CN1802386B - Glp-1类似物融合蛋白质 - Google Patents
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/26—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against hormones ; against hormone releasing or inhibiting factors
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- C07K2319/00—Fusion polypeptide
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Abstract
本发明提供与特定IgG4-Fc衍生物融合的特定GLP-1类似物。这些融合蛋白质具有增加的半寿期、降低的免疫原性,并降低了效应子活性。这些融合蛋白质可用于治疗糖尿病、肥胖、肠易激综合征以及将通过降低血浆葡萄糖、抑制胃和/或肠运动和抑制胃和/或肠排空、或抑制食物摄入而受益的其他疾病。
Description
发明领域
本发明涉及胰高血糖素样肽类似物,该胰高血糖素样肽类似物与具有延长其体内半寿期作用的蛋白质融合。该融合蛋白质可用于治疗糖尿病及多种其他疾病或紊乱。
胰高血糖素样肽-1(GLP-1)类似物及衍生物在2型糖尿病治疗的临床试验中表现出前景。GLP-1诱导多种生物学效应,例如刺激胰岛素分泌、抑制胰高血糖素分泌、抑制胃排空、抑制胃运动或肠运动及诱导重量减轻。GLP-1的显著特征是其刺激胰岛素分泌而不伴有低血糖相关危险的能力,所述低血糖相关危险见于使用胰岛素治疗或通过增加胰岛素表达起作用的某些类型的口服治疗时。
GLP-1(1-37)活性很低,并且GLP-1(7-37)OH和GLP-1(7-36)NH2这两种天然存在的截短的肽在体内被迅速清除并且具有极短的体内半寿期这一事实限制了涉及GLP-1肽的治疗的有效性。已知内源性产生的二肽基肽酶IV(DPP-IV)通过去除N末端组氨酸和丙氨酸残基而失活循环GLP-1肽,是体内短半寿期的主要原因。
已经采取多种途径在保持生物学活性的同时延长GLP-1肽的清除半寿期或降低该肽从机体的清除。一条途径涉及将GLP-1肽与免疫球蛋白Fc部分融合。免疫球蛋白一般在体内具有长循环半寿期。例如,IgG分子在人体具有高达23天的半寿期。免疫球蛋白Fc部分是这种体内稳定性的部分原因。在保留GLP-1分子生物学活性的同时,GLP-1-Fc融合蛋白质具有由免疫球蛋白Fc部分提供的稳定性而保留GLP-1分子的生物活性的优点。
尽管此途径对于GLP-1疗法(见WO 02/46227)是可行的,但是关于多种融合蛋白质长时期反复施用时的抗原性存在普遍的顾虑。对于GLP-1-Fc融合物疗法,这尤其是一个顾虑,因为糖尿病患者一旦经诊断为该疾病,必须一生接受治疗。此外,如果Fc部分保留不需要的效应子功能,Fc融合蛋白质疗法可能是一个顾虑。
通过鉴定反复和长期施用后诱导免疫反应风险降低并且不再具有效应子功能的特定GLP-1-Fc融合蛋白质,本发明尝试克服与施用GLP-1-Fc融合物相关的关于潜在免疫原性和效应子活性的问题。这些特定的融合蛋白质在分子的GLP-1部分和Fc部分中具有多个位置的替换。此处所述的替换提供更大的潜能、增加的体内稳定性、效应子功能的消除以及该分子被免疫系统适应元件识别的可能性降低。
本发明的化合物包括包含GLP-1类似物的异源融合蛋白质,其中GLP-1类似物包含的序列选自
a)(SEQ ID NO:1)
His-Xaa8-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Gly-Gly
其中Xaa8选自Gly和Val;
b)(SEQ ID NO:2)
His-Xaa8-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Lys-Asn-Gly-Gly-Gly
其中Xaa8选自Gly和Val;
c)(SEQ ID NO:3)
His-Xaa8-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Gly-Pro
其中Xaa8选自Gly和Val;
d)(SEQ ID NO:4)
His-Xaa8-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Lys-Asn-Gly-Gly-Pro
其中Xaa8选自Gly和Val;
e)(SEQ ID NO:5)
His-Xaa8-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Gly
其中Xaa8选自Gly和Val;
f)(SEQ ID NO:6)
His-Xaa8-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Lys-Asn-Gly-Gly
其中Xaa8自Gly和Val;
GLP-1类似物与免疫球蛋白Fc部分融合,其中免疫球蛋白Fc部分包含SEQ ID NO:7的序列
Ala-Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Pro-Cys-Pro-Ala-Pro-
Xaa16-Xaa17-Xaa18-Gly-Gly-Pro-Ser-Val-Phe-Leu-Phe-Pro-Pro-Lys-Pro-
Lys-Asp-Thr-Leu-Met-Ile-Ser-Arg-Thr-Pro-Glu-Val-Thr-Cys-Val-
Val-Val-Asp-Val-Ser-Gln-Glu-Asp-Pro-Glu-Val-Gln-Phe-Asn-Trp-
Tyr-Val-Asp-Gly-Val-Glu-Val-His-Asn-Ala-Lys-Thr-Lys-Pro-Arg-
Glu-Glu-Gln-Phe-Xaa80-Ser-Thr-Tyr-Arg-Val-Val-Ser-Val-Leu-Thr-
Val-Leu-His-Gln-Asp-Trp-Leu-Asn-Gly-Lys-Glu-Tyr-Lys-Cys-Lys-
Val-Ser-Asn-Lys-Gly-Leu-Pro-Ser-Ser-Ile-Glu-Lys-Thr-Ile-Ser-
Lys-Ala-Lys-Gly-Gln-Pro-Arg-Glu-Pro-Gln-Val-Tyr-Thr-Leu-Pro-
Pro-Ser-Gln-Glu-Glu-Met-Thr-Lys-Asn-Gln-Val-Ser-Leu-Thr-Cys-
Leu-Val-Lys-Gly-Phe-Tyr-Pro-Ser-Asp-Ile-Ala-Val-Glu-Trp-Glu-
Ser-Asn-Gly-Gln-Pro-Glu-Asn-Asn-Tyr-Lys-Thr-Thr-Pro-Pro-Val-
Leu-Asp-Ser-Asp-Gly-Ser-Phe-Phe-Leu-Tyr-Ser-Arg-Leu-Thr-Val-
Asp-Lys-Ser-Arg-Trp-Gln-Glu-Gly-Asn-Val-Phe-Ser-Cys-Ser-Val-
Met-His-Glu-Ala-Leu-His-Asn-His-Tyr-Thr-Gln-Lys-Ser-Leu-Ser-
Leu-Ser-Leu-Gly-Xaa230(SEQ ID NO:7)
其中
16位的Xaa为Pro或Glu;
17位的Xaa为Phe、Val、或Ala;
18位的Xaa为Leu、Glu、或Ala;
80位的Xaa为Asn或Ala;以及
230位的Xaa为Lys或不存在。
本发明的异源融合蛋白质GLP-1类似物部分的C末端和Fc部分的N末端优选通过富含G的肽接头的1、1.5或2个重复融合在一起,其中肽接头具有序列Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly
-Ser(SEQ ID NO:8)。
本发明还包括编码本发明异源融合蛋白质的多核苷酸以及包含这些多核苷酸的载体和宿主细胞。本发明也包括治疗患有非胰岛素依赖型和胰岛素依赖型糖尿病、肥胖及多种其他疾病和病症的患者的方法,其包括施用在此讨论的异源融合蛋白质。
本发明的异源融合蛋白质包括GLP-1类似物部分和Fc部分。GLP-1类似物部分和Fc部分分别包含对天然GLP-1序列和人IgG4序列的替换,这些替换为该蛋白质提供与天然GLP-1或没有与Fc序列融合的GLP-1类似物相比增加的潜能和体内稳定性,而降低了对人长期和反复施用后诱导抗体形成的潜力。
天然GLP-1在体内经过加工,从而从该分子切割前6个氨基酸。因此,按照本领域习惯将GLP-1的氨基端指定为7号,羧基端为37号。如SEQID NO:9中所示对该多肽中的其他氨基酸连续编号。例如,第8位为丙氨酸,第22位为甘氨酸。经过加工的肽可以在体内得到进一步修饰,从而去除C末端甘氨酸残基并以酰胺基替代。因此,GLP-1(7-37)OH和GLP-1(7-36)酰胺代表该分子的两种天然形式。GLP-1(7-37)OH具有SEQID NO:9的氨基酸序列:
7His-Ala-Glu-10Gly-Thr-Phe-Thr-Ser-15Asp-Val-Ser-Ser-Tyr-20Leu-Glu-Gly-Gln-Ala-25Ala-Lys-Glu-Phe-Ile-30Ala-Trp-Leu-Val-Lys-35Gly-Arg-37Gly
(SEQ ID NO:9)
