结合人IL-5的单克隆抗体及其应用的制作方法

本发明属于生物医药领域，具体涉及一种结合人il-5的单克隆抗体及其应用。
背景技术：
：哮喘是肺部气道常见的长期炎症性疾病，通常是由遗传和环境因素相结合引起的，其特征是可变的和反复出现的症状，可逆的气流阻塞和支气管痉挛。症状包括喘鸣，咳嗽，气短和胸闷。这些症状可能每天发生几次或每周发生几次。目前，这种广泛的气道紊乱性疾病在全球影响超过3亿人，到2020年可能会增加到4亿以上。而对于其中一小部分哮喘患者，哮喘偶尔会出现急性症状，导致致命或接近致命的恶化。哮喘发病过程中气道重塑是其中特征性表现，而参与气道重塑的炎症细胞以嗜酸性粒细胞为主。正常情况下，嗜酸性粒细胞在健康肺中通常不存在，但在嗜酸性粒细胞性哮喘病变过程中，因为th2细胞的过度活化，产生大量il-5引起嗜酸性粒细胞的活化、趋化。与此同时，嗜酸性粒细胞会发挥释放碱性蛋白质，脂质介质，细胞因子和趋化因子等细胞毒素及促炎性介质的作用。这些释放的细胞介质会引起一系列的组织损伤，如上皮细胞的缺损，血浆外渗，气道高反应性及组织炎症的启动和维持。除此以外，嗜酸性粒细胞也具有作为抗原呈递细胞的能力，从而导致t细胞增殖和活化，增强炎症反应。因此，对嗜酸性粒细胞的分化、成熟进行干预可以起到治疗或者缓解哮喘的作用。研究表明，白细胞介素-5(il-5)对嗜酸性粒细胞在骨髓中的发育起着至关重要的作用，它可以调节嗜酸性粒细胞的募集、活化、分化和生长。人源白细胞介素-5(il-5)是由134个氨基酸组成，其中包含有19个氨基酸组成的信号肽以及两个潜在的糖基化位点。最初il-5是以可促进小鼠淋巴b细胞成长分化为可分泌抗体的细胞而被人们所熟知的。而后有研究发现，小鼠倘若在il-5或il-5受体呈现出基因缺陷型，那么将会在其b细胞和嗜酸性粒细胞谱系中出现许多发育和功能损伤。因此，il-5除了刺激淋巴b细胞成长分化以外还可以刺激嗜酸性粒细胞或者嗜碱性粒细胞的分化和成熟。而与小鼠不同的是在人类中il-5生物学效应的最明显表征则体现在嗜酸性粒细胞的增殖、迁移和活化。因此，il-5作为嗜酸性粒细胞的谱系特异性细胞因子，在与嗜酸性粒细胞增多相关的疾病如哮喘中发挥重要作用。动物模型和临床试验的大量数据也已经证实，il-5拮抗剂已经是治疗哮喘，特别是严重哮喘的有效方法。英国制药公司葛兰素史克(gsk)开发的抗人il-5蛋白抑制剂美泊利单抗(mepolizumab，商品名nucala)是全球获批的首个靶向白细胞介素-5(il-5)的生物疗法，于2015年底连获美欧2大市场批准，该药同时也是获批治疗重度嗜酸性粒细胞性哮喘的首个il-5单抗。nucala可显著提高患者生活质量和肺功能。在美国，nucala获批用于12岁-17岁青少年及18岁及以上成人重度嗜酸性粒细胞性哮喘患者。在欧盟，nucala获批用于成人重度嗜酸性粒细胞性哮喘患者。目前在国内还没有针对人il-5蛋白的国产治疗性抗体上市，开发国产的抗人il-5单克隆抗体，利用抗体的特异性强、灵敏度高的特点，达到中和il-5的生物学效应的目的，进而起到治疗或缓解哮喘的作用，对于解决国内哮喘病人的用药可及性具有十分重要的意义。技术实现要素：本申请的发明目的是提供一种结合人il-5的单克隆抗体及其应用。