CN108976299B - 一种改善抗体片段亲和力和体内半衰期的方法 - Google Patents
一种改善抗体片段亲和力和体内半衰期的方法 Download PDFInfo
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Abstract
本发明涉及一种改善抗体片段亲和力和体内半衰期的方法，具体是通过将所述抗体片段与铁蛋白融合表达，一方面提高抗体片段的亲和力，另一方面延长抗体片段在体内的半衰期，同时形成一种可以在其空腔内容纳药物的靶向性载体，用于疾病的治疗或诊断。
Description
技术领域
本发明属于纳米技术、生物仿生和生物医药的交叉领域。特别地，本发明属于优化纳米抗体的亲和力和半衰期的新技术。具体的说，提供一种可以提高纳米抗体亲和力，延长其在体内半衰期的一种多功能自组装展示平台及方法。
背景技术
纳米抗体(Nanobody，Nb)，也称为单域抗体，是一种分子量仅约为15kD的基因工程抗体，源于所有抗体的重链可变区。纳米抗体具有以下主要特征：①分子小；②仅仅重链可变区就可发挥结合抗原的功能；③稳定性好；④可溶性好；⑤易于基因工程操作等1。大量的研究显示出纳米抗体具有很好的应用前景，尤其是在很多疾病的诊断和治疗方面，纳米抗体都有着不俗的表现2,3。其中比利时的Ablynx公司研发的抗血栓形成的纳米抗体已经进入临床II期，并显示出良好的效果4。目前，利用纳米抗体对生物标志物的特异靶向性，以及纳米材料独特的理化性质，将两者整合到一起，发展了很多新型的疾病诊断方法5,6。
然而，因为纳米抗体的本身结构特点，使得其在应用中存在以下的问题。①亲和力低。纳米抗体仅由重链可变区部分组成，其亲和力相对于完整抗体来说，是低几个数量级的7。②体内半衰期短。纳米抗体的分子量为15kD左右，远低于肾脏清除蛋白的截留分子量(50kDa)，因此，纳米抗体在体内的半衰期非常短8，这也大大制约了纳米抗体在体内的治疗效果。
解决这两个问题需要一个理想的纳米抗体展示平台和方法来实现对纳米抗体的多价化9和分子量的提高3。目前，常用的解决方案是通过纳米抗体与纳米颗粒共价连接的方法，来实现其分子量和亲和力的改善。比如将纳米抗体与PLGA10、胶束11、白蛋白12或者金纳米颗粒5共价偶联。但是化学偶联过程不容易控制，易造成纳米抗体失效，并会改变纳米材料本身的特性，从而加大了纳米抗体临床转化的难度。因此，一种理想的纳米抗体展示平台亟待开发。
参考文献
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发明内容
基于上文问题和需要，本发明一方面提供一种改善抗体片段对抗原的亲和力和/或延长其体内半衰期的方法，所述方法包括将所述抗体片段与铁蛋白或其亚基去除了其短α-螺旋的截短体融合表达。
在优选的实施方案中，所述抗体片段与铁蛋白通过接头融合表达。
在优选的实施方案中，所述接头的氨基酸序列如SEQ ID NO：4所示。
在优选的实施方案中，所述抗体片段选自由以下各项组成的组：scFc，Fab，Fv，sdAb，F(ab')2或纳米抗体。
在优选的实施方案中，所述铁蛋白源自选自以下各项中任一项的物种：人、鼠、激烈火球菌(pyrococcus furiosus)等。
在优选的实施方案中，所述铁蛋白包括人重链铁蛋白(human HFn)、人轻链铁蛋白(human LFn)，激烈火球菌铁蛋白。
本文中“铁蛋白”的“截短体”是指铁蛋白亚基去除了短α-螺旋后形成的截短体。
在本领域中，技术人员知晓铁蛋白亚基的三级结构是高级保守的，包含四个长α-螺旋，一个短α-螺旋及在第二、三个α-螺旋之间的长环。从激烈火球菌的铁蛋白到人的铁蛋白，均具有这种结构。其中所述短α-螺旋，即第五个α-螺旋在铁蛋白亚基的C末端。