相对于天然GLP-1(7-37),异源融合蛋白质的GLP-1类似物部分包括第8、22和36位的三个初步替换。第8位的替换降低内源性酶二肽基肽酶IV(DPP-IV)失活该类似物的速率。DPP-IV在第二和第三个氨基酸之间(第8和第9位之间)切割天然GLP-1,产生的分子活性较低。因此,本发明的异源融合蛋白质是抵抗DPP-IV的。第22位的替换降低分子聚集的潜力,并增加分子的潜能。具有8和22位变化的类似物以及在整个融合蛋白质中第36位的替换降低融合蛋白质在人体反复和长期施用后诱导中和性免疫应答的危险。
产生体液和细胞介导的免疫应答二者中的中心事件是T辅助(TH)细胞的活化和克隆扩增。在抗原呈递细胞(APC)的存在下,T细胞受体(TCR)-CD3复合物和与II类主要组织相容性(MHC)分子结合的经过加工的抗原肽相互作用,启动TH细胞活化。TH细胞与抗原的相互作用启动了生物化学事件的级联,其诱导剩余TH细胞进入细胞周期(G0向G1过渡)的。活化的T细胞通过细胞周期,增殖并分化为记忆细胞或效应子细胞。
分析下列序列以确定可能的表位:
His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Ala-Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro(SEQ ID NO:10)
相对于天然序列,该序列是在第8和22位具有改变的GLP-1类似物序列,继之为2个拷贝的富含G的接头序列,然后是来自人IgG4的Fc区的前10个氨基酸。此处所使用的表位指抗体可以结合的蛋白质分子区域。将免疫原性表位定义为当整个蛋白质为免疫原时引起抗体反应的蛋白质部分。表位作图涉及使用滑动的九个氨基酸窗口扫描序列,加上高级统计分析技术提取这些模式中含有的信息。使用被称为EpiMatrixTM的专有软件包分析序列,并确定向T细胞呈递时很可能引发免疫应答的肽。8个非常常见的等位基因用于II类MHC受体相互作用分析。这些等位基因包括DRB1*0101、DRB1*0301、DRB1*0401、DRB1*0701、DRB1*0801、DRB1*1101、DRB1*1301和DRB1*1501。
预测强表位位于GLP-1类似物部分C末端与接头开始的连接处。该表位序列是与DRB1*0801相互作用的Trp-Leu-Val-Lys-Gly-Arg-Gly-Gly-Gly(SEQ ID NO:11)。本发明也包含发现可以通过将GLP-1类似物C末端改变为下列序列之一而消除该表位:Trp-Leu-Val-Lys-Gly-Gly-Gly (SEQ ID NO:12);Trp-Leu-Lys-Asn-Gly-Gly-Gly (SEQ ID NO:13);Trp-Leu-Val-Lys-Gly-Gly-Pro (SEQ ID NO:14);Trp-Leu-Lys-Asn-Gly-Gly-Pro (SEQ ID NO:15);Trp-Leu-Val-Lys-Gly-Gly(SEQ ID NO:16);和Trp-Leu-Lys-Asn-Gly-Gly(SEQ ID NO:17)。
本发明的异源融合蛋白质含有来自人类IgG4,但是与野生型人类序列相比包括一个或多个替换的Fc部分。如此处所使用,免疫球蛋白的Fc部分具有免疫学领域通常赋予该术语的意义。该术语尤其指不含有来自该抗体两个抗原结合区(Fab片段)的抗体片段。Fc部分由通过非共价相互作用和二硫键结合的抗体的两个重链恒定区组成。Fc部分可以包含铰链区,并经CH2和CH3结构域延伸到抗体C末端。Fc部分还可以包含一个或多个糖基化位点。
有五种类型的具有不同效应子功能和药物动力学特性的人类免疫球蛋白。IgG是五种类型中最稳定的,在人体具有约23天的血清半寿期。有四个IgG亚类(G1、G2、G3和G4),各亚类具有称作效应子功能的不同生物学功能。这些效应子功能通常由与Fc受体(FcγR)的相互作用或通过结合C1q和固定补体介导。与FcγR的结合可以导致抗体依赖细胞介导的细胞裂解,而与补体因子的结合可以导致补体介导的细胞裂解。在仅利用Fc部分延长半寿期的能力的异源Fc融合蛋白质的设计中,将效应子功能最小化是重要的。因此,本发明的异源融合蛋白质来自人类IgG4 Fc区域,因其与其他IgG亚型相比结合FcγR和补体因子的能力降低。然而,已经表明IgG4耗竭人体中靶细胞[Issacs等人,(1996)Clin.Exp.Immunol.106:427-433]。因为本发明的异源融合蛋白质靶定胰腺β细胞以诱导胰岛素表达,所以在Fc融合蛋白质中使用来自IgG4的区域可以通过融合蛋白质与存在于胰腺β细胞上的GLP-1受体的相互作用引起针对胰腺β细胞的免疫应答。因此,作为本发明的异源融合蛋白质一部分的IgG4 Fc区域含有消除效应子功能的替换。本发明的融合蛋白质的IgG4 Fc部分可以含有一个或多个下列替换:在残基233处以脯氨酸替换谷氨酰胺,在残基234处以丙氨酸或缬氨酸替换苯丙氨酸,以及在残基235处以丙氨酸或谷氨酰胺替换亮氨酸(EU编号方式,Kabat,E.A.等人,(1991)《Sequences of Proteinsof Immunological Interest》,第五版,U.S.Dept.of Health and HumanServices,Bethesda,MD,NIH出版91-3242)。这些残基对应于SEQ ID NO:7中的第16、17和18位。另外,在对应于SEQ ID NO:7第80位的第297个残基(EU编号)以Ala替换Asn而去除IgG4 Fc区域中N-连接的糖基化位点是保证消除异源融合蛋白质消除残留效应子活性的另一条路径。
另外,本发明异源融合蛋白质的IgG4 Fc部分含有稳定重链二聚体结构和防止半IgG4 Fc链形成的替换。本发明的异源融合蛋白质优选以通过二硫键和多种非共价相互作用连接的二聚体存在。野生型IgG4含有在残基224处(EU编号)开始的Pro-Pro-Cys-Pro-Ser-Cys(SEQ ID NO:18)基序。单个GLP-1类似物-Fc链中的该基序与另一条GLP-1类似物-Fc链中的对应基序形成二硫键。然而,该基序中丝氨酸的存在引起单链融合蛋白质的形成。本发明包含异源Fc融合蛋白质,其中IgG4序列经过进一步修饰,从而以脯氨酸替换第228位(EU编号)的丝氨酸(SEQ ID NO:7中的第11个氨基酸残基)。
在此讨论的异源融合蛋白质的IgG4衍生的Fc部分中可以缺失存在于天然分子中的C末端赖氨酸残基(SEQ ID NO:7第230位;将缺失的赖氨酸称为des-K)。某些细胞型(例如NS0细胞)表达的、其中赖氨酸由C末端密码子编码的融合蛋白质是异质的,因为一部分分子将赖氨酸作为C末端氨基酸,而一部分缺失赖氨酸。该缺失归因于某些类型的哺乳动物细胞表达过程中蛋白酶的作用。因此,为了避免这种异质性,Fc融合表达构建体优选缺乏C末端赖氨酸密码子。
在此讨论的GLP-1类似物部分的C末端氨基酸优选通过富含甘氨酸的接头与IgG4 Fc类似物部分的N末端融合。可以通过加入小的肽接头防止潜在的不必要的结构域相互作用来优化本发明的异源融合蛋白质的体内功能和稳定性。另外,富含甘氨酸的接头提供了一定的结构柔性,使GLP-1类似物部分可以与靶细胞例如胰腺β细胞上的GLP-1受体有效地相互作用。然而,这些接头可以显著增加融合蛋白质体内免疫原性的危险。因此,优选不多于必要的长度以防止不必要的结构域相互作用和/或优化生物学活性和/或稳定性。优选的富含甘氨酸的接头包括序列:Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser(SEQ IDNO:8)。尽管在本发明的异源融合蛋白质中可以使用该接头的更多拷贝,优选使用该接头的单拷贝以最小化与长期和反复施用有关的免疫原性危险。
本发明优选的GLP-1-Fc异源融合蛋白质包括下列蛋白质:Gly8-Glu22-Gly36-GLP-1(7-37)-1L-IgG4S228P)、Gly8-Glu22-Gly36-GLP-1(7-37)-1L-IgG4S228P、 F234A、 L235A)、Gly8-Glu22-Gly36-GLP-1(7-37)-1L-IgG4(S228P,N297A)、Gly8-Glu22-Gly36-GLP-1(7-37)-1L-IgG4(S228P、F234A、L235A、N297A)、Gly8-Glu22-Gly36-GLP-1(7-37)-1.5L-IgG4(S228P)、Gly8-Glu22-Gly36-GLP-1(7-37)-1.5L-IgG4(S228P、F234A、L235A)、Gly8-Glu22-Gly36-GLP-1(7-37)-1.5L-IgG4(S228P、N297A)、Gly8-Glu22-Gly36-GLP-1(7-37)-1.5L-IgG4(S228P、F234A、L235A、N297A)、Gly8-Glu22-Gly36-GLP-1(7-37)-2L-IgG4(S228P)、Gly8-Glu22-Gly36-GLP-1(7-37)-2L-IgG4(S228P、F234A、L235A)、Gly8-Glu22-Gly36-GLP-1(7-37)-2L-IgG4(S228P、N297A)、以及Gly8-Glu22-Gly36-GLP-1(7-37)-2L-IgG4(S228P、F234A、L235A、N297A)、以及上述所有蛋白质的Val8和des-K形式。
