CN103906530B - 成纤维细胞生长因子21变体 - Google Patents
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Abstract
本发明涉及药理学上有效力的和/或稳定的人成纤维细胞生长因子21(FGF21)的变体,含有FGF21变体的药物组合物,以及使用该变体用于治疗2型糖尿病、肥胖、异常脂血症、或代谢综合征、或其任意组合的方法。
Description
本发明涉及人成纤维细胞生长因子21(FGF21)的变体,含有FGF21变体的药物组合物,以及用于治疗2型糖尿病、肥胖、异常脂血症、或代谢综合征、或其任意组合的方法。
FGF21是一种激素,其作为葡萄糖和脂质内稳态的重要代谢调节物发挥作用。通过上调GLUT1表达,FGF21促进脂肪细胞中的葡萄糖摄取,其机制不同于胰岛素。在糖尿病啮齿动物和猴子中,人FGF21降低葡萄糖空腹血清浓度,并降低甘油三酯、胰岛素和胰高血糖素的空腹血清浓度。此外,在饮食诱导的肥胖啮齿类动物模型中,施用FGF21导致累计体重呈剂量依赖性丧失。因此,FGF21具有用于治疗糖尿病、肥胖、异常脂血症、代谢综合征的潜在效用。
在WO2010/065439、WO2006/028595和WO2005/061712中已经描述了人FGF21的变体。
与人野生型FGF21和已知的人FGF21变体相关的问题是分子的低的药理学效力和/或低的药物稳定性。因此,仍然需要一种有效和/或稳定的替代性FGF21变体。
本发明提供了替代性的人FGF21变体,其具有人野生型FGF21和本领域公开的已知的人FGF21变体没有的优点。这些优点包括改善的药理学效力和/或改善的药物稳定性。本发明的某些FGF21变体具有一种或多种有利的生理化学特征,这些特征对作为治疗性蛋白质的有效制备和/或制剂是有用的,包括在体内降低蛋白质水解降解、降低对氧化的易感性、减少在高浓度聚集的倾向、降低在哺乳动物细胞系统中生产期间的翻译后修饰水平、增加与某些防腐剂的相容性、和/或改善化学稳定性。另外,本发明的FGF21变体对2型糖尿病、肥胖、异常脂血症、或代谢综合征、或其任意组合的治疗具有潜在的作用。
本发明提供了人成纤维细胞生长因子21(FGF21)的变体,其中氨基酸序列是
HPIPDSSPLLQFGGQVRQRYLYTDDAQQTECHLEIREDGTVGCAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFREX1LLEDGYNVYQSEAHGLX2LHLPGDKSPHRKPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPX3X4LVX5PSQLLSPSFLG(SEQIDNO:13)
其中X1是L或D,X2是P或W,X3是L或Y,X4是S或R,且X5是G或E。
本发明提供了人成纤维细胞生长因子21(FGF21)的变体,其中氨基酸序列是
HPIPDSSPLLQFGGQVRQRYLYTDDAQQTECHLEIREDGTVGCAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFREDLLEDGYNVYQSEAHGLPLHLPGDKSPHRKPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLRLVEPSQLLSPSFLG(SEQIDNO:1)。
本发明还提供了药物组合物,其包含如文中所述本发明的人FGF21变体、和至少一种可药用的载体、稀释剂或赋形剂,以及任选地其他治疗成分。
本发明还提供了治疗患者2型糖尿病、肥胖、异常脂血症、或代谢综合征、或其任意组合的方法,包括向所述患者施用如文中所述的本发明的人FGF21变体。
本发明还提供了治疗患者2型糖尿病、肥胖、异常脂血症、或代谢综合征、或其任意组合的方法,包括向所述患者施用如文中所述的本发明的药物组合物。
进一步地,本发明提供了如文中所述的本发明的人FGF21变体用于治疗。优选地,本发明提供了如文中所述的本发明的人FGF21变体用于治疗2型糖尿病、肥胖、异常脂血症、或代谢综合征、或其任意组合。
进一步地,本发明提供了如文中所述的本发明的人FGF21变体在制备用于治疗2型糖尿病、肥胖、异常脂血症、或代谢综合征、或其任意组合的药物中的用途。
全长人野生型FGF21是包含27个氨基酸信号肽的208个氨基酸的多肽。成熟人野生型FGF21包含不具有27个氨基酸信号肽的全长多肽,因而其为181个氨基酸的多肽(SEQIDNO:2)。本发明FGF21变体中氨基酸位置的改变由成熟人野生型FGF21(SEQIDNO:2)多肽中的氨基酸位置决定。因此,文中描述的取代“A31C”指在成熟人野生型FGF21蛋白质的位置31处由氨基酸Cys取代野生型氨基酸Ala。
