CN108640998A - Fgf1-fgf21嵌合蛋白及其治疗糖尿病的应用 - Google Patents
Fgf1-fgf21嵌合蛋白及其治疗糖尿病的应用 Download PDFInfo
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Abstract
本发明提供了FGF1‑FGF21嵌合蛋白，其包括FGF1突变体和FGF21的C尾，其减少促细胞分裂和增殖副作用，并且具有长效的降糖、降脂等活性。
Description
技术领域
本发明属于蛋白质技术领域，具体而言，本发明涉及长效FGF21的激动剂，其是FGF1-FGF21嵌合蛋白。另外本发明还涉及该蛋白的应用等，如治疗糖尿病的应用。
背景技术
成纤维细胞生长因子(Fibroblast Growth Factor，FGF)中共有18个家族成员，被分为5个旁分泌和1个内分泌亚家族。FGF作用的发挥则是通过与细胞表面具有酪氨酸激酶活性的FGF受体(FGF Receptor，FGFR)相互作用进而引起受体的二聚化并进一步激活下游级联信号来实现的。其中，成纤维细胞生长因子21(FGF21)是内分泌作用的FGF19亚家族的成员。
由于FGF21在代谢和安全性方面的优点，它被寄予作为糖尿病、肥胖和脂肪代谢异常(dyslipidemia)的药物的厚望。然而它的体内药效和半衰期却不理想，因此现有技术对它的研究包括，将FGF21与Fc融合，定点突变FGF21上蛋白酶裂解位点，FGF21与靶向性抗体融合，PEG化。
本发明人跳出了现有技术研究的框架，经过长期研究，令人意外地发现了一种长效FGF21的激动剂，其是FGF1-FGF21嵌合蛋白，但是并不包括FGF1-FGF21的全部部分，不但引入有限的突变，即可使得其仅能与FGFR形成较弱的二元复合物即可启动代谢调控的信号通路，而基本丧失了促细胞分裂和增殖的功能，同时，只使用了一小部分FGF21，又提供了长效的降糖等药理作用。
发明内容
本发明要解决的技术问题在于提供新的FGF1-FGF21嵌合蛋白，其基本消除了促细胞分裂和增殖的能力，不但仍旧保留了FGF21的降血糖、降血脂等功能，而且可以长效地起效。另外，本发明还提供了该突变体的编码基因、表达载体和宿主细胞以及医药用途等。
具体而言，在第一方面，本发明提供了FGF1-FGF21嵌合蛋白，其包括FGF1突变体和FGF21的C尾，其中FGF1突变体的促细胞分裂和增殖的能力减少。本发明第一方面的嵌合蛋白减少(优选消除)促细胞分裂和增殖的能力，不但仍旧保留了FGF21的降血糖、降血脂等功能，而且可以长效地起效。
优选本发明第一方面的嵌合蛋白由FGF1突变体和FGF21的C尾组成，更优选其从N端到C端由FGF1突变体和FGF21的C尾组成。
优选在本发明第一方面的嵌合蛋白中，FGF1突变体具有Lys127、Lys128和/或Lys133突变，优选具有Lys127Asp、Lys128Gln和/或Lys133Val突变。在本文中，突变的表示采用本领域技术人员所熟知的方式。例如，Lys127表示对第127位上的Lys进行突变；而Lys128Gln表示对第128位上的Lys进行突变，突变为Gln。突变可以是添加、缺失和/或取代。本领域技术人员知晓，通过改变已知多肽的编码基因序列并将其导入表达载体，可以制备出取代、添加和/或缺失了氨基酸残基的多肽或蛋白质，这些方法广泛记载于《分子克隆实验指南》等本领域公知的文献中。在本发明的具体实施方式中，突变是取代。在本发明的具体实施方式中，该突变体(仅)具有Lys127Asp、Lys128Gln和Lysl33Val突变。
优选在本发明第一方面的嵌合蛋白中，FGF21的C尾包括FGF21第142-182位。在本发明的具体实施方式中，本发明第一方面的嵌合蛋白的氨基酸序列如SEQ ID NO：1所示。
