CN107698684A - 包含突变的免疫球蛋白Fc部分的GLP‑1融合蛋白 - Google Patents
包含突变的免疫球蛋白Fc部分的GLP‑1融合蛋白 Download PDFInfo
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- CN107698684A CN107698684A CN201710630742.6A CN201710630742A CN107698684A CN 107698684 A CN107698684 A CN 107698684A CN 201710630742 A CN201710630742 A CN 201710630742A CN 107698684 A CN107698684 A CN 107698684A
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Abstract
本发明涉及包含GLP‑1或其类似物、肽接头和突变的免疫球蛋白Fc部分的融合蛋白,其具有增加的半衰期。本发明还提供生产所述融合蛋白的方法和所述融合蛋白在制备药物中的应用。
Description
技术领域
本发明涉及胰高血糖素样肽-1(glucagon-like peptide-1,GLP-1)的融合蛋白,其包含突变的免疫球蛋白Fc部分,因而具有延长的体内半衰期。该融合蛋白可用于治疗糖尿病、肥胖及其他相关疾病或病症。
背景技术
胰高血糖素样肽-1(glucagon-like peptide-1,GLP-1)最初是从肠黏膜中被分离提取出来,是回肠内分泌细胞分泌的一种脑肠肽,目前主要作为2型糖尿病药物作用的靶点。由于GLP-1可抑制胃排空,减少肠蠕动,故有助于控制摄食,减轻体重。
肠道最初产生的GLP-1是37肽,是无活性的肽链,需酶解切除N端6肽,成为具有生物活性的GLP-1(7~37),其C末端甘氨酸可以作为酰胺化酶的底物,这样,肠道中天然产生的GLP-1有80%左右为GLP-1(7~36)酰胺,其序列在目前已研究的哺乳类动物中均相同。C末端酰胺化增加了GLP-1的体内稳定性。
天然的GLP-1(7-37)的氨基酸序列为:
HisAlaGluGlyThrPheThrSerAspValSerSerTyrLeuGluGlyGlnAlaAlaLysGluPheIleAlaTrpLeuValLysGlyArgGly
GLP-1有N端和C端,N端与其生理活性有关,C端与受体结合有关。二肽酶Ⅳ(dipeptidyl peptidase-Ⅳ,DPPⅣ)能催化水解GLP-1N端第2位丙氨酸,形成的GLP-1(9~36)NH2失去活性,为GLP-1R的体内天然拮抗剂。GLP-1的生物半衰期较短,为1~1.5min,很快被二肽酶Ⅳ所降解,因此,临床上很难检测其在血液中的浓度。因此对GLP-1进行结构修饰,形成具有同样药理活性的GLP-1类似物,并掩盖DPP-Ⅳ的结合位点,延长半衰期是该类药物研发的主要课题。
在过去的几年中,礼来,诺和诺德,GSK等竞相改造该蛋白,以期获得长效GLP-1类降糖药物。
艾塞那肽(exenatide)是从蜥蜴唾液腺中提取的生物活性肽,其氨基酸序列与GLP-1有53%同源性。研究表明其给药周期可以延长至每日两次。艾塞那肽的氨基酸序列如下所示:
H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2
由于其N端第二位由Gly代替了GLP-1中Ala,不被DPP-Ⅳ降解,而具有较长的半衰期和较强的生物活性。
利拉鲁肽(liraglutide)是对GLP-1蛋白进行脂肪酸链修饰的药物,给药周期延长至每日一次。利拉鲁肽为GLP-1(7-37)链上34位Lys被Arg取代,在26位的Lys上接入经十六烷酸修饰的谷氨酰胺。GLP-1经脂肪链修饰后的,增加了与白蛋白之间的亲和力,从而降低了被DPP‐Ⅳ的水解速率和肾清除率,延长生物半衰期。
利司那肽(Lixisenatide)(商品名:lyxumia)是由法国Sanofi Aventis和Zealand公司共同开发。利司那肽氨基酸序列如下所示:
H-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Ser-Lys-Lys-Lys-Lys-Lys-Lys-NH2
从结构上看,利司那肽是Exendin-4去掉38位的Pro,并在39位的Ser接6个Lys。经过了结构修饰,Lixisenatide的半衰期相对Exenatide有所延长,可每日一次皮下注射。
阿必鲁肽(Albiglutide)(商品名:Eperzan)是由GlaxoSmithKline研发的每周一次皮下注射的长效GLP-1类似物。从结构上看,Albiglutide是将GLP-1(7-36)链上8位上的Ala替换成了Gly,再将两条经修饰过的GLP-1肽链串联融合在一个含有585个残基的血清白蛋白上,这样大大延长了半衰期。
杜拉鲁肽(Dulaglutide)是由Lily公司研发的每周一次皮下注射的长效GLP-1类似物。从结构上看,Dulaglutide是将GLP-1(7-37)链上8位的Ala替换成了Gly,22位的Gly替换成了Glu,36位的Arg替换成了Gly,再通过“GGGGSGGGGSGGGGSA”偶联桥融合到重组IgG4免疫白蛋白(含227个氨基酸Fc片段)的216位的谷氨酸上,平均生物半衰期长达90小时。
索玛鲁肽(Semeglutide)是由Nove Nordisk公司研发的每周一次皮下注射的长效GLP-1类似物。从结构上看Semeglutide是将GLP-1(7-37)链上8位的Ala替换成Aib,34位的Lys替换成Arg,26位的Lys接上十八烷酸脂肪链。与利拉鲁肽相比,索玛鲁肽脂肪链更长,疏水性增加,但是索玛鲁肽经过短链的PEG修饰,亲水性大大增强。PEG修饰后不但可以与白蛋白紧密结合,掩盖DPP-4酶水解位点,还能降低肾***,可延长半衰期,达到长循环的效果。
目前市场上最为长效的GLP-1类药物,其注射频率为每周一次。开发作用时间更优的长效GLP-1类药物,有利于减少频繁的药物注射,提高患者的依从性。
发明内容
在本发明的一个方面,提供了一种融合蛋白,其包含或由以下组成:GLP-1或其类似物;肽接头;和免疫球蛋白Fc部分,其中所述免疫球蛋白Fc部分在434位点(根据EU编号***编号)的氨基酸N被弱疏水性氨基酸替换,其中所述疏水性氨基酸选自丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、色氨酸和蛋氨酸,优选为丙氨酸。由此具有在动物(优选哺乳动物,例如小鼠,更优选人)体内的增加的半衰期。