在优选的实施方案中，所述人重链铁蛋白的截短体氨基酸序列如下所示：
MTTASTSQVRQNYHQDSEAAINRQINLELYASYVYLSMSYYFDRDDVALKNFAKYFLHQSHEEREHAEKLMKLQNQRGGRIFLQDIKKPDCDDWESGLNAMECALHLEKNVNQSLLELHKLATDKNDPHLCDFIETHYLNEQVKAIKELGDHVTNLRKMGAPE(SEQ ID NO：1)。
在优选的实施方案中，所述激烈火球菌铁蛋白的截短体的氨基酸序列如下所示：
MLSERMLKALNDQLNRELYSAYLYFAMAAYFEDLGLEGFANWMKAQAEEEIGHALRFYNYIYDRNGRVELDEIPKPPKEWESPLKAFEAAYEHEKFISKSIYELAALAEEEKDYSTRAFLEWFINEQVEEEASVKKILDKLKFAKD(SEQ ID NO：2)。
在优选的实施方案中，所述人轻链铁蛋白的截短体的氨基酸序列如下所示：
MSSQIRQNYSTDVEAAVNSLVNLYLQASYTYLSLGFYFDRDDVALEGVSHFFRELAEEKREGYERLLKMQNQRGGRALFQDIKKPAEDEWGKTPDAMKAAMALEKKLNQALLDLHALGSARTDPHLCDFLETHFLDEEVKLIKKMGDHLTNLHRLGG(SEQ ID NO：3)。
本发明的另一方面提供一种纳米载体，所述纳米载体包括铁蛋白或其亚基去除了短α-螺旋的截短体自组装形成的空腔蛋白壳，以及展示在所述蛋白壳表面的抗体片段。
在优选的实施方案中，其中所述空腔可以容纳药物等。
在优选的实施方案中，所述药物包括小分子化合物、寡核苷酸、功能性肽段或纳米酶。在本文中，纳米酶是指具有内在天然酶活性的纳米材料，比如四氧化三铁纳米颗粒，三氧化二铁纳米颗粒。
在优选的实施方案中，所述载体通过将铁蛋白或其亚基去除了短α-螺旋的截短体和抗体片段融合表达来获得。
本发明的另一方面提供一种融合蛋白，所述融合蛋白的结构由下式表示：
X-任选的L-Z，
其中，X和Z任选地分别是铁蛋白或其亚基去除了短α-螺旋的截短体和抗体片段，即抗体片段可以连接在铁蛋白或其亚基去除了短α-螺旋的截短体的C末端，也可以连接在铁蛋白或其亚基去除了短α-螺旋的截短体的N末端；L是接头。
在优选的实施方案中，所述抗体片段与铁蛋白或其亚基去除了短α-螺旋的截短体通过接头融合表达。
在优选的实施方案中，所述接头的氨基酸序列如SEQ ID NO：4所示。
在优选的实施方案中，所述抗体片段选自由以下各项组成的组：scFc，Fab，Fv，sdAb，F(ab')2或纳米抗体。
在优选的实施方案中，所述铁蛋白源自选自以下各项中任一项的物种：人、鼠、激烈火球菌(pyrococcus furiosus)等。
在优选的实施方案中，所述铁蛋白包括人重链铁蛋白(human HFn)、人轻链铁蛋白(human LFn)，激烈火球菌铁蛋白。
本发明的另一方面提供所述载体或融合蛋白用于靶向输送药物、体内成像、手术导航或用于疾病诊断的用途。
在优选的实施方案中，所述靶向由抗体片段决定和/或由铁蛋白决定。
本发明的展示平台和方法可以将抗体片段可溶性重组表达，并能够将其对抗原的亲和力提高超过三百倍；另外，本发明的展示平台和方法可以将抗体片段的体内半衰期延长10倍；此外，作为平台技术，在本发明得到的载体或融合蛋白中的空腔内可以装载不同的物质，例如装在药物来治疗疾病，装载纳米酶，用于疾病的诊断等。
附图说明