在第二方面，本发明提供了编码本发明第一方面的嵌合蛋白的多核苷酸。在本文中，多核苷酸可以是DNA形式，也可以是RNA形式，优选DNA形式。DNA形式包括天然cDNA和人工合成的cDNA，DNA可以是编码链或模板链。通过常规技术，如PCR方法、重组法或人工合成的方法，本领域技术人员可以很容易获得编码本发明的突变体的核酸分子多核苷酸或其片段。这些序列一旦获得，就可以将其克隆入载体，再转化或转染入相应的细胞，然后通过常规的宿主细胞进行增殖，从中分离得到大量的核酸分子。
在第三方面，本发明提供了一种载体，其含有本发明第二个方面所述的多核苷酸。在本文中，载体包括表达载体和克隆载体，是指本领域中常用的细菌质粒、粘粒、噬菌粒、酵母质粒、植物细胞病毒、动物病毒及其它各种病毒载体。本发明中适用的载体包括但不限于：在细菌中表达用的载体(原核表达载体)、在酵母中表达用的载体(如毕赤酵母载体、汉逊酵母载体等)、在昆虫细胞中表达的杆状病毒载体、在哺乳动物细胞中表达用的载体(痘苗病毒载体、逆转录病毒载体、腺病毒载体、腺伴病毒载体等)、在植物中表达用的植物病毒载体以及在哺乳动物乳腺中表达用的各种载体。总之，只要能在宿主细胞中稳定复制，任何质粒和载体都可使用。优选表达载体包含选择标记基因，如细菌的氨苄青霉素抗性基因、四环素抗性基因、卡那霉素抗性基因、链霉素抗性基因、氯霉素抗性基因；酵母菌的新霉素抗性基因、Zeocin抗性基因，酵母菌的缺陷选择标志，如His，Leu，Trp等；真核细胞的新霉素抗性基因、Zeocin抗性基因、二氢叶酸还原酶基因及荧光蛋白标记基因等。在本发明的具体实施方式中，使用的是已经商品化的pET质粒。
在第四方面，本发明提供了一种细胞，如宿主细胞，其含有本发明第二个方面所述的多核苷酸。本发明的细胞可以含有本发明第三方面的载体，也可以用本发明第二方面的多核苷酸转化或转染。宿主细胞可以是原核细胞，也可以是真核细胞，如，细菌细胞、酵母细胞、植物细胞、昆虫细胞、哺乳动物细胞等。宿主细胞在转化或转染含本发明所述编码融合蛋白的基因序列后，即构成工程化细胞或细胞株，可用于生产所需融合蛋白。本领域技术人员能够恰当地选择适当的载体、宿主细胞，并熟知如何将载体高效地转化或转染入宿主细胞中，所用方法包括但不限于：氯化钙法、电穿孔法用于细菌细胞，电穿孔法和原生质体融合法用于酵母细胞，脂质体包裹、磷酸钙共沉淀、电融合法以及显微注射法用于哺乳动物细胞等真核细胞。优选本发明的宿主细胞是大肠杆菌BL21(DE3)。
在第五方面，本发明提供了药物组合物，其包含本发明第一方面的突变体以及药学上可接受的载体。本发明的药物组合物能治疗糖尿病、降血糖、治疗或预防肥，并且其减少(甚至消除)促细胞增殖的副作用，而且还能够长效地起效。在本文中，长效指的是具有比野生型FGF21更长体内半衰期。
在本文中，药学上可接受的载体指无毒的填充剂、稳定剂、稀释剂、佐剂或其他制剂辅料。根据本领域的公知技术，可以根据治疗目的、给药途径的需要将药物组合物制成各种剂型，优选该组合物为单位剂量形式，如冻干剂、片剂、胶囊、粉剂、乳液剂、注射剂或喷雾剂，更优选该药物组合物为注射剂型，如冻干粉针剂。也优选该药物组合物是液体制剂，如可以包含缓冲液(如磷酸盐缓冲液、Tris-HCl缓冲液)。
在第六方面，本发明提供了本发明第一方面的嵌合蛋白在制备减少促细胞分裂和增殖副作用的长效FGF21药物中的应用。在本文中，FGF21药物指的是用语预防或治疗野生型FGF21可用于预防或治疗的疾病的药物。本发明的嵌合蛋白及其药物能够显著降低(甚至基本消除)这样的副作用，从而能够长期安全地给药。
本发明的嵌合蛋白及其药物基本保留了的降糖的功能。因此，优选在本发明第六方面的应用中，药物用于降血糖。也优选在本发明第六方面的应用中，药物用于治疗糖尿病，尤其是2型糖尿病。