在本发明的融合蛋白的一个实施方案中,所述GLP-1或其类似物的C末端通过所述肽接头与所述Fc部分的N末端融合,和/或其中所述免疫球蛋白Fc部分来自人类IgG1、IgG2、IgG3或IgG4。
在本发明的融合蛋白的另一个实施方案中,所述免疫球蛋白Fc部分还包含一个或多个(例如,1、2、3、4、5、6、7或8个)选自由以下组成的组的氨基酸替换(根据EU编号***编号):S228P,F234A,L235A,M252Y,T256E,T307A,E380A和M428L,优选包含S228P,F234A,L235A和任选地一个或多个(例如1、2、3、4或5个)选自由以下组成的组的氨基酸替换(根据EU编号***编号):M252Y,T256E,T307A,E380A和M428L,更优选包含S228P,F234A,L235A和任选地一个或多个(例如1、2、3、4或5个)选自由以下组成的组的氨基酸替换(根据EU编号***编号):T307A,E380A和M428L。
在本发明的融合蛋白的另一个实施方案中,所述免疫球蛋白Fc部分包含选自以下的氨基酸替换(根据EU编号***编号)的组合:
1)S228P+F234A+L235A+N434A;
2)S228P+F234A+L235A+M428L+N434A;
3)S228P+F234A+L235A+T307A+E380A+N434A;和
4)S228P+F234A+L235A+M252Y+T256E+N434A。
在本发明的融合蛋白的另一个实施方案中,所述融合蛋白具有一个或多个选自以下的特征:
1)所述GLP-1或其类似物具有一个或多个(2个或3个)选自以下的氨基酸替换(根据EU编号***编号):A8G,G22E和R36G;
2)所述GLP-1或其类似物具有在1-5个(例如1、2、3、4或5个)氨基酸残基上的(C12-24,如C12、C14、C16或C18长链)脂肪酸修饰;和
3)所述融合蛋白的GLP-1或其类似物部分的第27位氨基酸和免疫球蛋白Fc部分的第216位氨基酸(根据EU编号***编号)均带有负电荷,而GLP-1或其类似物部分的第34位氨基酸和免疫球蛋白Fc部分的第218位氨基酸位点(根据EU编号***编号)均带有正电荷,并且GLP-1或其类似物与免疫球蛋白Fc部分中间包含(10-30个氨基酸残基的)连续的极性氨基酸残基片段。
在本发明的融合蛋白的另一个实施方案中,所述融合蛋白的氨基酸序列选自SEQID NO.1、SEQ ID NO.2、SEQ ID NO.3和SEQ ID NO.4组成的组;和/或其中所述GLP-1或其类似物的氨基酸序列如SEQ ID NO.6所示;和/或其中所述肽接头的氨基酸序列如SEQ IDNO.7所示。
在本发明的融合蛋白的另一个实施方案中,所述融合蛋白以二聚体的形式的形式存在,优选的以同源二聚体的形式存在。
在本发明的另一个方面,提供多核苷酸,其编码本发明的融合蛋白。
在本发明的另一个方面,还提供了载体,其包含根据本发明的多核苷酸。
在本发明的另一个方面,还提供了宿主细胞,其包含根据本发明的载体,优选所述宿主细胞为CHO细胞。
在本发明的另一个方面,还提供了一种生产根据本发明的融合蛋白的方法,所述方法包括在宿主细胞中表达根据本发明的载体。
在本发明的另一个方面,还提供了本发明的融合蛋白在制备药物中的应用,优选所述药物用于治疗糖尿病或肥胖。
附图说明
图1.本发明的包含突变的免疫球蛋白Fc部分的GLP-1融合蛋白的示意图(从左到右:N端至C端);
图2.包含Fc突变体1的融合蛋白(Na)的氨基酸序列(SEQ ID NO.1);
图3.包含Fc突变体2的融合蛋白(MNa)的氨基酸序列(SEQ ID NO.2);
图4.包含Fc突变体3的融合蛋白(TENa)的氨基酸序列(SEQ ID NO.3);
图5.包含Fc突变体4的融合蛋白(MTNa)的氨基酸序列(SEQ ID NO.4);
图6.杜拉鲁肽的氨基酸序列(SEQ ID NO.5);
图7.YES的氨基酸序列(S228P,F234A,L235A,M252Y,T256E,N434S)(SEQ IDNO.8);
图8.YTE的氨基酸序列(S228P,F234A,L235A,M252Y,S254T,T256E)(SEQ IDNO.9);
图9.YTELS的氨基酸序列(S228P,F234A,L235A,M252Y,S254T,T256E,M428L,N434S)(SEQ ID NO.10);
图10.YE的氨基酸序列(S228P,F234A,L235A,M252Y,T256E)(SEQ ID NO.11);
图11.本发明融合蛋白的体外活性测定;和
图12.本发明实施例同源二聚体融合蛋白的设计示意图,其中Fc融合蛋白包括抗体Fc区域和独特的药物融合片段(GLP-1或其类似物),该独特的药物融合片段含有一段约5nm的柔性区域,GLP-1蛋白的E27位点和Fc蛋白的E216位点(根据EU编号***编号)均带有负电荷,而GLP-1蛋白的K34位点与Fc蛋白的K218位点(根据EU编号***编号)均带有正电荷,该结构使得该约5nm长的药物融合片段与Fc片段上具有的电荷残基和极性残基存在潜在的相互作用,进一步影响药物片段的空间状态。
具体实施方式
Fc融合蛋白类药物是利用基因工程等技术,将功能蛋白与免疫球蛋白Fc片段相融合的新功能重组蛋白。Fc融合蛋白和抗体属于不同类型蛋白。其本质区别在于:抗体包括两个重链和两个轻链,Fc片段位于重链的恒定区;而Fc融合蛋白包括功能蛋白和Fc片段。Fc融合蛋白的该特征也使得其保留了功能蛋白的生物学活性,并且还具有长效半衰期等抗体的性质。
本研究通过对该类Fc融合蛋白的Fc区域进行结构改造,获得药代半衰期具有明显优势的长效药物。
更具体地,本发明提供一种具有长效降糖能力的GLP-1类Fc融合蛋白,该蛋白由GLP-1类似物与Fc突变体通过连接肽获得,其中Fc突变体至少具有一个突变:氨基酸N434被A434取代(该编号是根据EU索引)。
根据本发明的一个具体实施方式,提供了一种融合蛋白,其包含或由以下组成:GLP-1或其类似物;肽接头;和免疫球蛋白Fc部分,其中所述免疫球蛋白Fc部分在434位点(根据EU编号***编号)的氨基酸N被弱疏水性氨基酸替换,优选所述免疫球蛋白Fc部分包括氨基酸替换N434A(根据EU编号***编号)(注:在本文中,免疫球蛋白Fc部分的氨基酸替换使用以下命名:初始氨基酸,位置(根据EU编号***编号),替换氨基酸。用加号(+)分隔多个突变)。
