CN103509118A - 胰岛素-Fc融合蛋白 - Google Patents
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Abstract
本发明属于生物技术领域，更具体地，本发明公开了一种胰岛素-Fc融合蛋白、其制备方法和用途。本发明公开的胰岛素-Fc融合蛋白具有半衰期长，可减少胰岛素使用的间隔时间，减少病人的用药痛苦等优点。
Description
技术领域
本发明属于生物技术领域，更具体地，本发明公开了一种融合蛋白、其制备方法和用途。
背景技术
糖尿病是一种日渐普遍的流行病，据估计，到2025年，糖尿病将影响3亿以上的人口，因此急需一种有效的药物治疗方法。2型糖尿病占所有病例的90-95％。因血糖水平持续升高引起的并发症包括心血管疾病、肾病、神经病和视网膜病。此外，在2型糖尿病晚期时，胰腺的β细胞死亡，从而停止分泌胰岛素。目前对糖尿病的治疗会产生多种有害的副作用，其中包括低血糖和体重增加。此外，目前对2型糖尿病的治疗并不能治愈这种疾病，而只是延缓时间，直至患者需要接受胰岛素治疗。
胰岛素这样的天然或者重组蛋白在水溶液状态下半保存期通常比较短，由于这些分子制剂的不稳定性使之必须冻干保存，给运输和保存带来不便。
胰岛素类的重组治疗蛋白的体内半衰期也很短，为了使用药间隔延长，必须生产体内半衰期长的药物。
发明内容
本发明公开了一种胰岛素-Fc融合蛋白，其通式为
B-C-A-Fc，其中A为胰岛素A链，B为胰岛素的B链，C为4-50个氨基酸的C肽连接序列，优选为6-30个氨基酸，Fc为免疫球蛋白Fc段或其突变体。
优选地，C为GGGPGKR、GGGPQT、GGGPGAG、AAGGGPSVR之一；C可以是天然或者人工序列，序列长度在4-50之间，主要为6-30个氨基酸。天然序列可以通过替代，增减修饰形成人工序列。为了减少免疫反应，C优选包含人血液中的天然人蛋白序列，例如天然胰岛素C肽序列(RREAEDLQVGQVELGGGPGAGSLQPLALEGSLQKR)、IGF-I C肽连接(GYGSSRRAPQT)，抗体重链铰链区序列(EPKSCDKTHTCPPCPAP)等。在有些结构中连接序列也可以只包含部分的上述序列，或者替代、增减修饰上述序列中一个或者几个氨基酸。连接序列可以被增减某个特定氨基酸。但是在一些结构中，连接序列不能包含两个连续的碱性氨基酸，例如KK，RR，RK。选择没有赖氨酸或者精氨酸的序列，或者选择没有酪氨酸的序列作为连接序列。
融合蛋白序列还包含一个N端的引导序列或者信号肽。例如抗体的信号肽，胰岛素的引导序列，酵母a因子信号序列，白蛋白信号肽序列，胰岛素样生长因子结合蛋白4信号肽序列，酸性磷酸酶信号肽序列等等。
在本发明中，该构造的抗体Fc部分保留了结合蛋白质A的能力并且可以利用蛋白质A亲和色谱法进行纯化。通过重组方法合成，并且可以代表任何人类或动物抗体的任一亚型。公众可从某些出版物(例如Kabat，et al.Sequences ofProteins of Immunological Interest，U.S.Dept.Health(1983)(Kabat等人，免疫学相关蛋白质序列，美国卫生部，1983年))或在线获得人和动物免疫球蛋白的序列。抗体的Fc区域通过对其Fc受体结合区域(H235-H236)进行突变例如从亮氨酸突变为丙氨酸，可降低补体结合、抗体依赖细胞介导的细胞毒性(ADCC)以及通过将该二聚体结构的亚区与免疫细胞表面受体(Fcγ受体)结合来介导的其他功能。