需要注意的是在特定变体中一个氨基酸残基的取代可以影响变体作为一个整体的特征,其总体效果可能对药理学效力和/或药物稳定性有益或有害。例如,一个氨基酸取代,P115W,增加FGF21变体的效力,但是,也认为P115W导致引起聚集的自身相互作用(参见实施例5)。因而,其总体效果对变体是有害的,所以,P115W取代不包含在本发明优选的FGF21变体中。
某些本发明的人FGF21变体是有效的生物活性蛋白质,如实施例2和3中所示的SEQIDNO:1。本发明优选的FGF21变体包含一些氨基酸取代,所有这些取代不仅仅改善药理学效力,而且与其他氨基酸改变是相容的,这反过来提供了改善的生理化学性质,并增加了体内稳定性。在本发明的优选FGF21变体中改善效力的一组氨基酸取代包含D127K、S167R、G174L、R175L和A180L(参见,实施例2和3)。
人野生型FGF21的浓缩蛋白质溶液暴露于药物防腐剂、例如间甲酚时,增加蛋白质形成聚集体的倾向。通过引入额外的二硫键产生的结构稳定性改善了人野生型FGF21的防腐剂相容性和热稳定性。本发明的FGF21变体并入了氨基酸取代A31C和G43C,其极大地改善了热稳定性和防腐剂相容性,而不影响生物活性。之前也曾经描述过包含A31C/G43C取代的高效力FGF21变体。那些报道的变体与野生型FGF21相比,呈现显著改善的防腐剂相容性,但是其仍然容易聚集。已知聚集将增加免疫原性风险,因而降低了这样的变体作为治疗性蛋白质的可接受性。
为了尽量减少该有害的聚集,本发明优选的变体包含氨基酸取代L98D,其导致高浓度下高分子量聚集体形成的显著减少(参见实施例5)。有利地,氨基酸取代L98D不降低变体的效力。
用于制备本发明FGF21变体的优选的商售表达系统是哺乳动物CHO-K1细胞系。然而,哺乳动物细胞系CHO-K1和HEK293可能通过位置179的酪氨酸侧链硫酸盐化作用引起成熟的人野生型FGF21翻译后修饰。在位置179和180(如果存在的话)的酪氨酸残基的硫酸盐化作用降低了效力,并且是不想要的产品异质性的来源。因此,当在位置179和/或180具有Tyr的FGF21蛋白质从CHO-K1或HEK293细胞系表达时,一定比例的表达蛋白质可能在位置179被硫酸盐化,另一些可能在位置180被硫酸盐化,而另一些可能在两个位置均被硫酸盐化,而另一些在任何位置均未被硫酸盐化。这导致了效力降低的异质的且不可预知的蛋白质群。
通过向变体引入氨基酸取代Y179F,本发明的FGF21变体解决了该有害的硫酸盐化作用。Y179F消除了在CHO-K1和HEK293细胞中生产产生的硫酸盐化作用(参见实施例4)。而且,氨基酸取代Y179F与本发明其他有利的氨基酸取代是相容的,且确认了其不影响效力。
人野生型FGF21易感于体内的蛋白质水解降解。小鼠或食蟹猴静脉内或皮下注射后,从血清回收的主要蛋白质水解片段是中止于位置171的片段。在之前的体外效力测定中,确定了跨越残基1至171的FGF21片段效力低~100倍。消除该蛋白质水解切割位点通过增加活性药物的暴露而改善了药效。表明氨基酸取代G170E在小鼠中显著减缓切割(数据未显示),且表明在食蟹猴中在24小时后实质上消除了在位置171处的蛋白质水解(参见实施例6)。G170E取代不影响效力,且与期望的生理化学稳定性特性相容。因此,氨基酸取代G170E并入本发明优选的FGF21变体中。
人野生型FGF21对CHO-K1制备中产生的羧肽酶易感,氨基酸取代S181G减缓了该过程,因而降低了所表达的蛋白质长度的异质性(即,在由细胞系表达的成熟蛋白质中氨基酸残基数目的异质性)。尽管氨基酸取代S181G并不能消除哺乳动物细胞表达中的C末端蛋白质水解,其非常有效地减缓蛋白质水解,并同时维持本发明FGF21变体中存在其它所需氨基酸取代的条件下所期望的效力。鉴于该有利的特征,氨基酸取代S181G并入本发明的FGF21变体。
本发明还包含编码上述变体的多核苷酸,其可以是RNA形式,或DNA形式,其中DNA包括cDNA和合成的DNA。DNA可以是双链或单链。由于遗传密码子的冗余或简并,编码本发明变体的编码序列可以不同。
编码本发明变体的多核苷酸可以包含如下:仅仅编码变体的序列,编码变体的序列和编码其他(如功能多肽)的序列,或引导序列或分泌序列或前蛋白质序列;变体的编码序列和非编码序列,如内含子或变体编码序列的5’和/或3’非编码序列。因此,术语“编码变体的多核苷酸”可不仅仅包括变体编码序列的多核苷酸,还包括其他的编码序列和/或非编码序列的多核苷酸。