天然GLP-1在体内经过加工,其前6个氨基酸被切除,因此本领域通常将GLP-1的氨基端(N端)指定为7位,羧基端(C端)为37位。
保持GLP-1天然生物活性的其他GLP-1类似物是本领域技术人员公知的或者可以通过常规实验确定。
优选地,本发明中所述的GLP-1类似物包含在天然GLP-1序列上的1-10个(例如1、2、3、4、5、6、7、8、9或10个)氨基酸置换(例如保守氨基酸置换)、缺失或***,从而延长GLP-1的体内半衰期,并且同时保留GLP-1的天然生物活性。优选地,例如CN1802386A中公开的GLP-1类似物,特别是CN1802386A中公开的SEQ ID NOs.1-6。
优选地,本发明中所述的GLP-1类似物包括在天然GLP-1序列的1-5个(例如1、2、3、4或5个)氨基酸残基上的(C12-24,如C12、C14、C16或C18长链)脂肪酸修饰,从而延长体内半衰期。
在本发明的一个优选实施方案中,本发明中所述的GLP-1类似物选自由艾塞那肽(Exenatide),利拉鲁肽(Liraglutide),利司那肽(Lixisenatide),阿必鲁肽(Albiglutide)、杜拉鲁肽(Dulaglutide)和索玛鲁肽(Semeglutide)组成的组。其中所述阿必鲁肽(Albiglutide)和杜拉鲁肽(Dulaglutide)是指其结构中的GLP-1部分。
本发明中所述的GLP-1类似物的一个代表性序列(从左到右:N端到C端)如下所示:
HisGly8GluGlyThrPheThrSerAspValSerSerTyrLeuGluGlu22GlnAlaAlaLysGluPheIleAlaTrpLeuValLysGlyGly36Gly(SEQ ID NO:6)
与天然人体内活性GLP-1(7-37)相比,有三个氨基酸进行了替换:A8G,G22E,R36G,目的在于降低内源性酶对该类似物的降解,降低分子聚集的潜力和/或降低免疫原性。在该序列中,第27位氨基酸E27带有负电荷,而第34位氨基酸K34带有正电荷。这些残基的分布展示了GLP-1活性蛋白残基的特有电荷分布特征。
本发明中所述的GLP-1融合蛋白具有以下特征:GLP-1蛋白第27位氨基酸(如E27)和免疫球蛋白Fc的第216位氨基酸(根据EU编号***编号,如E216)带有负电荷,而GLP-1蛋白第34位氨基酸(如K34)和免疫球蛋白Fc的218位氨基酸(根据EU编号***编号,如K218)带有正电荷,GLP-1蛋白与免疫球蛋白Fc中间为一段连续的极性残基片段(如GGGGSGGGGSGGGGS)。
本发明的融合蛋白中的肽接头可以选择本领域熟知的肽接头(例如CN1802386A中公开的肽接头,特别是其中公开的SEQ ID NOs.8,19和21),只要其对于融合蛋白中GLP-1的活性和/或融合蛋白的稳定性没有不利影响。用于本发明的融合蛋白中的代表性的优选肽接头的氨基酸序列是由(GGGGS)重复序列加A组成,代表序列为:
GlyGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySerAla(SEQ ID NO:7)
根据本发明的一个具体实施方式,所述GLP-1或其类似物的C末端通过所述肽接头与所述Fc部分的N末端融合。
本发明的Fc部分可以来自人类IgG1、IgG2、IgG3或IgG4。
优选本发明的Fc部分被突变从而将效应子功能最小化(例如L235A,F234A,根据EU编号***编号,参见Kabat,E.A.等人,(1991),Sequences of Proteins of ImmunologicalInterest,第五版,U.S.Dept.of Health and Human Services,Bethesda,MD,NIH出版91-3242)。另外,优选本发明的Fc部分被突变(例如S228P)从而能够形成稳定二聚体结构。
本发明人通过对融合蛋白Fc区段内的残基进行***的改造研究后,进一步发现N434A位点的突变,可以显著增加GLP-1/Fc在血液中的药代半衰期,有望发展为一种更为长效的GLP-1类降糖药物。
根据本发明的一个具体实施方式,除了N434A替换以外,本发明融合蛋白中的所述免疫球蛋白Fc部分还包含一个或多个(例如,1、2、3、4、5、6、7或8个)选自由以下组成的组的氨基酸替换(根据EU编号***编号):S228P,F234A,L235A,M252Y,T256E,T307A,E380A和M428L,优选包含S228P,F234A,L235A和任选地另外的一个或多个(例如1、2、3、4或5个)选自由以下组成的组的氨基酸替换(根据EU编号***编号):M252Y,T256E,T307A,E380A和M428L,更优选包含S228P,F234A,L235A和任选地一个或多个(例如1、2、3、4或5个)选自由以下组成的组的氨基酸替换(根据EU编号***编号):T307A,E380A和M428L。
在本发明的一个优选实施方案中,所述免疫球蛋白Fc部分包含选自由以下组成的组的氨基酸替换(根据EU编号***编号)的组合:
1)S228P+F234A+L235A+N434A;
2)S228P+F234A+L235A+M428L+N434A;
3)S228P+F234A+L235A+T307A+E380A+N434A;和
4)S228P+F234A+L235A+M252Y+T256E+N434A。
在本发明的一个优选实施方案中,所述融合蛋白具有一个或多个选自以下的特征:
1)所述GLP-1或其类似物具有一个或多个(2个或3个)选自以下的氨基酸替换(根据EU编号***编号):A8G,G22E和R36G,以改善GLP-1或其类似物的生物半衰期;
2)所述GLP-1或其类似物具有在1-5个(例如1、2、3、4或5个)氨基酸残基上的(C12-24,如C12、C14、C16或C18长链)脂肪酸修饰,以改善GLP-1或其类似物的生物半衰期;和
3)所述融合蛋白的GLP-1或其类似物部分的第27位氨基酸和免疫球蛋白Fc部分的第216位氨基酸(根据EU编号***编号)均带有负电荷,而GLP-1或其类似物部分的第34位氨基酸和免疫球蛋白Fc部分的第218位氨基酸(根据EU编号***编号)均带有正电荷,并且GLP-1或其类似物与免疫球蛋白Fc部分之间包含(例如,10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29或30个氨基酸残基的)连续的极性氨基酸残基或主要由其构成的柔性片段。