融合蛋白的基因可以连入真核表达载体pcdna3.1，pngfpN，PEE144等真核载体，转染CHO，NS0，SP2/0等细胞进行表达，或者连入pet22，pet32，Pgex等质粒转染BL21(DE3)，ROSETTA，AD494等菌株进行表达，或者连入pPICZ等载体转染毕赤酵母进行酵母表达。
融合蛋白可以通过proteinA亲和层析柱，proteinG亲和层析柱以及其它层析柱纯化获得高纯度的蛋白分子。
本发明所述的胰岛素-Fc融合蛋白具有胰岛素活性，能够结合胰岛素受体，刺激表达胰岛素受体的细胞摄取葡萄糖。胰岛素-Fc融合蛋白包含通过连接序列连接的A链和B链，例如胰岛素-Fc融合蛋白包括抗体的Fc段或其突变体通过N端或者C端与胰岛素类似物连接，其胰岛素类似物公式为-B-C-A-，其中C为C肽连接序列。在一些实例中，连接序列为6-30个氨基酸，一般情况下，连接序列可以使A链和B链形成具有胰岛素活性的分子。
本发明公开的胰岛素-Fc融合蛋白为治疗性蛋白，可为具有胰岛素-Fc融合蛋白活性的胰岛素、胰岛素类似物以及胰岛素片段和突变体与免疫球蛋白及其片段连接形成的融合蛋白，通过与Fc蛋白连接形成的融合蛋白，可以极大的改善胰岛素的药代动力学表现，提高胰岛素的体内半衰期，减少胰岛素使用的间隔时间，减少病人的用药痛苦。
附图说明
图1 pDR1表达载体结构示意图，hCMV表示人巨细胞病毒Major Immediate早期启动子；BGH pA表示Bovine growth hormone多腺苷化信号；SV40 ori表示SV40病毒早期复制起点；DHFR表示二氢叶酸还原酶基因；pUC origin为质粒复制起点；AMP为氨苄青霉素抗性基因。
图2.胰岛素-Fc融合蛋白基因结构示意图；信号肽是指胰岛素的信号肽或其他蛋白的信号肽基因，A链B链分别是指胰岛素的A链基因和B链基因，Fc是指突变后抗体的Fc段基因，C连接序列是指专利中提到的各种连接序列基因。
图3.胰岛素-Fc融合蛋白结构示意图，A链B链分别是指胰岛素的A和B链，Fc是指突变后抗体的Fc段。
图4.胰岛素-Fc融合蛋白与胰岛素受体结合力实验。
图5.胰岛素-Fc融合蛋白与胰岛素受体的竞争抑制曲线。
图6.胰岛素-Fc融合蛋白在用cAMP检测分析法测量时的活性。
图7.胰岛素-Fc融合蛋白体内血药浓度时间曲线，AUC是指药物的曲线下面积。
具体实施方式
以下实施例、实验例仅仅对本发明进行进一步说明，不应构成对本发明的限制。
下述实施例、实验例中的原材料如果没有特别说明，均为市售。
实施例1：胰岛素-Fc融合蛋白的构建和表达
用淋巴细胞分离液分离健康人淋巴细胞，用Trizol试剂(Invitrogen公司产品)提取总RNA，根据文献(Cell，1980，22：197-207)和文献(Nucleic AcidsResearch，1982，10：4071-4079)报道的序列分别设计引物FCM SENSE：GAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCAGCAGGGGGACCGTCAGTCTTCC和FCANTISENSE：CGAATTCTCAT TTACCCGGAGACAGG扩增抗体Fc区，FCMSENSE中引入突变点，使H235，H236的两个亮氨酸突变为两个丙氨酸。反应均采用热启动，反应条件：94℃5分钟；94℃45秒，60℃45秒，72℃1分10秒，30个循环；72℃10分钟。全基因合成胰岛素B链、C分别为1、GGGPGKR，2、GGGPQT，3、GGGPGAG，4、AAGGGPSVR)和A链基因，并以overlap PCR(引物分别为FCM ANTISENSE和Insu-Fc SENSE：aagcttgccgccaccatggccctgtggatgcgcctcc连接到Fc基因5‘端，PCR产物经琼脂糖凝胶电泳纯化回收并克隆到pGEM-T载体中，测序验证后确认获得了正确的克隆。