序列被有效地连接至表达调控序列后,本发明的多核苷酸在宿主细胞中表达。表达载体通常作为附加体或作为宿主染色体DNA的整体部分在宿主生物体中复制。一般地,该表达载体含有选择标记物,例如,四环素、新霉素、和二氢叶酸还原酶,以允许选择被期望DNA序列转化的那些细胞。
本发明的FGF21变体可以容易地在如下细胞中生产:在哺乳动物细胞中如CHO、NS0、HEK293或COS细胞;在细菌细胞中如大肠杆菌、枯草芽孢杆菌(Bacillussubtilis)、荧光假单胞菌(Pseudomonasfluorescence);或在真菌或酵母细胞中。使用本领域已知的技术培养宿主细胞。优选的哺乳动物宿主细胞是含有谷氨酰胺合成酶(GS)表达系统的CHOK1SV细胞系(参见US5,122,464)。
可以通过熟知的方法将含有目的多核苷酸序列(例如FGF21变体和表达调控序列)的载体转移至宿主细胞中,这样的方法取决于宿主细胞的类型。例如,氯化钙转染通常用于原核细胞,而磷酸钙处理或电穿孔可用于其他宿主细胞。
可采用多种蛋白质纯化的方法,这些方法是本领域公知的,例如,在Deutscher,MethodsinEnzymology182:83-89(1990)和Scopes,ProteinPurification:PrinciplesandPractice,3rdEdition,Springer,NY(1994)中描述的。
可通过本领域已知的任何方法施用本发明的FGF21变体药物组合物,实现通常预期的治疗2型糖尿病、肥胖、异常脂血症、或代谢综合征、或其任意组合的目的。优选的给药途径是胃肠外途径。给药剂量取决于接收者的年龄、健康状态和体重,如果有,还取决于同时治疗的种类,治疗的频率,以及期望效果的性质。典型的剂量水平可使用标准临床技术优化,其取决于给药模式和患者状态,可由具有本领域常规技术的人员确定。
根据已知的方法配制本发明的FGF21变体,以制备可药用的组合物。期望的制剂是稳定的冻干产品,其与合适的高纯度稀释剂或水性溶液重建,所述高纯度稀释剂或水性溶液任选带有药学上可接受的载体、防腐剂、赋形剂或稳定剂[Remington,TheScienceandPracticeofPharmacy,19thedition,Gennaro,ed.,MackPublishingCo.,Easton,PA1995]。
本发明的FGF21变体可以与药学上可接受的缓冲液组合,调节pH以提供可接受的稳定性,以及给药可接受的pH。另外,本发明的FGF21变体可放置于容器内,如药瓶、药筒、笔递送装置、注射器、静脉内给药管或静脉内给药袋,其中容器是单位剂量容器。
术语“异常脂血症”指脂蛋白质代谢紊乱,包括脂蛋白质过量产生或不足。异常脂血症可表现为血液中总胆固醇、低密度脂蛋白质(LDL)胆固醇和甘油三酯浓度的升高和/或高密度脂蛋白质(HDL)胆固醇浓度的降低。
术语“代谢综合征”的特征在于一个个体中的一组代谢风险因子。它们包括:腹部肥胖-在大多数男人中,有40英寸或更大的腰围;高血糖-禁食后至少110毫克每分升(mg/dl);高甘油三酯-血流中至少150mg/dL;低HDL-少于40mg/dl;和/或血压130/85或更高。
术语“肥胖”定义为其中与瘦体重相比具有过量皮下脂肪的状态(Stedman’sMedicalDictionary28thedition,2006,LippincottWilliams&Wilkins)。
“患者”是哺乳动物,优选人。
术语“治疗”指减缓、降低或逆转症状、紊乱、状态或疾病的进展或严重性。
术语“治疗有效量”指本发明FGF21变体的量或剂量,其通过单次或多次剂量给药于患者提供期望的治疗。
术语“2型糖尿病”特征在于尽管有可利用的胰岛素,仍然产生过量的葡萄糖,且由于葡萄糖清除不足导致循环的葡萄糖水平持续过高。
以下的实施例基本上如下述进行。
实施例1
CHOK1SV细胞中FGF21变体的表达
使用中国仓鼠卵巢(CHOK1SV)细胞在哺乳动物细胞表达系统中产生本发明的FGF21变体。将编码FGF21变体的基因亚克隆至含有谷氨酰胺合成酶(GS)的表达质粒骨架中(基于pEE12.4的质粒)。编码FGF21变体的cDNA序列与优选信号肽序列的编码序列符合读框的融合,以增加期望的产品分泌到组织培养基中。优选信号肽序列是如氨基酸序列SEQIDNO:3、SEQIDNO:4、SEQIDNO:5或SEQIDNO:6所示的多肽。
由巨细胞病毒(CMV)启动子驱动表达。使用电穿孔以及适当量的重组表达质粒稳定转染CHOK1SV细胞,转染的细胞以足够的细胞密度维持在悬浮培养中。通过在含有甲硫氨酸砜亚胺(methioninesulfoximine,MSX)、不含血清的培养基中生长完成转染细胞的选择,并将其孵育在35-37℃和5-7%CO2中。