在本发明的一个优选实施方案中,所述的融合蛋白以二聚体的形式存在;在本发明的一个特别优选实施方案中,所述的融合蛋白以同源二聚体的形式存在。
在本发明的一个特别优选实施方案中,所述免疫球蛋白Fc部分的氨基酸序列选自SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3和SEQ ID NO.4组成的组;和/或其中所述GLP-1或其类似物的氨基酸序列如SEQ ID NO.6所示;和/或其中所述肽接头的氨基酸序列如SEQ IDNO.7所示。
本发明的免疫球蛋白Fc部分的突变体可以使用本领域中已知的任何诱变方法来制备,例如定点诱变、合成基因构建、半合成基因构建、随机诱变、改组等等。
定点诱变是其中在编码亲本的多核苷酸中的一个或多个限定位点制造一个或多个(若干个)突变的技术。
定点诱变能在体外通过PCR完成,所述PCR涉及包含期望诱变的寡核苷酸引物的使用。定点诱变也能在体外通过盒诱变进行,所述盒诱变涉及限制性酶在包含编码亲本的多核苷酸的质粒中的一个位点上裂解以及随后连接在多核苷酸中包含突变的寡核苷酸。通常消化质粒和寡核苷酸的限制性酶是相同的,使得质粒的粘性末端和***序列彼此连接。定点诱变也可通过本领域已知的方法在体内完成。
本发明能够使用任何定点诱变程序。有许多可利用的商品化试剂盒能够用于制备变体。
诱变/改组方法可以和高通量的、自动的筛选方法联合,以检测宿主细胞表达的克隆的、诱变处理的多肽的活性。编码活性多肽的诱变DNA分子可从宿主细胞中回收并使用本领域的标准方法快速测序。这些方法允许多肽中单个氨基酸残基重要性的快速确定。
半合成基因构建通过组合使用合成基因构建、和/或定点诱变、和/或随机诱变、和/或改组来完成。半合成构建的典型是利用合成的多核苷酸片段的方法与PCR技术的组合。因此可从头合成限定的基因区域,然而其他区域可使用位点特异性诱变引物进行扩增,而其他区域可经过易错PCR或非易错PCR进行扩增。然后多核苷酸亚序列可以被改组。
本发明还提供了编码本发明的融合蛋白的多核苷酸,包含该多核苷酸的载体(特别是表达载体),已经包含该载体的宿主细胞(优选CHO细胞)。术语“多核苷酸”、“载体”和“宿主细胞”具有本领域公知的含义,除非另外指明。
本发明还提供了生产本发明所述的融合蛋白的方法,所述方法包括在宿主细胞中表达包含编码本发明的融合蛋白的多核苷酸的载体。该方法可以按照本领域技术人员公知的重组蛋白表达方法进行。
另外,本发明还提供本发明的融合蛋白在制备药物中的应用,优选所述药物用于治疗糖尿病或肥胖。
本发明还涉及药物组合物,其包括本发明的融合蛋白,和任选地,至少一种药用载体、稀释剂或赋形剂。
本发明还提供一种治疗糖尿病或肥胖的方法,所述方法包括向需要其的受试者(例如哺乳动物,优选任何灵长类动物,但是特别地是人)施用治疗有效量的本发明的融合蛋白。
以下通过下列非限制性实验部分和附图的方式,进一步描述本发明。技术路线概述:
1.设计免疫球蛋白Fc部分的突变位点,将GLP-1类似物通过肽接头与IgG-Fc突变体融合;所述突变体融合蛋白包括三个结构域,如图1所示,其中,GLP-1为GLP-1类活性蛋白序列,代表序列为:
HisGly8GluGlyThrPheThrSerAspValSerSerTyrLeuGluGlu22GlnAlaAlaLysGluPheIleAlaTrpLeuValLysGlyGly36Gly
与天然人体内活性GLP-1相比,有三个氨基酸进行了替换:A8G,G22E,R36G,
肽接头序列由(GGGGS)重复序列加A组成,代表序列为:
GlyGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySerAla
Fc代表免疫球蛋白Fc突变体。
所述突变体融合蛋白的代表序列为SEQ ID NOs.1-4,其中:
SEQ ID NO.1中Fc部分对应的EU编号为E216-G446,包括以下氨基酸替换:S228P,F234A,L235A,N434A;
SEQ ID NO.2中Fc部分对应的EU编号为E216-G446,包括以下氨基酸替换:S228P,F234A,L235A,M428L,N434A;
SEQ ID NO.3中Fc部分对应的EU编号为E216-G446,包括以下氨基酸替换:S228P,F234A,L235A,T307A,E380A,N434A;
SEQ ID NO.4中Fc部分对应的EU编号为E216-G446,包括以下氨基酸替换:S228P,F234A,L235A,M252Y,T256E,N434A;
2.对设计的突变体融合蛋白进行基因设计,并进行全基因合成(委托金斯瑞);
3.对全合成的基因采用分子克隆方法,构建到真核表达载体(pcDNA3.3,Invitrogen)中,获得表达构建体(Plasmid-X);
4.表达构建体(Plasmid-X)采用电转化方法转染宿主细胞(CHO细胞),并用800μg/mL的G418(Geneticin,遗传霉素)进行加压筛选;
5.进行混合克隆株的Fc突变体融合蛋白表达;
6.对Fc突变体融合蛋白采用Protein A填料进行亲和层析纯化;
7.对收集的蛋白进行质谱鉴定,确认产物的正确性,同时进行SDS-PAGE研究,确定纯化蛋白的纯度;
8.对纯化的融合蛋白用10mM的PBS稀释至浓度0.1mg/ml,按0.1mg/kg大鼠的注射量,每只大鼠约注射0.03-0.04mg蛋白。并在0h,2h,5h,8h,24h,48h,72h,120h,168h取样,采用GLP-1试剂盒检测,最后计算药代半衰期。
实施例1载体的构建
基于上述技术路线,采用分子克隆的方法,构建了本发明的以下(从N端至C端)的各种表达载体:
GLP-1类似物(氨基酸序列如SEQ ID NO.6所示)+肽接头(氨基酸序列如SEQ IDNO.7所示)+突变的免疫球蛋白Fc部分。
构建得到的融合蛋白的氨基酸序列分别如SEQ ID NOs.1-4、8-11所示。
作为对照,构建编码以下(从N端至C端)的载体:
杜拉鲁肽(WT),氨基酸序列如SEQ ID NO.5所示。
本发明的突变的免疫球蛋白Fc部分总结如下:
表1