为实现上述发明目的，本申请的技术方案如下：一种结合人il-5的单克隆抗体，包含轻链可变区和重链可变区中的至少一个氨基酸片段，所述的重链可变区具有含有如(1)-(10)组中其中一组所示氨基酸序列的重链cdr区：(1)与seqidno.1具有至少50％同源性的氨基酸序列、与seqidno.2具有至少50％同源性的氨基酸序列、与seqidno.3具有至少50％同源性的氨基酸序列；(2)与seqidno.11具有至少50％同源性的氨基酸序列、与seqidno.12具有至少50％同源性的氨基酸序列、与seqidno.13具有至少50％同源性的氨基酸序列；(3)与seqidno.21具有至少50％同源性的氨基酸序列、与seqidno.22具有至少50％同源性的氨基酸序列、与seqidno.23具有至少50％同源性的氨基酸序列；(4)与seqidno.31具有至少50％同源性的氨基酸序列、与seqidno.32具有至少50％同源性的氨基酸序列、与seqidno.33具有至少50％同源性的氨基酸序列；(5)与seqidno.21具有至少50％同源性的氨基酸序列、与seqidno.41具有至少50％同源性的氨基酸序列、与seqidno.42具有至少50％同源性的氨基酸序列；(6)与seqidno.50具有至少50％同源性的氨基酸序列、与seqidno.51具有至少50％同源性的氨基酸序列、与seqidno.52具有至少50％同源性的氨基酸序列；(7)与seqidno.50具有至少50％同源性的氨基酸序列、与seqidno.60具有至少50％同源性的氨基酸序列、与seqidno.61、seqidno.69、seqidno.76或seqidno.84具有至少50％同源性的氨基酸序列；(8)与seqidno.91具有至少50％同源性的氨基酸序列、与seqidno.92具有至少50％同源性的氨基酸序列、与seqidno.93具有至少50％同源性的氨基酸序列；(9)与seqidno.101具有至少50％同源性的氨基酸序列、与seqidno.102具有至少50％同源性的氨基酸序列、与seqidno.103具有至少50％同源性的氨基酸序列；(10)与seqidno.109具有至少50％同源性的氨基酸序列、与seqidno.110具有至少50％同源性的氨基酸序列、与seqidno.111具有至少50％同源性的氨基酸序列；所述的轻链可变区具有含有如下列(11)-(23)组中其中一组所示氨基酸序列的轻链cdr区：(11)与seqidno.5具有至少50％同源性的氨基酸序列、与seqidno.6具有至少50％同源性的氨基酸序列、与seqidno.7具有至少50％同源性的氨基酸序列；(12)与seqidno.15具有至少50％同源性的氨基酸序列、与seqidno.16具有至少50％同源性的氨基酸序列、与seqidno.17具有至少50％同源性的氨基酸序列；(13)与seqidno.25具有至少50％同源性的氨基酸序列、与seqidno.26具有至少50％同源性的氨基酸序列、与seqidno.27具有至少50％同源性的氨基酸序列；(14)与seqidno.35具有至少50％同源性的氨基酸序列、与seqidno.36具有至少50％同源性的氨基酸序列、与seqidno.37具有至少50％同源性的氨基酸序列；(15)与seqidno.44具有至少50％同源性的氨基酸序列、与seqidno.45具有至少50％同源性的氨基酸序列、与seqidno.46具有至少50％同源性的氨基酸序列；(16)与seqidno.54具有至少50％同源性的氨基酸序列、与seqidno.55具有至少50％同源性的氨基酸序列、与seqidno.56具有至少50％同源性的氨基酸序列；(17)与seqidno.63具有至少50％同源性的氨基酸序列、与seqidno.64具有至少50％同源性的氨基酸序列、与seqidno.65具有至少50％同源性的氨基酸序列；(18)与seqidno.63具有至少50％同源性的氨基酸序列、与seqidno.71具有至少50％同源性的氨基酸序列、与seqidno.72具有至少50％同源性的氨基酸序列；(19)与seqidno.78具有至少50％同源性的氨基酸序列、与seqidno.79具有至少50％同源性的氨基酸序列、与seqidno.80具有至少50％同源性的氨基酸序列；(20)与seqidno.63具有至少50％同源性的氨基酸序列、与seqidno.86具有至少50％同源性的氨基酸序列、与seqidno.87具有至少50％同源性的氨基酸序列；(21)与seqidno.95具有至少50％同源性的氨基酸序列、与seqidno.96具有至少50％同源性的氨基酸序列、与seqidno.97具有至少50％同源性的氨基酸序列；(22)与seqidno.105具有至少50％同源性的氨基酸序列、与seqidno.106具有至少50％同源性的氨基酸序列、与seqidno.107具有至少50％同源性的氨基酸序列；(23)与seqidno.113具有至少50％同源性的氨基酸序列、与seqidno.114具有至少50％同源性的氨基酸序列、与seqidno.115具有至少50％同源性的氨基酸序列。本发明制备的单克隆抗体与il-5具有良好的亲和力，同时表现出与阳性对照抗体同一水平的中和活性，为制备il-5拮抗剂提供了优异的基础原料，对于解决国内哮喘病人的用药可及性具有十分重要的意义。显然，本发明中，“与seqidno.x具有至少50％同源性的氨基酸序列”应当是包含“seqidno.x”本身在内的。作为优选，上述的结合人il-5的单克隆抗体包含重链可变区和轻链可变区，所述的重链可变区具有含有如(7)组所示氨基酸序列的重链cdr区，所述的轻链可变区具有含有如(17)组所示氨基酸序列的轻链cdr区。作为进一步优选，所述的重链可变区的三个重链cdr区分别具有：与seqidno.50具有至少50％同源性的氨基酸序列、与seqidno.60具有至少50％同源性的氨基酸序列、与seqidno.61具有至少50％同源性的氨基酸序列。或者，作为优选，上述的结合人il-5的单克隆抗体包含重链可变区和轻链可变区，所述的重链可变区具有含有如(9)组所示氨基酸序列的重链cdr区，所述的轻链可变区具有含有如(22)组所示氨基酸序列的轻链cdr区；或者，所述的重链可变区具有含有如(10)组所示氨基酸序列的重链cdr区，所述的轻链可变区具有含有如(23)组所示氨基酸序列的轻链cdr区。本申请所述的结合人il-5的单克隆抗体可以为人源化抗体、人鼠嵌合抗体或者单链抗体结合片段。作为优选，所述的结合人il-5的单克隆抗体为人源化抗体，所述的重链可变区具有与seqidno.9、seqidno.19、seqidno.29、seqidno.39、seqidno.48、seqidno.58、seqidno.67、seqidno.74、seqidno.82、seqidno.89、seqidno.99和seqidno.117中的至少一种具有至少80％同源性的氨基酸序列；所述的轻链可变区具有与seqidno.10、seqidno.20、seqidno.30、seqidno.40、seqidno.49、seqidno.59、seqidno.68、seqidno.75、seqidno.83、seqidno.90、seqidno.100和seqidno.118中的至少一种具有至少80％同源性的氨基酸序列。作为进一步优选，所述的结合人il-5的单克隆抗体为人源化抗体，所述的重链可变区具有与seqidno.67具有至少80％同源性的氨基酸序列；所述的轻链可变区具有与seqidno.68具有至少80％同源性的氨基酸序列。或者，所述的重链可变区具有与seqidno.117具有至少80％同源性的氨基酸序列；所述的轻链可变区具有与seqidno.118具有至少80％同源性的氨基酸序列。将如seqidno.119所示的重链恒定区、如seqidno.120所示的轻链恒定区分别与上述的重链可变区、轻链可变区重组，即可获得完整的人源化抗体。本发明还提供了所述的结合人il-5的单克隆抗体的编码基因；以及含有所述的结合人il-5的单克隆抗体的编码基因的表达载体；以及表达所述的结合人il-5的单克隆抗体的重组细胞。