此处使用的提及特定异源融合蛋白质的命名法定义如下:融合蛋白质GLP-1部分的特定替换以被替换的特定氨基酸跟随残基编号来表示。GLP-1(7-37)表示成熟融合蛋白质的GLP-1部分以第7位的His开始,以第37位的Gly结束。L指具有序列Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser(SEQ IDNO:8)的接头。直接在L前面的数字指将GLP-1部分与Fc部分分开的接头的数目。指定为1.5L的接头指序列Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser(SEQ ID NO:19)。IgG4指如SEQ ID NO:7所指定的人IgG4 Fc序列的类似物。异源融合蛋白质IgG4 Fc部分的替换显示于圆括号中。野生型氨基酸以常用缩写表示,其后跟随使用EU编号系统的在整个IgG4序列中的位置号,随后为该位置以常用缩写表示的被替换的氨基酸。
尽管可以通过多种不同的方法制备本发明的异源融合蛋白质,但是由于融合蛋白质的大小,优选重组方法。对于在此处所公开并要求保护的本发明,下面定义下列常见分子生物学术语及缩写。
此处使用的“碱基对”或“bp”指DNA或RNA。当缩写A、C、G和T出现在DNA分子中时,分别相应于脱氧核糖核苷(脱氧)腺苷、(脱氧)胞苷、(脱氧)鸟苷和胸苷的5′-单磷酸形式。当缩写U、C、G和A出现在RNA分子中时,分别相应于核糖核苷尿嘧啶核苷、胞苷、鸟苷和腺苷的5′-单磷酸形式。在双链DNA中,碱基对可以指A与T或C与G的配对。在DNA/RNA中,异源双链体碱基对可以指A与U或C与G的配对。(见下文“互补”的定义。)
DNA的“消化”或“限制”指用限制酶的催化切割DNA,所述限制酶仅作用在DNA中特定序列(“序列特异性核酸内切酶”)。此处使用的多种限制性酶可以通过商业途径获得,并且它们的反应条件、辅因子和其他要求是本领域普通技术人员公知的。特定限制性酶的适宜缓冲液和底物量由制造商说明,或者易于在文献中找到。
“连接”指两个双链核酸片段之间形成磷酸二酯键的过程。除非另有提供,可以使用公知缓冲液和条件以DNA连接酶例如T4 DNA连接酶完成连接。
“质粒”指染色体外(通常)自我复制的遗传元件。
此处使用的“重组DNA克隆载体”指包含可以或已经加入一个或多个额外DNA片段的DNA分子的任何自主复制的介质,包括但是不限于质粒和噬菌体。
此处使用的“重组DNA表达载体”指整合了控制插入DNA转录的启动子的任何重组DNA克隆载体。
“转录”指将DNA核酸序列中含有的信息转移到互补RNA序列的过程。
“转染”指宿主细胞对表达载体的吸收,无论事实上是否表达任何编码序列。普通技术人员已知大量转染方法,例如磷酸钙共沉淀、脂质体转染和电穿孔。当该载体作用的任何指征出现在宿主细胞内时,一般认为成功转染。
“转化”指将DNA引入生物体,该DNA从而可以作为染色体外元件或通过染色体整合而复制。转化细菌和真核宿主的方法在本领域广为人知,在J.Sambrook等人,《Molecular Cloning:A Laboratory Manual》(1989)中总结了许多方法,例如核注射、原生质体融合或通过使用氯化钙的钙处理。通常,将DNA引入酵母时,术语转化与术语转染相对使用。
此处使用的“翻译”指使用信使RNA(mRNA)的遗传信息指定并指导多肽链合成的过程。
“载体”指用于基因操作中细胞转染和/或转化的核酸化合物,其携带对应于适当蛋白质分子的多核苷酸序列,其当与适当的控制序列组合时把特定性质赋予将被转染和/或转化的宿主细胞。质粒、病毒和噬菌体是适宜的载体。使用限制性酶和连接酶切割和连接不同来源的DNA分子构建人工载体。此处使用的术语“载体”包括重组DNA克隆载体和重组DNA表达载体。
此处使用的“互补”或“互补性”指双链核酸中通过氢键结合的碱基对(嘌呤和嘧啶)。下列碱基对是互补的:鸟嘌呤和胞嘧啶;腺嘌呤和胸腺嘧啶;以及腺嘌呤和尿嘧啶。
“引物”指起酶或合成延伸的启动底物功能的核酸片段。
“启动子”指指导DNA转录为RNA的DNA序列。
“探针”指与另一种核酸化合物杂交的核酸化合物或其片段。
“前导序列”指可以经酶或化学去除产生所需目的多肽的氨基酸序列。
“分泌信号肽”指通常出现在较大多肽N末端区域的氨基酸序列,其功能是启动所述多肽与细胞膜隔室例如内质网的结合以及该多肽通过质膜分泌。
可以从多种来源获得野生型人类IgG4蛋白质。例如,可以从以可检测水平表达目的mRNA的细胞制备cDNA文库以获得这些蛋白质。使用特定目的蛋白质的公开的DNA或蛋白质序列设计的探针可以对文库进行筛选。例如,在Adams等人,(1980)Biochemistry 19:2711-2719;Goughet等人,(1980)Biochemistry 19:2702-2710;Dolby等人,(1980)Proc.Natl.Acad.Sci.USA 77:6027-6031;Rice等人,(1982)Proc.Natl.Acad.Sci.USA79:7862-7862;Falkner等人,(1982)Nature 298:286-288;以及Morrison等人,(1984)Ann.Rev.Immunol.2:239-256中描述了免疫球蛋白轻或重链恒定区。
可以使用标准程序以所选探针筛选cDNA或基因组文库,例如在Sambrook等人,《Molecular Cloning:A Laboratory Manual》,Cold SpringHarbor Laboratory Press,NY(1989)中所述。分离编码免疫球蛋白蛋白质的基因的备选方法是使用PCR方法[Sambrook等人,如上;Dieffenbach等人,《PCR Primer:A Laboratory Manual》,Cold Spring HarborLaboratory Press,NY(1995)]。可以基于已发表的序列设计PCR引物。
从特定文库克隆的全长野生型序列通常可以作为产生本发明IgG4 Fc类似物片段的模板,其中IgG4 Fc类似物片段保留了赋予GLP-1类似物较长血浆半寿期的能力,GLP-1类似物是的融合蛋白质的一部分。可以使用PCR技术,用经设计与对应于IgG4 Fc类似物片段所需末端的序列杂交的引物产生该片段。也可以设计PCR引物产生限制性酶位点以便于克隆到表达载体中。
可以通过多种不同的方法产生编码本发明的GLP-1类似物的DNA,所述方法包括如上所述的那些克隆方法和化学合成的DNA。假设编码肽的长度短,化学合成是有吸引力的。GLP-1的氨基酸序列及前胰高血糖素原基因序列已经发表。[Lopez等人,(1983)Proc.Natl.Acad.Sci.,USA80:5485-5489;Bell等人,(1983)Nature,302:716-718;Heinrich,G.等人,(1984)Endocrinol,115:2176-2181;Ghiglione,M.等人,91984)Diabetologia27:599-600]。因此,可以基于天然序列设计引物以产生此处所述的编码GLP-1类似物的DNA。
然后可以通过将编码GLP-1类似物的DNA在框内与此处所述的编码IgG Fc蛋白质的DNA连接而构建编码融合蛋白质的基因。可以在连接前或在编码整个融合蛋白质的cDNA中突变编码野生型GLP-1和IgG4 Fc片段的DNA。多种诱变技术在本领域广为人知。编码GLP-1类似物的基因和编码IgG4 Fc类似物蛋白质的基因也可以通过编码富含G的接头肽的DNA在框内连接。在SEQ ID NO:20中提供编码本发明优选的异源融合蛋白质之一(Gly8-Glu22-Gly36 GLP-1(7-37)-1L-IgG4(S228P、F234A、L235A、des K))的优选DNA序列:
CACGGCGAGGGCACCTTCACCTCCGACGTGTCCTCCTATCTCGAGGAGCAGG
CCGCCAAGGAATTCATCGCCTGGCTGGTGAAGGGCGGCGGCGGTGGTGGTGG
CTCCGGAGGCGGCGGCTCTGGTGGCGGTGGCAGCGCTGAGTCCAAATATGGT
CCCCCATGCCCACCCTGCCCAGCACCTGAGGCCGCCGGGGGACCATCAGTCTT
CCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGG
TCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAA
CTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAG
GAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCA
GGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTC
CCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGC
CACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGT
CAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGT
GGGAAAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCT
GGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGC
AGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGC
ACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGT(SEQ ID NO:20)
以此处所述用于异源融合蛋白质产生的表达或克隆载体转染或转化宿主细胞,并在经改良适于诱导启动子、选择转化子或扩增编码所希望的序列的基因的常规营养培养基中培养。技术人员不用过多实验方法即可以选择例如培养基、温度、pH等培养条件。一般而言,可以在《Mammalian CellBiotechnology:A Practical Approach》,M.Butler编(IRL Press,1991)和如上之Sambrook等人著作中找到将细胞培养生产力最大化的原理、方法和实践技术。普通技术人员已知转染方法,例如CaPO4和电穿孔。在美国专利号4,399,216中描述了哺乳动物细胞宿主系统转化的一般方面。通常根据van Solingen等人,J Bact.130(2):946-7(1977)和Hsiao等人,Proc.Natl.Acad.Sci.USA 76(8):3829-33(1979)的方法进行酵母转化。然而,也可以使用将DNA引入细胞的其他方法,例如核微注射、电穿孔、细菌与完整细胞的原生质体融合、或聚阳离子,例如1,5-二甲基-1,5-二氮十一亚甲基聚甲溴化物(polybrene)或polyomithine。关于转化哺乳动物细胞的多种技术,见Keown等人,Methods in Enzymology 185:527-37(1990)和Mansour等人,Nature 336(6197):348-52(1988)。
克隆或表达此处载体中核酸(例如DNA)的合适的宿主细胞包括酵母或高等真核细胞。
真核微生物例如丝状真菌或酵母是融合蛋白质载体的合适的克隆或表达宿主。酿酒酵母(Saccharomyces cerevisiae)是常用的低等真核宿主微生物。其他包括粟酒裂殖酵母(Schizosaccharomyces pombe)[Beach和Nurse,Nature 290:140-3(1981);1995年5月2日公布的EP 139,383];Muyveromyces宿主[美国专利号4,943,529;Fleer等人,Bio/Technology9(10):968-75(1991)]例如乳酸克鲁维酵母(K.lactis)(MW98-8C、CBS683、CBS4574)[de Louvencourt等人,J.Bacteriol.154(2):737-42(1983)];脆壁克鲁维酵母(K.fiagilis)(ATCC 12,424)、保加利亚克鲁维酵母(K.bulgaricus)(ATCC 16,045)、威克克鲁维酵母(K wickeramii)(ATCC24,178)、K waltii(ATCC 56,500)、果蝇克鲁维酵母(K.drosophilarum)(ATCC 36.906)[Van den Berg等人,Bio/Technology 8(2):135-9(1990)];K.thermotoierans和马克斯克鲁维酵母(K.marxianus);yarrowia(EP402,226);巴斯德毕赤酵母(Pichia pastoris)(EP 183,070)[Sreekrishna等人,J.Basic Microbiol.28(4):265-78(1988)];假丝酵母属(Candida);Trichoderma reesia(EP 244,234);粗糙脉孢霉(Neurospora crassa)[Case等人,Proc.Natl.Acad Sci.USA 76(10):5259-63(1979)];许旺酵母属(Schwanniomyces),例如许旺酵母(Schwanniomyces occidentulis)(EP394,538,1990年10月31日发表);以及丝状真菌例如脉孢霉属(Neurospora)、青霉属(Penicillium)、Tolypocladium(WO 91/00357,1991年1月10日发表)以及曲霉属(Aspergillus)宿主,例如构巢曲霉(A.nidulans)[Balance等人,Biochem.Biophys.Res.Comm.112(1):284-9(1983)];Tilburn等人,Gene 26(2-3):205-21(1983);Yelton等人,Proc.Natl.Acad.Sci.USA 81(5):1470-4(1984)]和黑曲菌(A.niger)[Kelly和Hynes,EMBO J.4(2):475-9(1985)]。甲基营养酵母选自汉逊酵母属(Hansenula)、假丝酵母属(Candida)、克勒克酵母属(Kloeckera)、毕赤酵母属(Pichia)、酵母属、球拟酵母属(Torulopsis)和红酵母属(Rhodotorula)。可以在C.Antony,《The Biochemistry of Methylotrophs》,269(1982)中找到这类酵母示范性的特定种的名单。
表达本发明融合蛋白质的合适的宿主细胞来自多细胞生物。无脊椎动物细胞的实例包括昆虫细胞,例如果蝇S2和灰赤夜蛾(Spodoptera)Sp、灰赤夜蛾high5及植物细胞。有用的哺乳动物宿主细胞系的实例包括NSO骨髓瘤细胞、中国仓鼠卵巢(CHO)细胞、SP2和COS细胞。更特定的实例包括以SV40转化的猴肾脏CVl系(COS-7,ATCC CRL 1651);人胚胎肾系[293或在悬浮培养中生长的亚克隆293细胞,Graham等人,J.Gen Virol.,36(1):59-74(1977)];中国仓鼠卵巢细胞/-DHFR[CHO,Urlaub和Chasin,Proc.Natl.Acad.Sci.USA,77(7):4216-20(1980)];小鼠支持细胞[TM4,Mather,Biol.Reprod.23(1):243-52(1980)];人类肺细胞(W138.ATCCCCL 75);人类肝细胞(Hep G2、HB 8065);以及小鼠乳腺肿瘤(MMT 060562,ATCC CCL51)。产生本发明Fc融合蛋白质的优选细胞系是可从欧洲动物细胞保藏中心(European Collection of Cell Cultures)获得的NS0骨髓瘤细胞系(ECACC,目录号#85110503),其在Galfre,G.和Milstein,C.描述((1981)Methods in Enzymology 73(13):3-46;及《Preparation ofMonoclonal Antibodies:Strategies and Procedures》,Academic Press,N.Y.,N.Y.)中描述。
可以直接或作为具有信号序列或其他附加序列蛋白质重组产生本发明的融合蛋白质,所述其他附加序列在成熟融合蛋白质N末端产生特异剪切位点。一般,信号序列可以是载体组分,或可以是插入到载体中的编码融合蛋白质的DNA的一部分。对于酵母分泌,信号序列可以是例如酵母转化酶前导序列、α因子前导序列(包括酵母和克鲁维氏酵母cc-因子前导序列,后者在美国专利号5,010,182中有描述)、或酸性磷酸酶前导序列、白假丝酵母(C.albicans)葡糖淀粉酶前导序列(EP 362,179)或WO 90/13646中描述的信号。在哺乳动物细胞表达中,哺乳动物信号序列可以用于指导蛋白质的分泌,例如相同或相关物种分泌多肽的信号序列和病毒分泌性前导序列。
表达和克隆载体都含有使载体能够在一种或多种所选宿主细胞中复制的核酸序列。表达和克隆载体一般将含有选择基因,也称为选择标记。典型的选择基因编码(a)赋予抗生素或其他毒素(例如新霉素、氨甲蝶呤或四环素)抗性、(b)弥补自养缺陷、或(c)补充不能从复杂培养基获得的关键营养物(例如编码芽孢杆菌D-丙氨酸消旋酶的基因)的蛋白质。
哺乳动物细胞合适的选择标记的实例是能够鉴定有能力吸收融合蛋白质编码核酸的细胞的那些选择标记,例如DHFR或胸苷激酶。当使用野生型DHFR时,恰当的宿主细胞是如描述[Urlaub和Chasin,Proc.Natl.Acad.Sci.USA,77(7):4216-20(1980)]所描述的制备并增殖的DHFR活性缺陷的CHO细胞系。在酵母中使用的合适的选择基因是存在于酵母质粒YRp7中的trpl基因[Stinchcomb等人,Nature 282(5734):39-43(1979);Kingsman等人,Gene 7(2):141-52(1979);Tschumper等人,Gene 10(2):157-66(1980)]。Trpl基因为缺乏在色氨酸中生长的能力的酵母突变株例如ATCCNo.44076或PEPC1提供选择标记[Jones,Genetics 85:23-33(1977)]。
表达和克隆载体通常含有与融合蛋白质编码核酸序列有效连接以指导mRNA合成的启动子。由多种可能的宿主细胞识别的启动子广为人知。与酵母宿主使用的合适的启动子序列实例包括3-磷酸甘油酸激酶[Hitzeman等人,J.Biol.Chem.255(24):12073-80(1980)]或其他糖酵解酶[Hess等人,J.Adv.Enzyme Reg.7:149(1968);Holland,Biochemistry 17(23):4900-7(1978)],例如烯醇酶、甘油醛-3-磷酸脱氢酶、己糖激酶、丙酮酸脱羧酶、磷酸果糖激酶、葡萄糖-6-磷酸异构酶、3-磷酸甘油酸变位酶、丙酮酸激酶、丙糖磷酸异构酶、磷酸葡萄糖异构酶和葡糖激酶。其他酵母启动子是醇脱氢酶2、异细胞色素C、酸性磷酸酶、与氮素代谢有关的降解酶、金属硫蛋白、3-磷酸甘油醛脱氢酶和负责麦芽糖和半乳糖利用的酶的启动子区,所述启动子是可诱导的启动子,具有由生长条件控制转录的额外的优点。在EP 73,657中进一步描述了用于酵母表达的合适的载体和启动子。例如,通过启动子可以控制融合蛋白质编码mRNA从哺乳动物宿主细胞中载体的转录,所述启动子可以来自病毒基因组,例如多瘤病毒、禽痘病毒、腺病毒(例如腺病毒2)、牛乳头瘤病毒、鸟肉瘤病毒、巨细胞病毒、逆转录病毒、乙型肝炎病毒和猿病毒40(SV 40);异源哺乳动物启动子,例如肌动蛋白启动子或免疫球蛋白启动子,以及热休克启动子,条件是这些启动子与宿主细胞系统相容。