图1、人重链铁蛋白截短体-纳米抗体(下文中也称为Seq1-纳米抗体)的构建，其自组装成球形。A,SDS-PAGE分析表明，Seq1-纳米抗体重组表达成功。B，TEM电镜图对Seq1-纳米抗体及人重链铁蛋白(下文也称为Seq1)蛋白自组装成球形结构的分析。C，向Seq1-纳米抗体装铁成功。
图2、Seq1-纳米抗体的结构模拟及TEM电镜验证。不同侧面的蛋白壳显示，我们的模型构建成功。纳米抗体展示在蛋白壳表面后，不影响自己折叠，并互相成簇。
图3、对Seq1-纳米抗体及纳米抗体的亲和力分析。HAI为H5N1病毒的血凝集率，反映病毒的活力。
图4对Seq1-纳米抗体及纳米抗体的体内半衰期分析。
图5对激烈火球菌铁蛋白-纳米抗体(下文中也称为Seq2-纳米抗体)的TEM分析
图6对人轻链铁蛋白-纳米抗体(下文中也称为Seq3-纳米抗体)的TEM分析
具体实施方式
本发明中，以人重链铁蛋白截短体和纳米抗体(下文中使用的纳米抗体均为抗H5N1禽流感病毒抗体)为例举例说明本发明的平台和方法，但并不限于此。
实施例1：人重链铁蛋白展示纳米抗体的重组构建及表达和纯化。
本发明中，用到的纳米抗体为发明人所在实验室前期工作中鉴定出的针对H5N1禽流感病毒的纳米抗体，其氨基酸序列如下：
MAEVQLQASGGGLVQAGGSLRLSCAASGRTFSSGAMGWFRQAPGKEREFLSAIRWDGKIIRYADSVKGRFTISRDNAMNRVFLQMDSLKPEDTAVYYCAAGPDIITFDSWGQGTQVTVSS(SEQ ID NO：5)
首先，以人重链铁蛋白截短体-接头-纳米抗体的顺序合成Seq1-纳米抗体的融合基因，接头选择为GGGSGGGGSGGGS(SEQ ID NO：6)，序列由Genecreate公司(Wuhan,China)合成。基因序列随后克隆在大肠杆菌表达载体质粒pET-15b(Novagen)上。随后质粒载体转化大肠杆菌DH5α感受态细胞(TransGen Biotech，中国北京)。通过测序(Invitrogen)筛选得到阳性克隆。利用质粒小提试剂盒(TransGen Biotech)提取得到质粒。
Seq1-纳米抗体在大肠杆菌中表达并自组装成24聚体蛋白壳。表达菌在含100mg/ml氨苄抗生素的LB培养基中37℃培养至菌液OD600nm大于等于0.6，加入IPTG(Sigma-Aldrich)至终浓度为1mM，25℃诱导10h。诱导完成后，4,000g离心45min分离大肠杆菌，并用Tris缓冲液(20mM Tris,pH8.0)重悬菌体。
重悬的大肠杆菌4℃高压破碎后，12,000RPM离心30min收集上清液。上清液中含有His标签的Seq1-纳米抗体通过镍柱(Sangon Biotech,Shanghai,China)来纯化分离。使用300mM咪唑将蛋白Seq1-纳米抗体从镍柱上竞争性洗脱下来，并透析置换到Tris缓冲液(20mM Tris,pH 8.0)中。随后阴离子交换柱(Q-Sepharose Fast Flow,GE Healthcare)纯化分离Seq1-纳米抗体，透析置换到PBS缓冲液中。HFn蛋白浓度通过BCA蛋白定量试剂盒(Pierce)测定，使用BSA做蛋白标准品。
结果如图1A所示，单体的Seq1-纳米抗体的分子量为纳米抗体跟人重链铁蛋白截短体单体之和。TEM电镜结构显示，人重链铁蛋白截短体可以自组装为蛋白球，展示纳米抗体后，同样可以(图1B)。
实施例2：人重链铁蛋白截短体展示纳米抗体的生物矿化