使用流式细胞仪产生克隆来源的细胞系。在哺乳动物细胞中表达的FGF21变体通常产生天然的N末端序列,HPIP,即,在N末端没有甲硫氨酸残基,如氨基酸序列SEQIDNO:1所示的FGF21变体。
使用如下方法纯化从CHO细胞分泌至培养基的FGF21变体,其中在该方法中将澄清的细胞培养基加热至50-60℃,维持至多2小时,冷却,用洗涤剂(TritonX-100)处理失活病毒,并将其加载至CaptoMMC(GEHealthcare)混合模式层析柱。使用pH8的缓冲液从柱中洗脱FGF21变体,使用50mM柠檬酸、150mMNaCl溶液将洗脱后汇集的产品调节至3.2至3.5的pH范围,持续1小时使病毒失活。通过加入Tris缓冲液调节溶液pH至6.7到7.3之间,使用苯基琼脂糖高性能树脂(GEHealthcare)通过疏水性交换层析进一步纯化FGF21变体。在pH7下,用递减梯度的硫酸钠洗脱该疏水性相互作用柱。将HIC纯化的FGF21变体的缓冲液交换为pH8的含NaCl的Tris缓冲液,并由Source30Q树脂(GEHealthcare)上的阴离子交换层析进一步纯化。用pH8的递增浓度的氯化钠洗脱阴离子交换柱。使纯化的FGF21变体通过Planova20N(AsahiKaseiMedical)病毒滞留过滤器,而后在再生纤维素膜(Millipore)上通过切向流超滤浓缩/渗滤至pH为7的10mM柠檬酸、150mMNaCl中。
实施例2
3T3-L1-βKlotho成纤维细胞的葡萄糖摄取测定
3T3-L1-βKlotho成纤维细胞由3T3-L1成纤维细胞通过逆转录病毒转导CMV驱动的哺乳动物表达载体产生,所述CMV驱动的哺乳动物表达载体含有野生型小鼠βKlotho和杀稻瘟菌素抗性标记物的编码序列。在15μM杀稻瘟菌素存在下,生长14天后选择杀稻瘟菌素抗性细胞,应用抗βKlotho抗体通过免疫印迹证实βKlotho蛋白质表达。在含有10%小牛血清和15μM杀稻瘟菌素的Dulbecco'sModifiedEagle培养基(DMEM)中维持3T3-L1-βKlotho成纤维细胞,直至铺板用于实验用途。
对于葡萄糖摄取,3T3-L1-βKlotho成纤维细胞以20,000个细胞/孔铺板于96孔平板,在含有10%小牛血清的DMEM中孵育48小时。在含有或不含有目的FGF21变体、含有0.1%牛血清白蛋白质(BSA)的DMEM中孵育细胞3小时,而后在含有或不含有FGF21变体、含有100μM2-脱氧-D-(14C)葡萄糖的Krebs-Ringer磷酸盐(KRP)缓冲液(15mMHepes,pH7.4,118mMNaCl,4.8mMKCl,1.2mMMgSO4,1.3mMCaCl2,1.2mMKH2PO4,0.1%BSA)中孵育1小时。通过在含有1mM2-脱氧-D-(14C)葡萄糖的Krebs-Ringer重碳酸盐/Hepes(KRBH)缓冲液中孵育选择的孔确定非特异结合。通过向细胞加入20μM细胞松弛素B中止反应,使用液体闪烁计数器测量葡萄糖摄取。
在3T3-L1-βKlotho成纤维细胞的葡萄糖摄取测定中SEQIDNO:1的FGF21变体的体外效力是0.026nM。与已知的SEQIDNO:11的FGF21变体(公开于WO2006/028595)比较时,SEQIDNO:1的FGF21变体是有效的FGF21变体。在3T3-L1-βKlotho成纤维细胞的葡萄糖摄取测定中SEQIDNO:11的FGF21变体的体外效力是0.49nM。
实施例3
人293细胞βKlotho-SRE荧光素酶测定
293-βKlotho-SRE荧光素酶受体细胞的构建:
HEK293细胞(人胚肾细胞)培养在37℃,5%CO2下、含有10%胎牛血清(FBS)的Dulbecco'smodifiedEagle's的生长培养基中。用含有CMV启动子驱动的人βKlotho表达盒的质粒和含有血清反应元件(SRE)驱动的荧光素酶表达盒的质粒共同转染细胞。βKlotho表达质粒还含有SV40启动子驱动的新霉素磷酸转移酶表达盒,以提供对氨基糖苷类抗生素G418的抗性。用600μg/mLG418选择转染的HEK-293细胞,以选择其中转染的质粒被整合至基因组的细胞。选择的细胞被稀释克隆,并在添加FGF2124小时后测试荧光素酶产量的增加。选择具有最大FGF21依赖的荧光素酶增加的克隆作为用于测量相对FGF21变体活性的细胞系。
293-βKlotho-SRE荧光素酶FGF21活性测定:
漂洗293-βKlotho-SREluc细胞,将其置于CD293悬浮培养基中(Invitrogen)。在37℃,6%CO2,125rpm下、细胞在悬浮液中过夜生长。计数细胞、通过离心沉淀、并再悬浮于含有0.1%BSA的CD293培养基中。以每孔25,000个细胞将细胞放置于白色96孔平板。在CD293/0.1%BSA中制备各FGF21变体的四倍系列稀释液,以生成终浓度从100nM至0.006nM的8个稀释液。一式三份将稀释液加入细胞中,在37℃,5%CO2下孵育16-20小时。通过加入等体积OneGloTM荧光素酶底物(Promega)并测量相对发光测定荧光素酶水平。使用四参数logistic模型(XLfit版本5.1)分析数据,以拟合曲线并确定EC50。
在人293细胞βKlotho-SRE荧光素酶测定中,SEQIDNO:1的FGF21变体的体外效力是0.25nM。与已知的SEQIDNO:11的FGF21变体(公开于WO2006/028595)比较时,SEQIDNO:1的FGF21变体是有效的FGF21变体。在人293细胞βKlotho-SRE荧光素酶测定中SEQIDNO:11的FGF21变体的体外效力是22.39nM。
实施例4
在哺乳动物细胞生产期间酪氨酸的硫酸盐化作用
人野生型FGF21在CHOK1SV细胞中的哺乳动物蛋白质表达期间容易在位置179发生酪氨酸硫酸盐化作用(数据未表示)。该硫酸盐化作用导致产品异质性,这意味着产生不同形式的蛋白质(即,具有硫酸盐化和不具有非硫酸盐化)。产品同质化是期望的生物药学产品属性。发生在治疗性蛋白质生产期间的翻译后修饰并不是期望的,因为该修饰可能导致活性或其他生物药学性质的不同。
为了评估FGF21变体是否被硫酸盐化,1μL样品的等分试样与99μL0.1%三氟乙酸(TFA)混合。使用以下条件通过液体层析质谱(LC-MS)法分析样品:流动相A为0.1%TFA/10%乙腈,流动相B为0.1%TFA的乙腈溶液,柱是C8柱,3.5μm2.1×150mm,带有2.1×12.5mmC8防护装置,注射体积是12-20μL,其取决于样品浓度,以便注入大约1μg的蛋白质。
表1:液体层析分离的梯度条件
时间(min) | 0 | 12 | 15 | 15.1 | 20 | 21.1 | 30 |
%B | 10 | 50 | 60 | 90 | 90 | 10 | 10 |
流量(μL/min) | 200 | 200 | 200 | 200 | 200 | 200 | 200 |
WatersMicromassLCTPremierTM质谱仪设置为质谱范围400到1990amu之间、极性ES+、毛细管2000、采样锥40V、孔径1为30V、源温度为105℃、锥气流50L/小时、脱溶剂气温度(desolvationtemperature)为300℃、以及脱溶剂气流量(desolvationgasflow)是600L/小时。
表2:SEQIDNO:1的FGF21变体的LC/MS特征
产品 | 预期质谱 | 观察到的质谱 | %误差 | Rel% |
1-181 | 19633.3 | 19633.0 | 0.002 | 33.8 |
1-180 | 19576.2 | 19576.1 | 0.001 | 66.2 |
如表2所见,SEQIDNO:1的FGF21变体的预期质谱(没有硫酸盐化)几乎与其观察到的质谱相同,表明在SEQIDNO:1的FGF21变体中没有检测到硫酸盐化作用。因此,氨基酸取代Y179F防止了在SEQIDNO:1的FGF21变体位置179上发生硫酸盐化作用。该结果提供了更同质化的产品,使其作为治疗性蛋白质产品更能被接受。
实施例5
P115W促进聚集,而L98D增加制剂中的物理稳定性并增加与苯甲
醇的相容性
为了测量蛋白质自缔合和聚集的量,使用分析尺寸排阻层析(SEC)法测量高分子量(%HMW)聚集体的百分比。通过SEC表征蛋白质初始存储溶液以确定HMV的起始水平(表3)。
表3
FGF21变体 | HMW的起始水平(%) |
SEQ ID NO:10的FGF21变体 | 3.2 |
SEQ ID NO:1的FGF21变体 | 0.21 |
SEQ ID NO:8的FGF21变体 | 3.6 |
SEQ ID NO:9的FGF21变体 | 0.12 |
各蛋白质的样品通过在4℃下在样品缓冲液(在表4-6中所述)中过夜透析(使用具有分子量截留值10,000道尔顿的透析盒),浓度为2mg/mL。透析后,样品无菌过滤(0.22μm的膜),并通过280nm处的吸光度定量。然后,使用10,000MW截留值的离心过滤器在3000rpm在4℃下将样品浓缩至≥60mg/mL的目标浓度。浓缩样品后,在280nm吸光度处定量蛋白质浓度,使用SEC法测定%HMW。
