、甘氨酸42、甘氨酸43、谷氨酸50、谷氨酰胺54、亮氨酸58、缬氨酸62、亮氨酸66、甘氨酸67、赖氨酸69、精氨酸72、苯丙氨酸73、谷氨酰胺76、精氨酸77、天冬氨酸79、甘氨酸80、丙氨酸81、亮氨酸82、甘氨酸84、丝氨酸85、脯氨酸90、丙氨酸92、丝氨酸94、苯丙氨酸95、亮氨酸100、天冬氨酸102、酪氨酸104、酪氨酸107、丝氨酸109、谷氨酸110、脯氨酸115、组氨酸117、亮氨酸118、脯氨酸119、天冬酰胺121、赖氨酸122、丝氨酸123、脯氨酸124、组氨酸125、精氨酸126、天冬氨酸127、丙氨酸129、脯氨酸130、甘氨酸132、丙氨酸134、精氨酸135、亮氨酸137、脯氨酸138或亮氨酸139,其中氨基酸的编号基于SEQ ID NO:1,并且其中当在酵母中表达时,所述突变蛋白与野生型人FGF-21相比具有降低的O-糖基化能力。
11.分离的多核苷酸,其包含权利要求10的突变蛋白的编码核苷酸序列。
12.权利要求11的多核苷酸,其中所述核酸是DNA。
13.包含权利要求12的DNA的载体。
14.包含权利要求13的载体的宿主细胞。
15.产生多肽的方法,其包括从权利要求14的宿主细胞中表达所述DNA编码的多肽。
16.用于治疗表现出肥胖症、II型糖尿病、抗胰岛素性、高胰岛素血症、葡萄糖不耐受、高血糖症或代谢综合征中的一种或多种的患者的药物组合物,其包括下列:
(a)治疗有效量的权利要求10的人FGF-21突变蛋白;以及
(b)可药用的载体。
17.治疗表现出肥胖症、II型糖尿病、抗胰岛素性、高胰岛素血症、葡萄糖不耐受、高血糖症或代谢综合征中的一种或多种的患者的方法,其包括向需要此类治疗的所述患者施用治疗有效量的权利要求10的人FGF-21突变蛋白。
18.人FGF-21的突变蛋白或者其生物学活性肽,其包含除Ser或Thr外的任何氨基酸对Ser167的取代,以及带电的和/或极性却不带电的氨基酸对一个或多个以下位置上的氨基酸的取代:甘氨酸42、谷氨酰胺54、精氨酸77、丙氨酸81、亮氨酸86、苯丙氨酸88、赖氨酸122、组氨酸125、精氨酸126、脯氨酸130、精氨酸131、亮氨酸139、丙氨酸145、亮氨酸146、异亮氨酸152;丙氨酸154;谷氨酰胺156、甘氨酸161、丝氨酸163、甘氨酸170或丝氨酸172,其中氨基酸的编号基于SEQ ID NO:1,并且其中当在酵母中表达时,所述突变蛋白与野生型人FGF-21相比具有降低的O-糖基化能力。
19.分离的多核苷酸,其包含权利要求18的突变蛋白的编码核苷酸序列。
20.权利要求19的多核苷酸,其中所述核酸是DNA。
21.包含权利要求20的DNA的载体。
22.包含权利要求21的载体的宿主细胞。
23.产生多肽的方法,其包括从权利要求22的宿主细胞中表达所述DNA编码的多肽。
24.用于治疗表现出肥胖症、II型糖尿病、抗胰岛素性、高胰岛素血症、葡萄糖不耐受、高血糖症或代谢综合征中的一种或多种的患者的药物组合物,其包括下列:
(a)治疗有效量的权利要求18的人FGF-21突变蛋白;以及
(b)可药用的载体。
25.治疗表现出肥胖症、II型糖尿病、抗胰岛素性、高胰岛素血症、葡萄糖不耐受、高血糖症或代谢综合征中的一种或多种的患者的方法,其包括向需要此类治疗的所述患者施用治疗有效量的权利要求18的人FGF-21突变蛋白。
26.权利要求1、10或18中任一项的突变蛋白,其中所述突变蛋白在N端截短多达4个氨基酸。
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CN102625811A (zh) * | 2008-10-10 | 2012-08-01 | 安姆根有限公司 | Fgf21突变体及其用途 |
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EP1789442B1 (en) | 2009-09-30 |
US7582607B2 (en) | 2009-09-01 |
HRP20090597T1 (hr) | 2009-12-31 |
KR20070050454A (ko) | 2007-05-15 |
PT1789442E (pt) | 2009-11-11 |
WO2006028595A2 (en) | 2006-03-16 |
ATE444307T1 (de) | 2009-10-15 |
IL181573A0 (en) | 2007-07-04 |
EA200700533A1 (ru) | 2007-08-31 |
NO20071327L (no) | 2007-05-29 |
EP2161281A1 (en) | 2010-03-10 |
JP4809352B2 (ja) | 2011-11-09 |
WO2006028595A3 (en) | 2007-01-18 |
MX2007002616A (es) | 2007-05-16 |
SI1789442T1 (sl) | 2010-01-29 |
CA2575753A1 (en) | 2006-03-16 |
AU2005283025A1 (en) | 2006-03-16 |
DK1789442T3 (da) | 2009-11-16 |
PL1789442T3 (pl) | 2010-02-26 |
KR100854198B1 (ko) | 2008-08-26 |
US20080103096A1 (en) | 2008-05-01 |
EA011390B1 (ru) | 2009-02-27 |
JP2008511323A (ja) | 2008-04-17 |
CN101010338B (zh) | 2011-06-15 |
ES2332057T3 (es) | 2010-01-25 |
EP1789442A2 (en) | 2007-05-30 |
DE602005016946D1 (de) | 2009-11-12 |
AU2005283025B2 (en) | 2011-06-30 |
BRPI0514790A (pt) | 2008-06-24 |
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