可以通过向载体中插入增强子序列增加高等真核细胞对编码融合蛋白质的多核苷酸的转录。增强子是作用于启动子以增加其转录的DNA顺式作用元件,通常约10至300bp。已知许多来自哺乳动物基因(珠蛋白、弹性蛋白酶、白蛋白、a-酮蛋白(ketoprotein)和胰岛素)的增强子序列。然而,人们一般将使用真核细胞病毒启动子。实例包括复制起点后侧的SV40增强子(bp 100-270)、巨细胞病毒早期启动子增强子、复制起点后侧的多瘤病毒增强子、以及腺病毒增强子。可以将增强子在融合蛋白质编码序列的5′或3′位剪接在载体中,但是优选位于启动子5′位。
用于真核宿主细胞(酵母、真菌、昆虫、植物、动物、人类或其他多细胞生物的有核细胞)的表达载体也将含有转录终止和稳定mRNA所必需的序列。通常可以从真核或病毒DNA或cDNA的5′以及偶然从3′非翻译区获得这些序列。这些区域含有转录为编码融合蛋白质的mRNA中非翻译部分中聚腺苷酸化片段的核苷酸片段。
可以从培养基或宿主细胞裂解物中回收多种形式的融合蛋白质。如果是膜结合的,可以使用合适的去污剂溶液(例如Triton-X 100)或酶切割将其从膜上释放。可以通过多种物理或化学方法(例如循环冻融、超声处理、机械破碎或细胞裂解试剂)来破碎融合蛋白质表达中使用的细胞。
一旦在合适的宿主细胞中表达本发明的异源融合蛋白质,就可以分离并纯化类似物。下列步骤是适宜的纯化步骤的代表:羧甲基纤维素分级分离;凝胶过滤例如Sephadex G-75;阴离子交换树脂例如DEAE或Mono-Q;阳离子交换例如CM或Mono-S;金属鳌合柱以结合多肽的表位标签形式;反相HPLC;层析聚焦;硅胶;乙醇沉淀;以及硫酸铵沉淀。
可以使用多种蛋白质纯化方法,这类方法为本领域所公知并在例如Deutscher,Methods in Enzymology 182:83-9(1990)和《Scopes,ProteinPurification:Principles and Practice》,Springer-Verlag,NY(1982)中得到描述。所选的纯化步骤取决于使用的生产方法以及产生的特定融合蛋白质的性质。例如,使用蛋白质A或蛋白质G亲合基质可以有效纯化包含Fc片段的融合蛋白质。可以使用低或高pH缓冲液从亲合基质洗脱融合蛋白质。温和的洗脱条件将有助于防止融合蛋白质的不可逆变性。
可以用一种或多种赋形剂配制本发明的异源融合蛋白质。本发明的融合蛋白质可以与可药用的缓冲液、经调节提供可接受的稳定性的pH、以及可施用(例如胃肠外施用)的pH组合。任选地,可以添加一种或多种可药用的抗微生物剂。间甲酚和苯酚是优选的可药用抗微生物剂。可以添加一种或多种可药用盐溶液以调节离子强度或张力。可以添加一种或多种赋形剂以进一步调节制剂的等渗性。甘油是等渗性调节赋形剂的实例。可药用意味着适于施用于人类或其他动物,因此不含有毒性成分或不希望的污染物,并且不干扰其中活性化合物的活性。
可以以溶液制剂或能够用合适的稀释剂重构的冻干粉配制本发明的异源融合蛋白质。冻干剂型是其中融合蛋白质稳定的一种剂型,具有或不具有重构产品在预期的使用货架期内保持pH的缓冲能力。包含在此讨论的异源融合蛋白质的溶液在冻干前优选是等渗的,使之重构后能够形成等渗溶液。
本发明的异源融合蛋白质的可药用盐溶液形式在本发明范围内。常用于形成酸加成盐的酸为无机酸,例如盐酸、氢溴酸、氢碘酸、硫酸、磷酸等,以及有机酸,例如对甲苯磺酸、甲磺酸、草酸、对溴苯基-磺酸、碳酸、琥珀酸、柠檬酸、苯甲酸、乙酸等。优选的酸加成盐是与无机酸,例如盐酸和氢溴酸形成的盐。
碱加成盐包括从无机碱,例如铵、碱或碱土金属氢氧化物衍生的那些盐、碳酸盐、碳酸氢盐等。在制备本发明的盐溶液中有用的这类碱因此包括氢氧化钠、氢氧化钾、氢氧化铵、碳酸钾等。
本发明的异源融合蛋白质具有生物学活性。生物学活性指该融合蛋白质体内结合并激活GLP-1受体并引起反应的能力。反应包括但是不限于胰岛素的分泌、胰高血糖素的抑制、抑制食欲、体重减轻、诱导过饱、抑制凋亡、诱导胰腺β细胞增殖及胰腺β细胞分化。对有代表性的一些GLP-1融合蛋白质测试体外和体内活性。实施例1和2提供了基于该融合蛋白质与人类GLP-1受体相互作用并将其活化的能力的体外活性。在两组实验中使用过表达人类GLP-1受体的HEK293细胞。在这些细胞中,GLP-1受体的活化引起腺苷酸环化酶的活化,该酶的活化又诱导由环AMP应答元件(CRE)驱动的报告基因的表达。实施例1(表1)提供了其中报告基因为β内酰胺酶的数据,实施例2(表2)提供了其中报告基因为萤光素酶的数据。实施例3提供对大鼠施用本发明的异源融合蛋白质之一后产生的数据。这些数据共同表明融合蛋白质能够结合并活化GLP-1受体,并且在体外表现比Val8-GLP-1(7-37)OH更有效。另外,大鼠中产生的数据显示该融合蛋白质在体内具有活性,并且比天然GLP-1具有更长的半寿期。
可以通过具有普通技能的内科医生已知有效的任意途径施用该异源融合蛋白质。外周肠胃外一种此类方法。医学文献中通常将肠胃外施用理解为通过消毒注射器或一些其他机械装置例如注射泵向机体注射剂型。外周肠胃外可以包括静脉内、肌内、皮下和腹膜内施用途径。
本发明的异源融合蛋白质也可以进行经口、直肠、经鼻或下呼吸道途径施用,它们是非肠胃外途径。这些非肠胃外途径中,优选下呼吸道途径和经口途径。
本发明的融合蛋白质可以用于治疗多种疾病和病症。本发明的融合蛋白质首先通过作用于被称为“GLP-1受体”的受体而发挥其生物学作用。因此可以用本发明的GLP-1融合蛋白质治疗对GLP-1受体刺激或对GLP-1化合物施用有利应答的疾病和/病症的受试者。将这些受试者称为“需要GLP-1化合物治疗”或“需要GLP-1受体刺激”。包括非胰岛素依赖性糖尿病、胰岛素依赖性糖尿病、中风(见WO 00/16797)、心肌梗死(见WO 98/08531)、肥胖(见WO 98/19698)、手术后分解代谢改变(见美国专利号6,006,753)、功能性消化不良和肠易激综合征(见WO99/64060)。也包括要求以GLP-1化合物预防性治疗的受试者,例如具有发展为非胰岛素依赖性糖尿病危险的受试者(见WO 00/07617)。糖耐量损伤或空腹葡萄糖损伤的受试者、体重对于受试者身高和体液高出正常体重约25%的受试者、部分胰切除的受试者、父母一方或双方具有非胰岛素依赖性糖尿病的受试者、妊娠期糖尿病的受试者以及急性或慢性胰腺炎受试者具有发展为非胰岛素依赖性糖尿病的危险。
此处描述的GLP-1-Fc融合蛋白质的有效量是施用于需要GLP-1受体刺激的受试者时引起所希望的治疗和/或预防效果而不导致不可接受的副作用的量。“所希望的治疗性效果”包括下列一项或多项:1)疾病或病症相关症状的改善;2)疾病或病症相关症状发作的延迟;3)与没有治疗相比增长的寿命;以及4)与没有治疗相比更好的生活质量。例如,糖尿病治疗的GLP-1-Fc融合蛋白质的“有效量”是与没有治疗相比引起对血糖浓度的更佳控制,从而导致糖尿病并发症(例如视网膜病、神经病或肾脏疾病)发作延迟的量。预防糖尿病的GLP-1-Fc融合蛋白质的“有效量”是与没有治疗相比延缓血糖水平升高的发作的量,所述发作需要以抗低血糖药物例如磺酰脲类、噻唑烷二酮、胰岛素和/或双胍类治疗。
有效将患者血糖正常化的融合蛋白质的剂量取决于许多因素,其中包括但不限于受试者性别、体重和年龄、调节血糖无能的严重性、施用途径和生物利用率。融合蛋白质药物代谢动力学图谱、潜能和剂型。剂量可以为0.01至1mg/kg体重,优选0.05至0.5mg/kg体重。
优选每两周一次或每周一次施用本发明的融合蛋白质。取决于所治疗的疾病,可能有必要更频繁地施用该融合蛋白质,例如每周两至三次。
现在仅以非限制性实例参考下列实施例对本发明进行描述。
实施例
实施例1-体外GLP-1受体激活试验
使用CRE-BLAM系统将表达人类GLP-1受体的HEK-293细胞以20,000至40,000细胞/孔/100μl含10%FBS的DMEM培养基接种到多聚-d-赖氨酸包被的96孔黑色透明底板中。接种后一天,弹去(flick off)培养基,添加80μl无血清DMEM培养基。接种后第三天,将含0.5%BSA、含有不同浓度的多种GLP-1-Fc异源融合蛋白质的无血清DMEM培养基20μl加入各孔,以产生剂量反应曲线。通常,使用含有3纳摩尔至30纳摩尔异源GLP-1Fc融合蛋白质的14种稀释液来产生剂量反应曲线,从该曲线可以确定EC50值。以融合蛋白质孵育5小时后,加入20μl β-内酰胺酶底物(CCF2/AM,PanVera LLC),并持续培养1小时,此时在细胞荧光计上测定荧光。在Zlokarnik等人,(1998),Science,278:84-88中进一步描述了该测定。测试多种GLP-1-Fc融合蛋白质,EC50值在表1中给出。这些值是相对于Val8-GLP-1(7-37)OH的测定值,Val8-GLP-1(7-37)OH作为各实验内部对照。
表1
实施例2-体外GLP-1受体激活试验