编码WT融合蛋白(杜拉鲁肽)以及IgG4-Fc突变体融合蛋白的核苷酸序列是委托金斯瑞生物科技有限公司(中国南京)基于其编码的氨基酸序列通过化学合成获得的。所获得的合成序列通过双酶切后,***到真核表达载体的相同酶切位点间,构建Plasmid-GLP-1-Fc及突变体一系列载体。然后采用Invitrogen质粒提取试剂盒提取一系列经验证正确的表达载体,并用限制性内切酶进行线性化后纯化回收,-20℃保藏。
实施例2载体转染及在细胞中表达
将CHO宿主细胞用CHO培养基复苏培养后,当细胞密度约8x105个细胞/mL时收集细胞进行转染。转染细胞约1x107个细胞,质粒约40μg,通过电击方法转染(Bio-Rad,Genepulser Xcell)。电击后细胞于20mL CHO培养基中培养。培养第二天,离心收集细胞,并在加入G418(Geneticin,遗传霉素)至终浓度800μg/mL的20mL CHO培养基中重悬培养。当细胞密度约0.6x106个细胞/mL时,对获得的混合克隆株用CHO培养基进行传代,传代细胞密度约0.2x106个细胞/mL。当细胞存活率约90%时,收集细胞培养液。
实施例3从动物细胞培养液中纯化融合蛋白
对实施例1一系列的融合蛋白进行翻译水平上的检测。采用Protein A填料对少量细胞培养液进行富集,并收集融合蛋白,得到的融合蛋白是通过二硫键和多种非共价相互作用形成的同源二聚体,进行质谱检测,质谱检测分子量约62KD,与理论分子量一致。对收集的融合蛋白采用Protein A层析柱进行纯化。收集的样品经还原后通过10%SDS-PAGE电泳检测,电泳图谱显示单一条带,约36KD。纯化样品采用pH 7.2的10mM PBS缓冲液在4℃透析过夜。
实施例4融合蛋白在大鼠中的药代动力学
对纯化的一系列融合蛋白用10mM的PBS稀释至浓度0.1mg/mL。对通过体重的SD大鼠(0.3-0.4kg)随机选择,按每只SD大鼠注射0.1mg/kg的融合蛋白进行计算给药,每个融合蛋白皮下注射3只大鼠。
在给药前,分别从每只大鼠的颈静脉中取出约200μl的血,用EDTA-K2和DPP-4抑制剂进行抗凝处理,于-20℃保存。每只动物给药后2h,5h,8h,24h,48h,72h,120h,168h分别采血,同样进行抗凝处理后-20℃保存。
各血样中药物残留量采用GLP-1ELISA检测试剂盒(Millipore)检测,并进行药代数据计算,结果见下表:
表2
实施例5融合蛋白的体外活性测定
对纯化后的融合蛋白用BCA法进行定量,然后用Assay buffer(DMEM20ml,FBS 200μl,IBMX 20μl)进行三倍梯度稀释。用cAMP检测试剂盒(厂家cisbio)测定融合蛋白刺激GLP-1R/HEK293细胞后胞内的cAMP含量,即在一个384浅孔板中加入5μl稀释样品液,后加入5μl细胞悬液(细胞密度为100个/μl),于二氧化碳培养箱中温浴30min,再加入反应试剂反应后于多功能酶标仪中检测665nm、620nm的荧光值。根据cAMP标准品的浓度及其对应荧光值的比值绘制标准曲线,计算不同浓度下供试品刺激GLP-1R/HEK293细胞产生cAMP的数量。以供试品浓度的对数值为横坐标,以cAMP的nM值为纵坐标制作曲线。结果显示,经供试品刺激后胞内的cAMP含量曲线呈典型“S”型曲线,根据这些曲线计算EC50值。结果如下表和图11:
表3
融合蛋白 | WT | Na | TENa | MNa |
EC50值(nM) | 10.350 | 6.363 | 3.652 | 3.784 |
实施例6总结和讨论
抗体Fc区域与其受体的晶体结构(PDB:1FRT,4N0U)表明Fc区域通过其侧翼的氨基酸识别FcRn,这些侧翼的氨基酸包括:M252,S254,T256,M428,N434等(图12)。通过对这些侧翼氨基酸的定点突变可以改变Fc与其受体的结合力,如有研究组通过将M252,S254,T256突变为Y252,T254,E256,可以增加突变体与受体的结合力,该突变引入了负电荷残基E256和极性氨基酸T254。也有研究组将N434位点突变为S434等,引入极性氨基酸来增加突变体与受体的结合。
本发明设计的Fc融合蛋白包括了抗体Fc区域和独特的药物融合片段(GLP-1或其类似物)(图12)。其中该独特的药物融合片段含有一段约5nm的柔性区域。在Fc融合蛋白中,GLP-1蛋白的E27位点,与Fc蛋白的E216位点(根据EU编号***编号),均带有负电荷;而GLP-1蛋白的K34位点,与Fc蛋白的K218位点(根据EU编号***编号),均带有正电荷。并且,GLP-1蛋白与免疫球蛋白中间为一段连续的极性残基片段即所述肽接头(G38GGGSGGGGSGGGGS52)。这种特有的结构特征使得该5nm长的药物融合片段与Fc片段上新引入的电荷残基和极性残基存在潜在的相互作用,进一步影响药物片段的空间状态。
本发明的突变和药代实验也证实了这种观点(实验方法和步骤同之前实施例)。除了本发明的上述融合蛋白后,发明人还在本发明对应的融合蛋白药物中的Fc区域中引入负电荷残基E256,构建而成的四个候选药物YES(氨基酸序列如SEQ ID NO.8所示,Fc区域含有突变
S228P+F234A+L235A+M252Y+T256E+N434S),YTE(氨基酸序列如SEQ ID NO.9所示,Fc区域含有突变
S228P+F234A+L235A+M252Y+S254T+T256E),YTELS(氨基酸序列如SEQ ID NO.10所示,Fc区域含有突变
S228P+F234A+L235A+M252Y+S254T+T256E+M428L+N434S)和YE(氨基酸序列如SEQID NO.11所示,Fc区域含有突变
S228P+F234A+L235A+M252Y+T256E),其Cmax值减少,t1/2时间提前,并不具有潜在的长效药物成药性(与本发明融合蛋白的比较结果参见表2)。可以看出,在Fc区域引入极性残基S434,会造成t1/2的时间提前,不具有潜在的长效药物成药性。在药代实验过程中,我们筛选得到,在Fc区域引入弱疏水性残基A434,可以显著增加t1/2,其Cmax等指标也展示出药代优势。因此,本发明人出人意料地发现:在于对该药物Fc片段中引入弱疏水性残基(如A434)(而并不是极性或者带电荷的残基),能够减少与药物融合片段与Fc片段之间的结构自抑制,从而增加药代效果,从而完成了本发明。
虽然为了清楚的理解,已经借助于附图和实施例在一些细节上描述了上述发明,但是说明书和实施例不应当被视为限制本发明的范围。本文中引用的所有专利和科学文献的公开内容通过引用完整地清楚并入。