本发明的嵌合蛋白及其药物基本保留了的降脂的功能。因此，优选在本发明第六方面的应用中，药物用于降血脂(如，甘油三酯)。也优选在本发明第六方面的应用中，药物用于治疗或预防肥胖症。
本发明的嵌合蛋白具有长效的持久效果。因此，长效FGF21药物是具有比野生型FGF21更长体内半衰期的FGF21药物。
本发明的有益效果在于：本发明的嵌合蛋白基本丧失了促细胞分裂和增殖的功能，同时又保持了降糖等药理作用，而且还具有长效的持久作用，所以具安全、高效的成药前景。
本发明引用了公开文献，这些文献是为了更清楚地描述本发明，它们的全文内容均纳入本文进行参考，就好像它们的全文已经在本文中重复叙述过一样。
为了便于理解，以下将通过具体的实施例对本发明进行详细地描述。需要特别指出的是，这些描述仅仅是示例性的描述，并不构成对本发明范围的限制。依据本说明书的论述，本发明的许多变化、改变对所属领域技术人员来说都是显而易见了。
附图说明
图1和图2显示了FGF1-FGF21嵌合蛋白初步活性研究的实验结果图。
图3显示了FGF1-FGF21对db/db小鼠的血糖水平和胰岛素敏感性的影响的实验结果图。
图4显示了FGF1-FGF21对db/db小鼠的肝脏脂和糖代谢的长期影响的实验结果图。
图5显示了FGF1-FGF21对db/db小鼠的脂肪重塑和脂代谢的长期影响的实验结果图。
具体实施方式
以下通过实施例进一步说明本发明的内容。如未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段和市售的常用仪器、试剂，可参见《分子克隆实验指南(第3版)》(科学出版社)和CFDA的相关试验指引以及相应仪器和试剂的厂商说明书等参考。
实施例1本发明的FGF1-FGF21嵌合蛋白及其活性的初步研究
通过常规手段克隆、表达并纯化FGF1-FGF21嵌合蛋白(简称为FGF1ΔHBS-FGF21C -tail)。简而言之，将编码全长人野生型FGF1(简称为FGF1WT，第1-155位)的cDNA片段克隆入pET30a表达载体中，然后用QuikChange XL定点突变试剂盒(Stratagene，La Jolla，CA)导入Lys127Asp、Lys128Gln和Lys133Val这三个突变(即编码FGF1ΔHS)，然后导入编码FGF21的C尾(C-tail，第Gly142-Ser182位)部分的基因，获得嵌合构建体(其编码的嵌合蛋白的氨基酸序列如SEQ ID NO：1所示)。将带有嵌合构建体的表达载体转化入Escherichia coliBL21(DE3)中，于37℃培养，当A600达到0.5时，加入1mM IPTG继续培养4小时。菌体裂解后依次用阴离子交换层析柱(Source Q，GE Healthcare，Piscataway，NJ)和凝胶排阻层析柱(GEHealthcare，Piscataway，NJ)纯化，获得纯度＞98％的FGF1ΔHBS-FGF21C-tail。其他对照蛋白也可以用相似方法制备。
通过SPR生物感应芯片(BIAcore 2000system(GE Healthcare，Piscataway，NJ))检测，FGF1ΔHBS-FGF21C-tail具有很强的结合βklotho的ectodomain的能力(Kd＝0.768μM)(图1A)。