正确克隆以HinDI11和EcoRI酶切(Promega公司产品)，经琼脂糖凝胶电泳纯化回收获得1kb的酶切片断与同酶切的质粒pDR1(如图1所示)用T4 DNA连接酶(Invitrogen公司产品)进行连接，构建成真核表达载体，根据C的不同分别命名为pDR(Insu-Fc1)，pDR(Insu-Fc2)，pDR(Insu-Fc3)，pDR(Insu-Fc4)。Fc区的核苷酸和氨基酸序列如SEQ ID NO：1和SEQ ID NO：2所示，胰岛素-Fc1融合蛋白的核苷酸和氨基酸序列如SEQ ID NO：3和SEQ ID NO：4所示，胰岛素-Fc2融合蛋白的核苷酸和氨基酸序列如SEQ ID NO：5和SEQ ID NO：6所示，胰岛素-Fc3融合蛋白的核苷酸和氨基酸序列如SEQ ID NO：7和SEQ ID NO：8所示，胰岛素-Fc4融合蛋白的核苷酸和氨基酸序列如SEQ ID NO：9和SEQ ID NO：10所示。胰岛素-Fc融合蛋白的基因结构示意图如图2所示。
于3.5cm组织培养皿中接种3.5×105/孔的CHO-dhfr-细胞，细胞在含次黄嘌呤(H)、胸腺嘧啶脱氧核苷(T)和甘氨酸(G)的DMEM完全培养基中培养至90-95％融合时进行转染：分别取质粒pDR(Insu-Fc1)，pDR(Insu-Fc2)，pDR(Insu-Fc3)，pDR(Insu-Fc4)10μg和20μl Lipofectamine2000 Reagent[Invitrogen公司产品]分别溶于500μl无血清DMEM培养基，室温静置5分钟，将以上2种液体混合，室温孵育20分钟以使DNA-脂质体复合物形成，其间用3ml无血清的DMEM培养基替换培养皿中的含血清培养基，然后将形成的DNA-脂质体复合物加入到板中，CO2孵箱培养4小时后补加3ml含10％血清的DMEM完全培养基，置于CO2孵箱中继续培养。转染进行24小时后将细胞按0.8×105/皿传到10cm培养皿中，换不含H、T、G的选择培养基，待克隆形成后用二氢叶酸还原酶抑制剂氨甲喋呤(MTX)进行加压筛选，浓度从2×10-8mol/L到5×10-7mol/L梯度加压，用有限稀释法进行亚克隆。取细胞培养上清用ELISA检测筛选高表达克隆：羊抗人IgG(CH2)包被于ELISA板，4℃过夜，用2％BSA-PBS于37℃封闭2小时，加入待测的培养上清和标准品(Human myeloma IgG1，κ)，37℃孵育2小时，加入HRP-羊抗人IgG(CH3)进行结合反应，37℃孵育1小时，加入TMB于37℃作用5分钟，最后用H2SO4终止反应，测OD450值。。将筛选得到的高表达克隆用无血清培养基扩大培养，用ProteinA亲和柱(GE公司产品)分离纯化各胰岛素-Fc融合蛋白。将纯化融合蛋白用PBS进行透析，最后以紫外吸收法定量。图3为胰岛素-Fc融合蛋白结构示意图。
实施例2：胰岛素-Fc融合蛋白亲和力检测
ELISA鉴定胰岛素-Fc融合蛋白的结合活性：将可溶性胰岛素受体蛋白(Abnova公司产品)用0.05mmol/L碳酸钠.碳酸氢钠缓冲液(pH 9.6)稀释成2ug/ml，100ul/孔，4℃包被过夜。3％脱脂奶室温封闭2h后，加入不同浓度的胰岛素-Fc融合蛋白，对照抗体301，100ul/孔，每个浓度取3个平行孔，室温孵育2h。弃上清液，PBS洗涤3次，加入按效价稀释好的HRP标记的小鼠抗人IgG单抗(DAKO公司产品)，50u1/孑L，室温孵育45min。PBS充分洗涤后，OPD避光显色，加入2mol/L H2SO4终止反应后，置酶标仪中测定492nm吸光度(A492)值。如图4所示，各胰岛素-Fc融合蛋白均可以与胰岛素受体蛋白结合，其ED50值为～1.2μg/ml。