SEC法使用具有30cm×0.78cm尺寸的TosoHaas模式G2000SWXL柱。流动相是0.1M的磷酸钠,pH7.4,流速为0.5mL/分钟。低浓度样品作为10μL注射液加样,并在214nm处的吸光度波长下监测,而浓缩样品作为1μL注射液加样,并在280nm处监测。
表4报道了FGF21变体SEQIDNO:8和SEQIDNO:9浓缩溶液中的蛋白质浓度和%HMW。各变体以单体(非聚集形式)存在的百分比没有列于表中,但是其等于100%减去报道的%HMW。SEQIDNO:8的FGF21变体和SEQIDNO:9的FGF21变体仅仅在氨基酸位置115处不同,SEQIDNO:8的FGF21变体在115位具有增强效力的残基色氨酸(P115W),而SEQIDNO:9的FGF21变体含有野生型残基脯氨酸(P115P)。在多种制剂缓冲液条件下,SEQIDNO:8(P115W)的FGF21变体的%HMW显著高于SEQIDNO:9的FGF21变体,这证明了在位置115具有色氨酸对促进聚集和自缔合的因果作用。同样地,与在位置115上含有氨基酸脯氨酸的SEQIDNO:1的FGF21变体相比,在位置115上含有色氨酸残基的SEQIDNO:10的FGF21变体实质上具有增加的%HMW(表5)。
表4:由SEC测量的聚集倾向
表5:由SEC测量的聚集倾向
在SEC测定中将物理稳定性和与防腐剂水平浓度为0.9%的苯甲醇的相容性测量为%HMW,其中在存在或不存在0.02%Tween-80时、在具有150mMNaCl的pH为7.0的10mM组氨酸缓冲液中监测。样品制备为30mg/mL,并孵育在4℃、25℃以及40℃下,4周。通过SEC方法分析新鲜配制的FGF21变体(即,在时间点0)和那些孵育4周的变体的%HMW。表6总结了分析结果,比较了时间点0的样品(“初始”)和那些在40℃下孵育4周的样品。
表6:在30mg/mL制剂浓度下的聚集倾向和防腐剂相容性
SEQIDNO:1的FGF21变体含有氨基酸取代L98D。SEQIDNO:9的FGF21变体不含有氨基酸取代L98D,实际上其在位置98含有野生型氨基酸亮氨酸。当在配制条件下以30mg/mL配制各蛋白质时,观察到了氨基酸取代L98D的益处(表6)。在所有检测条件下,在40℃下应激FGF21变体4周使SEQIDNO:9的FGF21变体的%HMW基本上高于SEQIDNO:1的FGF21变体。而且,加入用于多种药物制备应用的普通防腐剂,0.9%苯甲醇,加重了SEQIDNO:9的FGF21变体%HMW的增加,但是并没有影响SEQIDNO:1的FGF21变体的%HMW。这种与苯甲醇的不相容性还在初始样品制备的分析中观察到,其中在存在0.9%苯甲醇时%HMW是11%,而在不存在苯甲醇时,其仅为0.97%。SEQIDNO:9的FGF21变体和SEQIDNO:1的FGF21变体均不含有P115W残基,因此,这些条件下差的物理稳定性不能归因于P115W。在制造了氨基酸取代L98D后,在0.9%苯甲醇存在时观察到了增强的物理稳定性。
这些数据表明某些取代能影响蛋白质整体的稳定性,其归因于聚集成高分子量的种类,特别是在某些防腐剂如苯甲醇存在时。对于治疗性蛋白质优选最小化这些HMW聚集体。其可通过FGF21变体蛋白质中的某些取代实现,如SEQIDNO:1所示的FGF21变体中的L98D取代。其他的取代,如P115W,可具有不利作用,如增加变体中聚集体的水平。
实施例6
体内蛋白质水解降解
雄性食蟹猴,n=2/组,一次皮下注射2mg/kg剂量的SEQIDNO:1的FGF21变体。在时间进程中收集血清(0.25至12小时后抽取(withdrawn),用于通过质谱法进行的24小时体内蛋白质水解评估,以定量活性化合物的量。
进行液相层析质谱(LC/MS)分析。使用共价结合到磁珠上的抗-FGF21单克隆抗体免疫沉淀每个血清样品的100μL等分试样。免疫沉淀的样品被分成单独的等分试样,使得检测完整蛋白质和胰蛋白酶消化的蛋白质。将完整蛋白质注射到Biowide孔柱,其内径为100×0.32mm,含涂层有C5的3μm的颗粒。胰蛋白酶消化的样品被注入到DiscoveryBiowide孔柱,其内径为100×0.32mm,含涂层有C18的3μm的颗粒。所有注射液的层析条件使用流动相A(0.1/100,甲酸:水)和流动相B(0.1/100,甲酸:乙腈)组成的二元梯度。从LC的流出液直接连接至MicromassQ-Tof质谱仪,用于正离子模式下的质谱检测。使用Masslynx(v4.1)和MaxEnt1反卷积软件采集来自Q-Tof质谱仪的数据。