通过HPLC-MALS分析，其中HPLC(Waters 1500pump with 2498UV detector and2707autosampler)偶联MALS(Wyatt miniDawn-Treos and Optilab rEX)，发现FGF1ΔHBS-FGF21C-tail能在溶液中与βklotho的ectodomain和FGFR1c形成1∶1∶1稳定的三元复合物(图1B)，这与FGF21的结果类似。单独表达FGFR1c或者共表达FGFR1c和βklotho的BaF3细胞系分别用野生型FGF21(FGF21WT)或FGF1ΔHBS-FGF21C-tail处理，细胞裂解液用Western blot分析，发现βklotho能极大地增强FGF1ΔHBS-FGF21C-tail激活FGFR1c的能力(图1C和1D)。因此，FGF1ΔHBS-FGF21C-tail保留了FGF21WT对Klotho共受体依赖的内分泌模式。
在表达内源βklotho的分化的3T3-L1脂肪细胞中，加入不同浓度的FGF1ΔHBS-FGF21C-tail，与加入不同浓度的FGF21WT类似，FGF1ΔHBS-FGF21C-tail留了类似于FGF1WT的刺激葡萄糖摄取的效应(图1E)；同样，FGF1ΔHBS-FGF21C-tail诱导的FGFR、IRS1和AKT磷酸化与FGF21WT类似(图1F)。
图2A显示了在FGF1上进行这三个突变可能会损害其与HS的相互作用能力，所以预计FGF1ΔHBS-FGF21C-tail也可能有这样的性质。通过基于荧光染料的热转变测试熔点温度(Tm)，发现FGF1ΔHBS-FGF21C-tail的比FGF21WT的高～15℃(图2B)；将相同浓度的FGF1ΔHBS-FGF21C-tail和FGF21WT分别注射入Sprague Dawley(SD)大鼠，发现FGF1ΔHBS-FGF21C-tail的血清浓度更高，其半衰期为～140min，是FGF21WT(～26min)的7倍(图2C)。这表明FGF1ΔHBS-FGF21C-tail无论在体内还是在体外，其生物稳定性都显著高于FGF21WT。
与图2A的模型分析一致，在与HS共存时，如图2D所示，FGF1ΔHBS-FGF21C-tail显示出可忽略不计的HS结合亲和力；如图2E所示，FGF1ΔHBS-FGF21C-tail无法诱导HS依赖的FGFR1c的二聚化。在NIH 3T3成纤维细胞中，FGF1ΔHBS-FGF21C-tail的诱导FGFR酪氨酸磷酸化及下游ERK1/2途径的能力至少一个数量级地弱于FGF1WT(图2F)；同样FGF1ΔHBS-FGF21C-tail的促进FGFR活化及下游的促有丝分裂活性活性也比FGF1WT弱得多(图2G)。
实施例2本发明的FGF1ΔHS的体内活性研究
(1)正常小鼠
每天对正常的C57BL/6J小鼠给药FGF1WT和FGF1ΔHBS-FGF21C-tail(2.0mg/kg体重)，持续20天，分别用Ki6免疫组化染色和Western blotting对小鼠的肝脏分析增生情况。FGF1WT被观察到能导致小鼠很明显的增生，而FGF1ΔHBS-FGF21C-tail相对于PBS的对照组，没有出现增生。
(2)FGF1ΔHBS-FGF21C-tail对db/db小鼠的血糖水平和胰岛素敏感性的影响
糖尿病模型(db/db)小鼠(C57BLKS/J-leprdb/leprdb)和其对照的表型正常的小鼠(db/m)均购自南京大学模型动物研究中心。
在db/db小鼠中，每天皮下注射FGF1WT和FGF1ΔHBS-FGF21C-tail(0.5mg/kg体重)，持续4周，注射后24小时内测量血糖。FGF1ΔHBS-FGF21C-tail能显著降低血糖水平，达到正常水平，但没有低至低血糖水平；而FGF21WT也能在24小时内降低一定的血糖水平，但无法是将血糖水平的降低到正常水平(图3A)。FGF21WT的这一现象与常用的胰岛素的降血糖情况相似。