在另一个分析检测中，即竞争性胰岛素封闭检测中，不同量的胰岛素-Fc融合蛋白与固定量的AP标记的胰岛素(50μg)混合并在室温温育1个小时。将混合物转移到用可溶性胰岛素受体蛋白(Abnova公司产品)包被的96孔板微量滴定并在室温继续温育2小时，然后将滴定板洗5次，加入AP的底物。对结合的胰岛素-AP分子定量，然后计算IC50，即50％抑制胰岛素与胰岛素受体结合所需要的V胰岛素-Fc融合蛋白浓度。如图5，胰岛素-Fc融合蛋白均可竞争性抑制胰岛素与可溶性胰岛素受体蛋白的结合，其IC50值约为1.0g/ml。
实施例3：用biocore检测胰岛素-Fc融合蛋白的亲和力常数
用Biacore T100检测各胰岛素-Fc融合蛋白与胰岛素受体的结合活性。将胰岛素受体通过氨基偶联的方法连接到CM5芯片上，然后根据操作说明在Biacore通用缓冲液(10mM Hepes pH 7.4，150mM NaCl，3mM EDTA，0.005％surfactant P-20)中分析胰岛素受体与各胰岛素-Fc融合蛋白的结合活性，流动池的流速为30ul/min。胰岛素受体耦联至反应值达到1500RU，分别检测胰岛素和各胰岛素-Fc融合蛋白(50ug/ml，25ug/ml，12.5ug/ml，6.25ug/ml，3.125ug/ml，1.56ug/ml，0.78ug/ml，0.39ug/ml)不同浓度时与胰岛素受体相互作用的信号活性。结合时间420s，解离时间600s，用10mM甘氨酸再生60s，基线平稳，并取1.56ug/ml做重复浓度来确保芯片再生活性。根据biacore evaluation software中affinity and kinetics 1：1结合模式运算获得结果如表1所示，胰岛素受体与胰岛素结合的亲和常数为1.47nM，而与各胰岛素-Fc融合蛋白亲和常数在1.4-17.6nM之间。
实施例4：用cAMP检测分析法检测胰岛素-Fc融合蛋白的活性
采用cAMP检测分析法测试各胰岛素-Fc融合蛋白的活性，该方法测量通过GPCR调节腺苷酸环化酶活性后产生的cAMP。cAMP检测分析法LANCETM cAMP检测分析法(Hemmila I.1999.LANCETM：Homogeneous AssayPlatform for HTS.J Biomol Screen.4(6)，303-308(Hemmila I.，1999年，LANCETM：用于HTS的均相检测分析平台，《生物分子筛选杂志》，第4卷第6期第303-308页))是一种均相时间分辨荧光共振能量转移(TR-FRET)免疫分析法。该检测分析法基于以下两者间的竞争：铕标记的cAMP示踪剂，以及用于结合通过Alexa 647染料标记的cAMP特异性抗体上的位点的样本cAMP。铕标记的示踪剂络合物由biotin-cAMP与用铕-W8044螯合物标记的链霉亲和素之间的紧密相互作用形成。当抗体与Eu-SA/b-cAMP示踪剂结合时，340nm的光脉冲会激发示踪剂的Eu螯合物分子。Eu螯合物发出的能量被转移到抗体上的Alexa分子，继而发出665nm的光。在存在cAMP的情况下，665nm下试验样本的荧光强度测量值将减小，所得的信号将与样本的cAMP浓度成反比(LANCE cAMP手册)。