发现在FGF21蛋白质位置171的切割大于100倍地降低了蛋白质的生物活性。在该位置降低蛋白质水解是可取的,以增加全活性药物的暴露。当在上述的LC/MS方法中分析时,在24小时的评估中,SEQIDNO:1的FGF21变体中没有检测到的蛋白质水解产物。这些数据表明,与不含有氨基酸取代G170E的SEQIDNO:7的FGF21变体相比,在SEQIDNO:1的FGF21变体中,G170E取代减少了雄性食蟹猴中24小时的体内蛋白质水解降解。
实施例7
Ob/ob小鼠模型中葡萄糖的降低
雄性ob/ob(B6.V-Lepob/Lep+/OlaHsd)小鼠和年龄相匹配的ob/m瘦型对照(B6.V-Lep+/OlaHsd)在收到时是7-8周龄,在初始处理时是10-11周龄。在收到时,所有小鼠每3只一笼,并在开始处理前使其适应环境3周。小鼠用PurinaRodentChow5015料饲育,并可自由饮水。小鼠饲养在12小时的亮/暗循环中,环境温度设置为70°F。在开始处理前一天,小鼠禁食2小时,通过尾巴放血将血液样品收集至肝素化毛细管中。用AscensiaContour血液葡萄糖测量仪测量血液葡萄糖水平,使用MesoScale小鼠/大鼠胰岛素测定试剂盒(MesoScaleDiscovery,Gaithersburg,MD)定量血浆胰岛素水平。在处理初始那天(第0天),根据前一天的体重、血葡萄糖、和血浆胰岛素将小鼠分类。用无菌盐水(0.9%NaCl)稀释FGF21变体,并通过微型渗透泵Alzet皮下施用。在第5天,在亮循环开始大约2小时后测量餐后血液葡萄糖和血浆胰岛素水平。在第5天,所有小鼠过夜禁食,并在第6天进行口服葡萄糖耐受实验(OGTT)。小鼠通过剪尾放血至肝素化毛细管,而后口服葡萄糖(2g/kg)。在口服葡萄糖后,在30、60、120分钟再收集血液样品。用来自CaymanChemicals的葡萄糖测定试剂盒测量血浆葡萄糖。在第6天归一化的葡萄糖AUC值上进行四参数logistic回归模型拟合。
在第5天,载体处理的小鼠血糖过高,测得的平均血液葡萄糖水平是240.4±15.0mg/dl(平均值±SEM),而ob/m瘦型对照小鼠血液葡萄糖水平是150.6±6.0mg/dl(平均值±SEM)。SEQIDNO:1的FGF21变体和SEQIDNO:11的FGF21变体均以剂量依赖的方式将血液葡萄糖降低至与ob/m瘦型对照可比较的水平。SEQIDNO:1的FGF21变体的ED50是0.7μg/kg/hr,而SEQIDNO:11的FGF21变体的ED50是3.1μg/kg/hr。SEQIDNO:1的FGF21变体在ob/ob小鼠中降低血液葡萄糖的能力比SEQIDNO:11的FGF21变体大约多4.4倍。因此,与已知的SEQIDNO:11的FGF21变体(公开于WO2006/028595)比较时,SEQIDNO:1的FGF21变体是有效力的FGF21变体。
序列
SEQIDNO:1-FGF21变体
HPIPDSSPLLQFGGQVRQRYLYTDDAQQTECHLEIREDGTVGCAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFREDLLEDGYNVYQSEAHGLPLHLPGDKSPHRKPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLRLVEPSQLLSPSFLG
SEQIDNO:2-野生型FGF21(人(HomoSapiens))
HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS
SEQIDNO:3-人转铁蛋白(hTrf)信号肽
MRLAVGALLVCAVLGLCLA
SEQIDNO:4-人成纤维细胞生长因子结合蛋白-1(hFGFP-1)信号肽
MKICSLTLLSFLLLAAQVLLVEG
SEQIDNO:5-牛溶菌酶信号肽
MKALVILGFLFLSVAVQG
SEQIDNO:6-鼠轻链(mκ)信号肽
METDTLLLWVLLLWVPGSTG
SEQIDNO:7-FGF21变体
HPIPDSSPLLQFGGQVRQRYLYTDDAQQTECHLEIREDGTVGCAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFREDLLEDGYNVYQSEAHGLPLHLPGDKSPHRKPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLRLVGPSQLLSPSFLG
SEQIDNO:8-FGF21变体
HPIPDSSPLLQFGGQVRQRYLYTDDAQQTECHLEIREDGTVGCAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLWLHLPGDKSPHRKPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPYSLVEPSQLLSPSFLG
SEQIDNO:9-FGF21变体
HPIPDSSPLLQFGGQVRQRYLYTDDAQQTECHLEIREDGTVGCAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGDKSPHRKPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPYSLVEPSQLLSPSFLG
SEQIDNO:10-FGF21变体
HPIPDSSPLLQFGGQVRQRYLYTDDAQQTECHLEIREDGTVGCAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLWLHLPGDKSPHRKPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLRLVEPSQLLSPSFLG
SEQIDNO:11-FGF21变体
DSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHCPGNKSPHRDPAPRGPCRFLPLPGLPPALPEPPGILAPQPPDVGSSDPLAMVGPSQGRSPSYAS
SEQIDNO:12-(DNA)FGF21变体
CACCCTATCCCTGACTCCAGCCCTCTGCTGCAGTTTGGCGGACAGGTCCGGCAGCGGTACCTGTACACCGACGACGCCCAGCAGACCGAGTGCCACCTGGAAATCCGGGAGGACGGCACCGTGGGCTGTGCCGCCGACCAGTCCCCTGAGTCCCTGCTGCAGCTGAAGGCCCTGAAGCCTGGCGTGATCCAGATCCTGGGCGTGAAAACCTCCCGGTTCCTGTGCCAGAGGCCTGATGGCGCCCTGTACGGCTCCCTGCACTTCGACCCTGAGGCCTGCTCCTTCCGGGAGGACCTGCTGGAAGATGGCTACAACGTGTACCAGTCCGAGGCTCACGGCCTGCCTCTGCATCTGCCTGGCGACAAGTCCCCCCACCGGAAGCCTGCTCCTAGGGGCCCTGCCAGATTCCTGCCACTGCCTGGCCTGCCTCCAGCTCTGCCTGAGCCTCCTGGCATCCTGGCCCCTCAGCCTCCAGACGTGGGCTCCTCCGACCCTCTGCGGCTGGTCGAGCCTTCCCAGCTGCTGAGCCCTAGCTTCCTGGGC
SEQIDNO:13-FGF21变体-共同
HPIPDSSPLLQFGGQVRQRYLYTDDAQQTECHLEIREDGTVGCAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFREX1LLEDGYNVYQSEAHGLX2LHLPGDKSPHRKPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPX3X4LVX5PSQLLSPSFLG
X1是L或D
X2是P或W
X3是L或Y
X4是S或R
X5是G或E
Claims (3)
1.一种人成纤维细胞生长因子21(FGF21)的变体,其氨基酸序列是
HPIPDSSPLLQFGGQVRQRYLYTDDAQQTECHLEIREDGTVGCAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFREDLLEDGYNVYQSEAHGLPLHLPGDKSPHRKPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLRLVEPSQLLSPSELG(SEQIDNO:1)。
2.一种药物组合物,其含有权利要求1的变体,和至少一种可药用的载体、稀释剂或赋形剂。
3.权利要求1的变体在制备用于治疗2型糖尿病、肥胖、异常脂血症、或代谢综合征、或其任意组合的药物中的用途。
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