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<110> 广东东阳光药业有限公司
<120> 包含突变的免疫球蛋白Fc部分的GLP-1融合蛋白
<130> IB178011
<150> CN 201610633073.3
<151> 2016-08-03
<160> 11
<170> PatentIn version 3.3
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<213> artificial sequence
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<210> 5
<211> 275
<212> PRT
<213> artificial sequence
<220>
<223> 杜拉鲁肽
<400> 5
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Gly Gly Gly
20 25 30
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu
35 40 45
Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala
50 55 60
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
65 70 75 80
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
85 90 95
Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu
100 105 110
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr
115 120 125
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
130 135 140
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser
145 150 155 160
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
165 170 175
Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val
180 185 190
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
195 200 205
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
210 215 220
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr
225 230 235 240
Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val
245 250 255
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
260 265 270
Ser Leu Gly
275
<210> 6
<211> 31
<212> PRT
<213> artificial sequence
<220>
<223> 代表性GLP-1类似物
<400> 6
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Leu Glu Phe Ile Ala Trp Leu Val Lys Gly Gly Gly
20 25 30
<210> 7
<211> 16
<212> PRT
<213> artificial sequence
<220>
<223> 接头
<400> 7
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala
1 5 10 15
<210> 8
<211> 275
<212> PRT
<213> artificial sequence
<220>
<223> YES的氨基酸序列
<400> 8
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Gly Gly Gly
20 25 30
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu
35 40 45
Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala
50 55 60
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
65 70 75 80
Tyr Ile Ser Arg Glu Pro Glu Val Thr Cys Val Val Val Asp Val Ser
85 90 95
Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu
100 105 110
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr
115 120 125
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
130 135 140
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser
145 150 155 160
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
165 170 175
Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val
180 185 190
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
195 200 205
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