我们利用人重链铁蛋白截短体展示纳米抗体的蛋白壳作为反应模板合成Fe3O4磁纳米颗粒(一种纳米酶)。将50ml含0.2mg/ml Seq1-纳米抗体的100mM NaCl溶液加入厌氧操作箱中，控制反应温度65摄氏度，pH值8.5。加入25mM六水合硫酸亚铁铵和8.33mM新鲜配置的过氧化氢溶液。通过计量控制装置(800Dosino)控制二价铁加入速率维持在100Fe/(protein min)。当加入二价铁量达到5000Fe/protein cage时，维持反应5min。最后，加入200μL 300mM柠檬酸钠溶液来螯合游离的铁原子。合成的M-Seq1-纳米抗体纳米粒(其中M代表矿化的)离心取上清后通过分子筛纯化，去除聚体纳米粒。M-Seq1-纳米抗体纳米粒的浓度测定方法和Seq1-纳米抗体一致，通过BCA蛋白定量试剂盒测定。纯化得到的M-Seq1-纳米抗体纳米粒的产率达到了90％。
结果如图1C所示，铁核成功的装载到Seq1-纳米抗体蛋白壳中。利用铁核的酶活性，可以构建多功能的用途，用于体外的疾病诊断、或者体内的ROS水平的调节。
实施例3：Seq1-纳米抗体和M-Seq1-纳米抗体纳米粒的负染电镜及结构模拟
制备得到的Seq1-纳米抗体和M-Seq1-纳米抗体纳米粒通过透射电子显微镜(TEM)和动态光散射(DLS)来进行表征。
透射电子显微镜(TEM)。将铜网用辉光(Plasma Cleaner HPDC32G)放电10秒预处理后，分别滴加20μL 0.25mg/ml的Seq1-纳米抗体和M-Seq1-纳米抗体纳米粒在铜网上，孵育1min后，用滤纸吸走多余的液滴，并水洗一次，用1％醋酸双氧铀染色60秒。使用JEM-1400电镜观察，电压80KV。
动态光散射(DLS)。将分子筛纯化得到的Seq1-纳米抗体和M-Seq1-纳米抗体蛋白样品置换到PBS缓冲液中，浓度0.25mg/ml。各取100μL样品在25摄氏度条件下在动态光散射分析仪(DynaPro Titan)中分析。
根据负染电镜的结果，利用蛋白质结构分析软件PyMOL Molecular GraphicsSystem重构分析了Seq1-纳米抗体的结构，并对自组装展示后的纳米抗体的折叠情况和组装情况进行了分析。
结果如图2所示，不同负染(图2A和2B)的Seq1-纳米抗体蛋TEM电镜图，对应于重组结构模型，从这些模型中可以发现：1.纳米抗体成功的展示在蛋白壳表面；2，纳米抗体自己会成簇，成簇后，不影响其彼此活性的发挥。
实施例4纳米抗体展示后亲和力的测定
本实施例使用双抗夹心法ELISA实验来验证Seq1-纳米抗体蛋白与流感病毒H5N1的结合能力。分别将用抗原包被液稀释的0.03μM的Seq1-纳米抗体，纳米抗体和Rabbitanti-H5N1单克隆抗体(义翘神州，克隆号：89，中国北京)包被96孔板，4摄氏度过夜。随后用5％脱脂奶粉37摄氏度封闭2小时。封闭后，用稀释浓度梯度的流感病毒H5N1在37摄氏度孵育1h，随后再用小鼠抗H5N1单克隆抗体(1:2000稀释，义翘神州，北京)37摄氏度孵育1h，最后用过氧化物酶偶联的抗小鼠二抗(1:3000稀释，GE，UK)37摄氏度孵育1.5h。每次孵育抗体后都用PBST洗4次，PBS洗一次。最后使用TMB作为显色底物来显色，并在562nm处读取光吸收值。
结果如图3所示，Seq1展示纳米抗体后，可以显著增强纳米抗体对抗原的亲和力，从89.3HAI提到到0.2429HAI，提高了367倍。
实施例5：纳米抗体体内半衰期的测定