使用CRE-荧光素酶系统将稳定表达人类GLP-1受体的HEK-293细胞以30,000个细胞/孔/80μl低血清DMEM F12培养基接种于96孔板中。接种后一天,将溶于0.5%BSA的待测蛋白质的20μl等分试样与细胞混合并孵育5小时。对于每种待测蛋白质,一般以5×浓度制备含有3pM至3nM的12个稀释物后加入细胞,从而产生剂量反应曲线,从中确定EC50值。孵育后,将100μl荧光素酶直接加入各板,轻轻混合2分钟。将板置入Tri-lux发光计并计算荧光素酶表达引起的光输出。测试多种GLP-1-Fc融合蛋白质,EC50值呈于表2中。这些值是相对于Val8-GLP-1(7-37)OH的测定值,Val8-GLP-1(7-37)OH作为各实验内部对照。由于下面测试的融合蛋白质为二聚体,考虑摩尔浓度2倍差异校正所述值。
表2
实施例3大鼠中静脉内葡萄糖耐量试验
在大鼠静脉内葡萄糖耐量测定中评价Fc融合蛋白质Gly8-Glu22-Gly36-GLP-1(7-37)-L-IgG4(S228P、F234A、L235A)。三组中每组包含至少四只大鼠。I组接受载体(表3),II组接受1.79mg/kg的Gly8-Glu22-Gly36-GLP-1(7-37)-L-IgG4(S228P、F234A、L235A)作为单次皮下注射(表4),III组接受0.179mg/kg的Gly8-Glu22-Gly36-GLP-1(7-37)-L-IgG4(S228P、F234A、L235A)作为单次皮下注射(表5)。在第一天早晨皮下注射大鼠。首次注射二十四小时之后,每克大鼠体重1μL葡萄糖(D50)以快速浓注灌注。在葡萄糖快速浓注灌注后的2、4、6、10、20和30分钟采集血液样品。
表3
表4
表5
实施例4单次皮下注射猕猴后的药物代谢动力学研究
以0.1mg/kg皮下(SC)注射雄性猕猴时,进行研究以表征Fc融合蛋白质Gly8-Glu22-Gly36-GLP-1(7-37)-L-IgG4(S228P、F234A、L235A)的药物代谢动力学(PK)。RIA抗体对GLP中部具有特异性。ELISA使用N末端特异性捕获抗体和Fc特异性检测抗体。使用ELISA和RIA二者得到的血浆浓度确定提出的药物代谢动力学参数值。
得到的PK参数值的代表总结于表6中。来自RIA的单剂SC PK与446.7ng/mL的平均Cmax及相应17.3小时的Tmax关联。平均清除半寿期约79.3小时(3.3天)。来自ELISA的PK与292.2ng/mL的平均Cmax及相应16.7小时的Tmax关联。评价清除半寿期约51.6小时(2.2天)。
表6
a观察到的最大血浆浓度。
b观察到的最大血浆浓度的时间。
c血浆浓度-时间曲线下测量的0至无穷大的面积。
d消除半寿期。
e总机体清除率关于生物利用率的函数。
f分布体积关于生物利用率的函数。
SD=标准差。
实施例5重复皮下注射后抗体潜在形成的评价
使用直接ELISA形式对指定猕猴血清样品测试抗Gly8-Glu22-Gly36-GLP-1(7-37)-L-IgG4(S228P、F234A、L235A)抗体的形成。以0.1μg/mL浓度的Gly8-Glu22-Gly36-GLP-1(7-37)-L-IgG4(S228P、F234A、L235A)包被微量滴定板。将猴血清样品50、500、1000和5000倍稀释到封闭溶液中,孵育0.05mL样品/孔约一小时。将二级抗体山羊<人Fab′2>-过氧化物酶(与人类75%的交叉反应性)10,000倍稀释于封闭溶液中,以0.05mL/孔加入并孵育约一小时。在450nm-630nm光密度读取使用四甲基联苯胺(TMB)底物的显色。将一式两份读数平均。使用GLP-1抗体作为阳性对照,山羊<兔>(H+L)-过氧化物酶缀合物是用于检测的二级抗体。在给药前、第二次给药24小时后、第一次和第二次皮下给药168小时后收集时点血清样品(Point serum samples)以评价可能的免疫原性。通过与给药前血清样品和阳性对照的比较解释对G8E22-CEX-L-hIgG4的抗体效价的存在。代表性结果呈于表7。
表7
剂量1动物# | 阳性对照 | IO7774 | IO7777 | IO7779 | IO7780 | ||||
采样时间: | 给药前 | 168h | 给药前 | 168h | 给药前 | 168h | 给药前 | 168h | |
50x | 2.854 | 0.268 | 0.268 | 0.160 | 0.128 | 0.144 | 0.152 | 0.264 | 0.224 |
500x | 2.270 | 0.117 | 0.133 | 0.052 | 0.069 | 0.065 | 0.061 | 0.067 | 0.061 |
1000x | 1.610 | 0.091 | 0.075 | 0.034 | 0.051 | 0.047 | 0.045 | 0.138 | 0.049 |
5000x | 0.525 | 0.056 | 0.048 | 0.032 | 0.037 | 0.029 | 0.033 | 0.051 | 0.039 |
剂量2动物# | 阳性对照 | IO7774 | IO7777 | IO7779 | IO7780 | ||||
采样时间: | 给药前 | 24h | 给药前 | 24h | 给药前 | 24h | 给药前 | 24h | |
50x | 3.056 | 0.298 | 0.231 | 0.164 | 0.159 | 0.227 | 0.176 | 0.211 | 0.192 |
500x | 2.247 | 0.120 | 0.119 | 0.048 | 0.045 | 0.061 | 0.060 | 0.056 | 0.057 |
1000x | 1.673 | 0.090 | 0.086 | 0.039 | 0.041 | 0.046 | 0.045 | 0.043 | 0.048 |
5000x | 0.534 | 0.039 | 0.042 | 0.030 | 0.034 | 0.033 | 0.036 | 0.033 | 0.034 |
剂量2动物# | 阳性对照 | IO7774 | IO7777 | IO7779 | IO7780 | ||||
采样时间: | 给药前 | 168h | 给药前 | 168h | 给药前 | 168h | 给药前 | 168h | |
50x | 3.075 | 0.413 | 0.270 | 0.174 | 0.182 | 0.185 | 0.190 | 0.224 | 0.191 |
500x | 2.173 | 0.097 | 0.103 | 0.042 | 0.051 | 0.056 | 0.057 | 0.048 | 0.053 |
1000x | 1.510 | 0.066 | 0.067 | 0.038 | 0.040 | 0.037 | 0.046 | 0.043 | 0.043 |
5000x | 0.474 | 0.042 | 0.042 | 0.033 | 0.046 | 0.033 | 0.033 | 0.036 | 0.041 |
实施例6禁食状态和分级静脉内葡萄糖注射期间单次皮下注射猕猴
后的药物动
力学研究
1期(研究第一天)施用载体的皮下灌注。灌注载体后立即施用5、10、和25mg/kg/min的分级静脉内葡萄糖(20%右旋糖)灌注。2期(研究第三天)施用GLP-1融合蛋白质(0.1mg/kg)的皮下灌注。3期在GLP-1融合蛋白质灌注后约96小时进行分级静脉内葡萄糖灌注。
在16小时过夜禁食后经过镇静的猴中进行分级静脉内葡萄糖灌注步骤。对于两次静脉内葡萄糖灌注,将每10分钟共20分钟取基线样品以定义基线。在+20分钟以5mg/kg/min的速率启动升高的葡萄糖灌注,随后为10mg/kg/min和25mg/kg/min的灌注。各灌注速率监测20分钟时程。以10分钟间隔采集血液样品以测量葡萄糖、胰岛素和胰高血糖素。在葡萄糖灌注前-20、-10分钟、0分钟以及1和3期葡萄糖灌注后10、20、30、40、50和60分钟收集约1.0mL血液。
数据显示于表8。
表8
以载体和GLP-1融合蛋白质给药的猴子之间胰高血糖素水平无统计学差异。
实施例7禁食状态和分级静脉内葡萄糖灌注期间三个不同剂量单次
皮下注射大鼠后的药物动力学研究
将长期置入导管的大鼠分配到载体对照(盐溶液)和三个治疗组之一(GLP-1融合蛋白质;0.0179mg/kg、0.179mg/kg、或1.79mg/kg)。通过皮下注射施用GLP-1融合蛋白质和载体。治疗二十四小时后,使过夜禁食(16h)的大鼠受到分级静脉内葡萄糖灌注测试。分级葡萄糖灌注测试由基线盐溶液灌注射期(20分钟)、随后的分别为5和15mg/kg/min的两个30分钟葡萄糖灌注期组成。在葡萄糖灌注前-20、-10分钟、0分钟(基线)以及10、20、30、40、50和60分钟收集血浆样品。
数据表示于表9中。
表9
*与载体相对P≤0.05
序列表
<110>伊莱利利公司
<120>GLP-1类似物融合蛋白质
<130>X-15984
<150>60/477880
<151>2003-06-12
<160>21
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Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro
100 105 110
Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
115 120 125
Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn
130 135 140
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
145 150 155 160
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
165 170 175
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg
180 185 190
Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Ash Val Phe Ser Cys
195 200 205
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
210 215 220
Ser Leu Ser Leu Gly Xaa
225 230
<210>8
<211>15
<212>PRT
<213>人工的
<220>
<223>合成构建体
<400>8
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10 15
<210>9
<211>31
<212>PRT
<213>人(Homo sapiens)
<400>9
His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly
20 25 30
<210>10
<211>71
<212>PRT
<213>人工的
<220>
<223>合成构建体
<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 Arg Gly Gly
20 25 30
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
35 40 45
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Ser
50 55 60
Lys Tyr Gly Pro Pro Cys Pro
65 70
<210>11
<211>9
<212>PRT
<213>人工的
<220>
<223>合成构建体
<400>11
Trp Leu Val Lys Gly Arg Gly Gly Gly
1 5
<210>12
<211>7
<212>PRT
<213>人工的
<220>
<223>合成构建体
<400>12
Trp Leu Val Lys Gly Gly Gly
1 5
<210>13
<211>7
<212>PRT
<213>人工的
<220>
<223>合成构建体
<400>13
Trp Leu Lys Asn Gly Gly Gly
1 5
<210>14
<211>7
<212>PRT
<213>人工的
<220>
<223>合成构建体
<400>14
Trp Leu Val Lys Gly Gly Pro
1 5
<210>15
<211>7
<212>PRT
<213>人工的
<220>
<223>合成构建体
<400>15
Trp Leu Lys Asn Gly Gly Pro
1 5
<210>16
<211>6
<212>PRT
<213>人工的
<220>
<223>合成构建体
<400>16
Trp Leu Val Lys Gly Gly
1 5
<210>17
<211>6
<212>PRT
<213>人工的
<220>
<223>合成构建体
<400>17
Trp Leu Lys Asn Gly Gly
1 5
<210>18
<211>6
<212>PRT
<213>人
<400>18
Pro Pro Cys Pro Ser Cys
1 5
<210>19
<211>22
<212>PRT
<213>人工的
<220>
<223>合成构建体
<400>19
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
1 5 10 15
Ser Gly Gly Gly Gly Ser
20
<210>20
<211>825
<212>DNA
<213>人
<400>20
cacggcgagg gcaccttcac ctccgacgtg tcctcctatc tcgaggagca ggccgccaag 60
gaattcatcg cctggctggt gaagggcggc ggcggtggtg gtggctccgg aggcggcggc 120
tctggtggcg gtggcagcgc tgagtccaaa tatggtcccc catgcccacc ctgcccagca 180
cctgaggccg ccgggggacc atcagtcttc ctgttccccc caaaacccaa ggacactctc 240
atgatctccc ggacccctga ggtcacgtgc gtggtggtgg acgtgagcca ggaagacccc 300
gaggtccagt tcaactggta cgtggatggc gtggaggtgc ataatgccaa gacaaagccg 360
cgggaggagc agttcaacag cacgtaccgt gtggtcagcg tcctcaccgt cctgcaccag 420
gactggctga acggcaagga gtacaagtgc aaggtctcca acaaaggcct cccgtcctcc 480
atcgagaaaa ccatctccaa agccaaaggg cagccccgag agccacaggt gtacaccctg 540
cccccatccc aggaggagat gaccaagaac caggtcagcc tgacctgcct ggtcaaaggc 600
ttctacccca gcgacatcgc cgtggagtgg gaaagcaatg ggcagccgga gaacaactac 660
aagaccacgc ctcccgtgct ggactccgac ggctccttct tcctctacag caggctaacc 720
gtggacaaga gcaggtggca ggaggggaat gtcttctcat gctccgtgat gcatgaggct 780
ctgcacaacc actacacaca gaagagcctc tccctgtctc tgggt 825
<210>21
<211>30
<212>PRT
<213>人工的
<220>
<223>合成构建体
<400>21
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
1 5 10 15
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
20 25 30
Claims (11)
1.异源融合蛋白质,其包含GLP-1类似物,其中GLP-1类似物由选自如下的序列组成:
a)(SEQ ID NO:1)
His-Xaa8-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu-
Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Gly-Gly
其中Xaa8选自Gly和Val;
b)(SEQ ID NO:2)
His-Xaa8-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu-
Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Lys-Asn-Gly-Gly-Gly
其中Xaa8选自Gly和Val;
c)(SEQ ID NO:3)
His-Xaa8-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu-
Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Gly-Pro
其中Xaa8选自Gly和Val;
d)(SEQ ID NO:4)
His-Xaa8-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu-
Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Lys-Asn-Gly-Gly-Pro
其中Xaa8选自Gly和Val;
e)(SEQ ID NO:5)
His-Xaa8-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu-
Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Gly
其中Xaa8选自Gly和Val;
f)(SEQ ID NO:6)
His-Xaa8-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu-
Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Lys-Asn-Gly-Gly
其中Xaa8选自Gly和Val;
GLP-1类似物与由SEQ ID NO:7序列组成的免疫球蛋白Fc部分通过肽接头融合