细胞与检测分析：在RPMI 1640/10％FBS/1％ L-谷氨酰胺/1％丙酮酸钠/1％非必需氨基酸/50μM β-巯基乙醇中培养INS-1E细胞(得自Claes Wollheim，Geneva，Switzerland.Endocrinology，1992，130(1)：167-178(《内分泌学》，1992年，第130卷第1期第167-178页))，将其保持在37℃、含5％CO2的增湿培养箱中。通过胰蛋白酶消化让细胞传代，每7天传代培养一次。为了进行检测分析，将INS-1E细胞在汇合时涂布于96孔板(Costar3610)上，让其在正常生长培养基中恢复4天。从孔中吸出培养基，并先后加入24ul Alexa647抗cAMP抗体(LANCE cAMP试剂盒，PerkinElmer(Boston，MA))和24ul系列稀释的供试品(溶于PBS/0.5％BSA/0.5mM IBMX中)。在室温下将细胞刺激7分钟，然后在含有Eu-SA/b-cAMP示踪剂的缓冲液中裂解。将板在室温下孵育1小时，然后测量665nm处的荧光强度。根据标准曲线确定cAMP浓度。结果：将各胰岛素-Fc融合蛋白对INS-1E细胞中cAMP的刺激能力与野生型胰岛素进行比较。如图6通过图解所示，结果表明各胰岛素-Fc融合蛋白的生物活性无明显损失。
实施例5：胰岛素-Fc融合蛋白的药代动力学检测
四周龄C57BL/6雌性小鼠(体重15-19g)皮下注射(s.c.)60μg(0.4μg/μl*150μl)/mice①Insu-Fc1、②Insu-Fc2、③Insu-Fc3、④Insu-Fc4，分别于注射后0.083hr，0.5hr，1hr，2hr，6hr，12hr，24hr，48hr，96hr后取血，每只小鼠取血约100μl(内眦静脉取血)加0.1％的肝素混匀，离心4000rpm*20min。取上清，弃沉淀。ELISA检测血浆中抗体的浓度(以小鼠抗人CH3单克隆抗体包被96孔板，加血浆后，以HRP标记的小鼠抗人CH2单克隆抗体检测)。检测结果如图7所示，各胰岛素-Fc融合蛋白的体内半衰期较长，可达3天左右，远远高于常规胰岛素的几小时。
Claims (7)
1.一种胰岛素-Fc融合蛋白，其通式为B-C-A-Fc，其中A为胰岛素A链，B为胰岛素的B链，C为4-50个氨基酸的C肽连接序列，Fc为免疫球蛋白Fc段或其突变体。
2.权利要求1所述的胰岛素-Fc融合蛋白，其中C为6-30个氨基酸的C肽连接序列。
3.权利要求2所述的胰岛素-Fc融合蛋白，其中C为GGGPGKR、GGGPQT、GGGPGAG、AAGGGPSVR之一。
4.权利要求1所述的胰岛素-Fc融合蛋白，其中Fc为免疫球蛋白Fc段H235、H236位从亮氨酸突变为丙氨酸。
5.权利要求1-4任一所述的胰岛素-Fc融合蛋白，其氨基酸序列为SEQ IDNO：2、SEQ ID NO：4、SEQ ID NO：6、SEQ ID NO：8、SEQ ID NO：10之一。
6.一种核酸分子，编码权利要求1-4任一所述的胰岛素-Fc融合蛋白。
7.权利要求6所述的核酸分子，其核苷酸序列为SEQ ID NO：1、SEQ ID NO：3、SEQ ID NO：5、SEQ ID NO：7、SEQ ID NO：9之一。
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| WO2016133372A3 (ko) * | 2015-02-17 | 2016-10-13 | 한미약품 주식회사 | 지속형 인슐린 또는 이의 아날로그 결합체 |
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