210 215 220
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr
225 230 235 240
Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val
245 250 255
Met His Glu Ala Leu His Ser His Tyr Thr Gln Lys Ser Leu Ser Leu
260 265 270
Ser Leu Gly
275
<210> 9
<211> 275
<212> PRT
<213> artificial sequence
<220>
<223> YTE的氨基酸序列
<400> 9
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Gly Gly Gly
20 25 30
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu
35 40 45
Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala
50 55 60
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
65 70 75 80
Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val Val Asp Val Ser
85 90 95
Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu
100 105 110
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr
115 120 125
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
130 135 140
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser
145 150 155 160
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
165 170 175
Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val
180 185 190
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
195 200 205
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
210 215 220
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr
225 230 235 240
Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val
245 250 255
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
260 265 270
Ser Leu Gly
275
<210> 10
<211> 275
<212> PRT
<213> artificial sequence
<220>
<223> YTELS的氨基酸序列
<400> 10
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Gly Gly Gly
20 25 30
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu
35 40 45
Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala
50 55 60
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
65 70 75 80
Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val Val Asp Val Ser
85 90 95
Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu
100 105 110
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr
115 120 125
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
130 135 140
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser
145 150 155 160
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
165 170 175
Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val
180 185 190
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
195 200 205
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
210 215 220
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr
225 230 235 240
Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val
245 250 255
Leu His Glu Ala Leu His Ser His Tyr Thr Gln Lys Ser Leu Ser Leu
260 265 270
Ser Leu Gly
275
<210> 11
<211> 275
<212> PRT
<213> artificial sequence
<220>
<223> YE的氨基酸序列
<400> 11