Ala-Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Pro-Cys-Pro-Ala-Pro-
Xaa16-Xaa17-Xaa18-Gly-Gly-Pro-Ser-Val-Phe-Leu-Phe-Pro-Pro-Lys-Pro-
Lys-Asp-Thr-Leu-Met-Ile-Ser-Arg-Thr-Pro-Glu-Val-Thr-Cys-Val-
Val-Val-Asp-Val-Ser-Gln-Glu-Asp-Pro-Glu-Val-Gln-Phe-Asn-Trp-
Tyr-Val-Asp-Gly-Val-Glu-Val-His-Asn-Ala-Lys-Thr-Lys-Pro-Arg-
Glu-Glu-Gln-Phe-Xaa80-Ser-Thr-Tyr-Arg-Val-Val-Ser-Val-Leu-Thr-
Val-Leu-His-Gln-Asp-Trp-Leu-Asn-Gly-Lys-Glu-Tyr-Lys-Cys-Lys-
Val-Ser-Asn-Lys-Gly-Leu-Pro-Ser-Ser-Ile-Glu-Lys-Thr-Ile-Ser-
Lys-Ala-Lys-Gly-Gln-Pro-Arg-Glu-Pro-Gln-Val-Tyr-Thr-Leu-Pro-
Pro-Ser-Gln-Glu-Glu-Met-Thr-Lys-Asn-Gln-Val-Ser-Leu-Thr-Cys-
Leu-Val-Lys-Gly-Phe-Tyr-Pro-Ser-Asp-Ile-Ala-Val-Glu-Trp-Glu-
Ser-Asn-Gly-Gln-Pro-Glu-Asn-Asn-Tyr-Lys-Thr-Thr-Pro-Pro-Val-
Leu-Asp-Ser-Asp-Gly-Ser-Phe-Phe-Leu-Tyr-Ser-Arg-Leu-Thr-Val-
Asp-Lys-Ser-Arg-Trp-Gln-Glu-Gly-Asn-Val-Phe-Ser-Cys-Ser-Val-
Met-His-Glu-Ala-Leu-His-Asn-His-Tyr-Thr-Gln-Lys-Ser-Leu-Ser-
Leu-Ser-Leu-Gly-Xaaa230(SEQ ID NO:7)
其中:
第16位的Xaa为Pro或Glu;
第17位的Xaa为Phe、Val或Ala;
第18位的Xaa为Leu、Glu或Ala;
第80位的Xaa为Asn或Ala;以及
第230位的Xaa为Lys或缺乏。
2.权利要求1的异源融合蛋白质,其中GLP-1类似物的C末端甘氨酸残基通过肽接头与Fc部分的N末端丙氨酸残基融合,其中肽接头是选自如下的序列:
a)Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser(SEQ ID NO:8);
b)Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser(SEQ ID NO:19);以及
c)Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser(SEQ IDNO:21)。
3.权利要求2的异源融合蛋白质,其中接头是SEQ ID NO:8的序列。
4.权利要求1至3中任一项的异源融合蛋白质,其中GLP-1类似物的Xaa8为Gly。
5.权利要求1至3中任一项的异源融合蛋白质,其中GLP-1类似物的Xaa8为Val。
6.权利要求1至3中任一项的异源融合蛋白质,其中GLP-1类似物的序列是SEQ ID NO:1。
7.异源融合蛋白质,其选自:
a)Gly8-Glu22-Gly36-GLP-1(7-37)-1L-IgG4;
b)Gly8-Glu22-Gly36-GLP-1(7-37)-1L-IgG4(F234A,L235A);
c)Gly8-Glu22-Gly36-GLP-1(7-37)-1L-IgG4(N297A);
d)Gly8-Glu22-Gly36-GLP-1(7-37)-1L-IgG4(F234A,L235A,N297A);
e)Gly8-Glu22-Gly36-GLP-1(7-37)-1.5L-IgG4;
f)Gly8-Glu22-Gly36-GLP-1(7-37)-1.5L-IgG4(F234A,L235A);
g)Gly8-Glu22-Gly36-GLP-1(7-37)-1.5L-IgG4(N297A);
h)Gly8-Glu22-Gly36-GLP-1(7-37)-1.5L-IgG4(F234A,L235A,N297A);
i)Gly8-Glu22-Gly36-GLP-1(7-37)-2L-IgG4;
j)Gly8-Glu22-Gly36-GLP-1(7-37)-2L-IgG4(F234A,L235A);
k)Gly8-Glu22-Gly36-GLP-1(7-37)-2L-IgG4(N297A);
l)Gly8-Glu22-Gly36-GLP-1(7-37)-2L-IgG4(F234A,L235A,N297A);
及其des-K形式,其中L、1.5L和2L分别为SEQ ID NO:8、19和21所示,IgG4为SEQ ID NO:7所示,des-K表示IgG4的C末端赖氨酸缺失,并且其中IgG4后括号内的氨基酸替代为根据EU编号,其中SEQ ID NO:7中一个氨基酸的位置加上217等于EU编号中该氨基酸的位置。
8.异源融合蛋白质,其选自:
a)Val8-Glu22-Gly36-GLP-1(7-37)-1L-IgG4;
b)Val8-Glu22-Gly36-GLP-1(7-37)-1L-IgG4(F234A,L235A);
c)Val8-Glu22-Gly36-GLP-1(7-37)-1L-IgG4(N297A);
d)Val8-Glu22-Gly36-GLP-1(7-37)-1L-IgG4(F234A,L235A,N297A);
e)Val8-Glu22-Gly36-GLP-1(7-37)-1.5L-IgG4;
f)Val8-Glu22-Gly36-GLP-1(7-37)-1.5L-IgG4(F234A,L235A);
g)Val8-Glu22-Gly36-GLP-1(7-37)-1.5L-IgG4(N297A);
h)Val8-Glu22-Gly36-GLP-1(7-37)-1.5L-IgG4(F234A,L235A,N297A);
i)Val8-Glu22-Gly36-GLP-1(7-37)-2L-IgG4;
j)Val8-Glu22-Gly36-GLP-1(7-37)-2L-IgG4(F234A,L235A);
k)Val8-Glu22-Gly36-GLP-1(7-37)-2L-IgG4(N297A);
l)Val8-Glu22-Gly36-GLP-1(7-37)-2L-IgG4(F234A,L235A,N297A);
及其des-K形式,其中L、1.5L和2L分别为SEQ ID NO:8、19和21所示,IgG4为SEQ ID NO:7所示,des-K表示IgG4的C末端赖氨酸缺失,并且其中IgG4后括号内的氨基酸替代为根据EU编号,其中SEQ ID NO:7中一个氨基酸的位置加上217等于EU编号中该氨基酸的位置。
9.权利要求1至8中任一项的异源融合蛋白质用于生产药物的用途。
10.权利要求1至8中任一项的异源融合蛋白质的用途,用于生产治疗非胰岛素依赖性糖尿病的药物。
11.权利要求1至8中任一项的异源融合蛋白质的用途,用于生产在超重受试者中治疗肥胖或诱导体重减轻的药物。
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CN101974090A (zh) * | 2003-06-12 | 2011-02-16 | 伊莱利利公司 | Glp-1类似物融合蛋白质 |
CN101974090B (zh) * | 2003-06-12 | 2015-06-17 | 伊莱利利公司 | Glp-1类似物融合蛋白质 |
US11123438B2 (en) | 2016-08-19 | 2021-09-21 | Ampsource Biopharma Shanghai Inc. | Linker peptide for constructing fusion protein |
US11471513B2 (en) | 2016-08-19 | 2022-10-18 | Ampsource Biopharma Shanghai Inc. | Highly glycosylated human blood-clotting factor VIII fusion protein, and manufacturing method and application of same |
US11472863B2 (en) | 2016-08-19 | 2022-10-18 | Ampsource Biopharma Shanghai Inc. | Human coagulation factor IX (FIX) fusion protein, preparation method therefor, and use thereof |
US11833212B2 (en) | 2016-08-19 | 2023-12-05 | Ampsource Biopharma Shanghai Inc. | Linker peptide for constructing fusion protein |
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