为研究纳米抗体及Seq1-纳米抗体在体内的半衰期，首先用FITC标记纳米抗体及Seq1-纳米抗体。按照说明书提供的标记方法，将NHS活化的FITC(GE Healthcare)标记到纳米抗体及Seq1-纳米抗体上。然后，我们将等量(1uM)的荧光标记的纳米抗体素和HFn-Dox纳米颗粒通过尾静脉注射到雌性BALB/c小鼠中(每组6只)。然后，在给药后10分钟、20分钟、30分钟、40分钟、50分钟、60分钟、120分钟、240分钟、360分钟、720分钟、1200分钟和1440分钟，通过静脉取血的方式取10～15μL小鼠的血样并立即加入含有肝素(1000U/mL)的PBS缓冲液中。接下来，在荧光读数仪器(Varioskan Flash Spectral Scanning Multimode Reader，ThermoFisher Scientific)，使用激发波长488nm,发射波长525nm来测定血样中的特异性荧光读数。通过与标准蛋白样品的荧光值曲线来测量出血样中不同蛋白的含量，从而绘制半衰期曲线。
结果如图4所示，通过本发明的方法展示纳米抗体后，可以显著增强纳米抗体的体内半衰期，从33.54min延长到326.3min。
实施例6：激烈火球菌铁蛋白截短体以及人轻链铁蛋白截短体对纳米抗体的展示及表征。
重组构建及表征参考实施例1进行。蛋白表达纯化后，Seq2-纳米抗体成功自组装为球形蛋白，组装过程与人重链铁蛋白(Hfn)的类似，而且其结构与Seq1-纳米抗体类似(图5)，Seq3-纳米抗体结果类似(图6)。
SEQUENCE LISTING
<110> 中科蕴达(北京)生物科技有限公司
<120> 一种改善抗体片段亲和力和体内半衰期的方法
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Claims (8)
1.一种改善抗体片段对抗原的亲和力和/或延长其体内半衰期的方法，所述方法包括将所述抗体片段与铁蛋白亚基去除了短α-螺旋的截短体融合表达；
所述抗体片段为纳米抗体，所述纳米抗体为抗H5N1禽流感病毒抗体；
所述铁蛋白亚基去除了短α-螺旋的截短体的氨基酸序列如SEQ ID NO:1所示；
所述抗H5N1禽流感病毒抗体的氨基酸序列如SEQ ID NO:5所示。
2.根据权利要求1所述的方法，所述抗体片段与铁蛋白亚基去除了短α-螺旋的截短体通过接头融合表达。
3.根据权利要求2所述的方法，所述接头的氨基酸序列如SEQ ID NO：4所示。
4.一种载体，所述载体包括权利要求1所述的铁蛋白亚基去除了短α-螺旋的截短体自组装形成的空腔蛋白壳，以及展示在所述蛋白壳表面的抗体片段；
所述铁蛋白亚基去除了短α-螺旋的截短体的氨基酸序列如SEQ ID NO:1所示；
所述抗体片段的氨基酸序列如SEQ ID NO:5所示。
5.根据权利要求4所述的载体，其中在所述空腔中容纳药物。
6.根据权利要求5所述的载体，所述药物包括小分子化合物、寡核苷酸、功能性肽段或纳米酶。
7.一种融合蛋白，所述融合蛋白的结构由下式表示：X-L-Z，其中，X和Z分别是权利要求1所述的铁蛋白亚基去除了短α-螺旋的截短体和权利要求1所述的抗体片段；L是接头；
所述铁蛋白亚基去除了短α-螺旋的截短体的氨基酸序列如SEQ ID NO:1所示；
所述抗体片段的氨基酸序列如SEQ ID NO:5所示。
8.权利要求4-6中任一项所述的载体或权利要求7所述的融合蛋白用于制备药剂的用途。
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| CN104013599A (zh) * | 2014-05-28 | 2014-09-03 | 中国科学院生物物理研究所 | 一种肿瘤特异性靶向给药的药物载体及其应用 |
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| CN104013599A (zh) * | 2014-05-28 | 2014-09-03 | 中国科学院生物物理研究所 | 一种肿瘤特异性靶向给药的药物载体及其应用 |
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