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Gly Gly Gly
20 25 30
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu
35 40 45
Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala
50 55 60
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
65 70 75 80
Tyr Ile Ser Arg Glu Pro Glu Val Thr Cys Val Val Val Asp Val Ser
85 90 95
Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu
100 105 110
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr
115 120 125
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
130 135 140
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser
145 150 155 160
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
165 170 175
Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val
180 185 190
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
195 200 205
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
210 215 220
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr
225 230 235 240
Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val
245 250 255
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
260 265 270
Ser Leu Gly
275
Claims (12)
1.融合蛋白,其包含或由以下组成:GLP-1或其类似物;肽接头;和免疫球蛋白Fc部分,其中所述免疫球蛋白Fc部分在434位点(根据EU编号***编号)的氨基酸(如氨基酸N)被疏水性氨基酸替换,其中所述疏水性氨基酸选自丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、色氨酸和蛋氨酸,优选为丙氨酸,最优选所述免疫球蛋白Fc部分包含氨基酸替换N434A。
2.根据权利要求1所述的融合蛋白,其中所述GLP-1或其类似物的C末端通过所述肽接头与所述Fc部分的N末端融合,和/或其中所述免疫球蛋白Fc部分来自人类IgG1、IgG2、IgG3或IgG4。
3.根据权利要求1或2所述的融合蛋白,其中所述免疫球蛋白Fc部分还包含一个或多个(例如,1、2、3、4、5、6、7或8个)选自由以下组成的组的氨基酸替换(根据EU编号***编号):S228P,F234A,L235A,M252Y,T256E,T307A,E380A和M428L,优选包含S228P,F234A,L235A和任选地一个或多个(例如1、2、3、4或5个)选自由以下组成的组的氨基酸替换(根据EU编号***编号):M252Y,T256E,T307A,E380A和M428L,更优选包含S228P,F234A,L235A和任选地一个或多个(例如1、2、3、4或5个)选自由以下组成的组的氨基酸替换(根据EU编号***编号):T307A,E380A和M428L。
4.根据权利要求3所述的融合蛋白,其中所述免疫球蛋白Fc部分包含选自以下的氨基酸替换(根据EU编号***编号)的组合:
1)S228P+F234A+L235A+N434A;
2)S228P+F234A+L235A+M428L+N434A;
3)S228P+F234A+L235A+T307A+E380A+N434A;和
4)S228P+F234A+L235A+M252Y+T256E+N434A。
5.根据权利要求1所述的融合蛋白,其中所述融合蛋白具有一个或多个选自以下的特征:
1)所述GLP-1或其类似物具有一个或多个(2个或3个)选自以下的氨基酸替换(根据EU编号***编号):A8G,G22E和R36G;
2)所述GLP-1或其类似物具有在1-5个(例如1、2、3、4或5个)氨基酸残基上的(C12-24,如C12、C14、C16或C18长链)脂肪酸修饰;和
3)所述融合蛋白的GLP-1或其类似物部分的第27位氨基酸和免疫球蛋白Fc部分的第216位氨基酸(根据EU编号***编号)均带有负电荷,而GLP-1或其类似物部分的第34位氨基酸和免疫球蛋白Fc部分的第218位氨基酸(根据EU编号***编号)均带有正电荷,并且GLP-1或其类似物与免疫球蛋白Fc部分中间包含(10-30个氨基酸残基的)连续的极性氨基酸残基片段。
6.根据权利要求4或5所述的融合蛋白,其中所述融合蛋白的氨基酸序列选自SEQ IDNO.1、SEQ ID NO.2、SEQ ID NO.3和SEQ ID NO.4组成的组;和/或其中所述GLP-1或其类似物的氨基酸序列如SEQ ID NO.6所示;和/或其中所述肽接头的氨基酸序列如SEQ ID NO.7所示。
7.根据权利要求1所述的融合蛋白,其以二聚体的形式存在,优选的以同源二聚体的形式存在。
8.多核苷酸,其编码根据权利要求1-7中任一项所述的融合蛋白。
9.载体,其包含根据权利要求8所述的多核苷酸。
10.宿主细胞,其包含根据权利要求9所述的载体,优选所述宿主细胞为CHO细胞。
11.一种生产根据权利要求1-7中任一项所述的融合蛋白的方法,所述方法包括在宿主细胞中表达根据权利要求9所述的载体。
12.根据权利要求1-7中任一项所述的融合蛋白在制备药物中的应用,优选所述药物用于治疗糖尿病或肥胖。
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