在4周的长期治疗中，FGF1ΔHBS-FGF21C-tail和FGF21WT都能显著降低db/db小鼠的体重(图3B和3C)。4周的长期治疗，在糖耐测试(GTT)中，注射FGF1ΔHBS-FGF21C-tail的db/db小鼠都能保持低的血糖水平(图3D和3E)，并在胰岛素耐受测试(ITT)中，注射FGF1ΔHBS-FGF21C -tail的db/db小鼠也显示出了显著的胰岛素敏感性的改善(图3F)，而且都比FGF21WT要更好。FGF1ΔHBS-FGF21C-tail的优势应该是因为它比FGF21WT更稳定、有更长的半衰期。
(3)FGF1ΔHBS-FGF21C-tail对db/db小鼠的肝脏脂和糖代谢的长期影响
前述长期给药FGF1ΔHBS-FGF21C-tail的db/db小鼠的肝脏大小和比例均有显著降低(图4A和4B)。前述长期给药的小鼠及其对照小鼠中取肝脏组织用不同染料染色，结果如图4C和4D所示，用FGF1ΔHBS-FGF21C-tail长期给药的db/db小鼠显著减轻了肝脂肪变性的程度。
同样，长期给药FGF1ΔHBS-FGF21C-tail的db/db小鼠的肝脏的甘油三酯水平也显著降低(图4F)，而肝(图4H和4I)和血清中丙氨酸转氨酶(ALT)和天冬氨酸转氨酶(AST)的水平也都显著降低。
小鼠的肝糖原水平通过PAS染色(图4E)和比色测定(图4G)，发现db/db小鼠相对于db/m小鼠提高了肝糖原水平，而用FGF1ΔHBS-FGF21C-tail长期给药的db/db小鼠的肝糖原水平进一步增加了。定量检测也显示了这样的结果(图4C)。表明FGF1ΔHBS-FGF21C-tail的降糖和提高胰岛素敏感性是通过增加肝糖原的合成和储存来实现的。FGF1ΔHBS-FGF21C-tail长期给药的db/db小鼠肝组织中的AKT磷酸化增加了并且降低了肝组织中FAS、SCD-1的水平(图4J和4K)。
(4)FGF1ΔHBS-FGF21C-tail对db/db小鼠的脂肪重塑和脂代谢的长期影响
肥胖症和2型糖尿病均油增多的脂肪储存和白色脂肪细胞(WAT)扩充的特征，而用FGF1ΔHBS-FGF21C-tail长期给药的db/db小鼠都减少了白色和棕色脂肪细胞(BAT)的大小(图5A-D)。多种脂肪生成有关的蛋白的表达减少了，表明WAT脂肪细胞大小的减少可能是因为FGF1ΔHBS-FGF21C-tail所导致的脂肪生成和脂肪储存的减少。
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Claims (10)
1.FGF1-FGF21嵌合蛋白，其包括FGF1突变体和FGF21的C尾，其中FGF1突变体的促细胞分裂和增殖的能力减少，优选其由FGF1突变体和FGF21的C尾组成，更优选其从N端到C端由FGF1突变体和FGF21的C尾组成。
2.权利要求1所述的嵌合蛋白，其中，FGF1突变体具有Lys127、Lys128和/或Lys133突变，优选具有Lys127Asp、Lys128Gln和/或Lys133Val突变。
3.权利要求1所述的嵌合蛋白，其中，FGF21的C尾包括FGF21第142-182位。
4.权利要求1-3之一所述的嵌合蛋白，其氨基酸序列如SEQ ID NO：1所示。
5.编码权利要求1-4之一所述的突变体的多核苷酸。
6.载体或细胞，其包含权利要求5所述的多核苷酸。
7.药物组合物，其包括权利要求1-4之一所述的嵌合蛋白以及药学上可接受的载体。
8.权利要求1-4之一所述的嵌合蛋白在制备减少促细胞分裂和增殖副作用的长效FGF21药物中的应用。
9.权利要求8所述的应用，其中药物用于治疗或预防糖尿病或肥胖症，尤其是2型糖尿病。
10.权利要求8所述的应用，其中长效是具有比野生型FGF21更长体内半衰期。
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