本发明还提供了所述的结合人il-5的单克隆抗体在制备il-5拮抗剂中的应用；以及采用所述的结合人il-5的单克隆抗体制成的il-5拮抗剂，该il-5拮抗剂可用于治疗哮喘、嗜酸性粒细胞增多综合征、嗜酸性肉芽肿血管炎、慢性阻塞性肺病或慢性鼻窦炎等。与现有技术相比，本发明的有益效果体现在：本发明制备的单克隆抗体与il-5具有优异的亲和力，同时表现出与阳性对照抗体同一水平的中和活性，为制备il-5拮抗剂提供了优异的基础原料，对于解决国内哮喘病人的用药可及性具有十分重要的意义。附图说明图1为亚克隆32d12c12与il-5的亲和力测试结果；其中，sample表示检测样本，sensorgram表示传感图，temperature表示温度，“model1:1binding”表示1:1结合模式；下同；图2为亚克隆64a5e1与il-5的亲和力测试结果；图3为人源化32d12c12抗体与il-5的亲和力测试结果；图4为抗体30e6d5和35c8f7对il-5与受体结合的半数抑制浓度测试结果；图5为人源化抗体30e6d5对il-5与受体结合的半数抑制浓度测试结果；其中，“absneutralizinghil5(1ng/ml)onctll2”表示在小鼠t细胞中抗体对人源化il5(1ng/ml)的中和结果；pc和igg均为阳性对照抗体，ic50表示半数抑制浓度；图6为人源化抗体30e6d5与il-5的亲和力测试结果。具体实施方式下面结合附图和具体实施方式对本发明的技术方案做进一步详细说明。实施例1抗体产生及结合人il-5蛋白的抗体筛选1)按照如下表1中所列的免疫方案对balb/c小鼠进行免疫：表12)细胞融合2.1融合前调整小鼠骨髓瘤sp2/0细胞的状态，保证其生长密度不超过5×106个细胞。2.2收集sp2/0细胞，轻轻晃动并弃掉培养基及漂浮细胞，每皿加入5ml基础培养基轻轻吹下细胞，将所有细胞收集于50ml离心管，1000rpm，离心5min。2.3离心结束后弃上清用1640培养基重悬并计数，细胞数量为4.78×108。2.4将balb/c小鼠摘眼球取血脱颈处死，用75％酒精浸泡消毒。2.5将滤网放在50ml离心管上，用无菌手术器械取出小鼠脾脏，脾脏置于滤网中，用研磨棒研磨脾脏，收集b细胞。加入1640培养基后1000rpm，离心5min。2.6离心结束后弃上清加入10mlacklysingbuffer重悬，静置5min。1000rpm，离心5min。2.7离心结束后弃上清用1640培养基重悬并计数，细胞数量为2.35×108。2.8将b细胞与sp2/0细胞以1:1比例混匀，1000rpm，离心5min。2.9离心结束后弃上清，取融合缓冲液mediumc20ml重悬细胞，1000rpm，离心5min，重复一次。2.10用mediumc调整细胞密度为1×107/ml，混匀后将细胞转入融合池中，8ml/池。2.11设定融合程序：75vac30s，1500v40μs1time，postfusionac3s，进行融合。2.12将融合后的细胞转入hat培养基中，培养箱放置30min。2.13铺板，共铺64块96孔板，37℃培养，培养十天后检测。3)elisa结合实验筛选阳性克隆3.1抗原包被：cbs稀释il-5蛋白使其终浓度为1μg/ml。elisa板每孔包被100μl，4℃过夜。3.2封闭：弃上清，每孔用200μlpbst清洗三次。加入200μl封闭液。37℃放置1h。3.3洗涤：每孔用200μlpbst清洗三次，拍干。3.4加细胞上清：取杂交瘤细胞上清80μl，阳性对照为稀释1000倍的免疫后的小鼠血清，阴性对照为pbs。37℃孵育2h。3.5加二抗：弃上清，每孔用200μlpbst清洗三次。加入带hrp标记的羊抗鼠二抗100μl。37℃放置1h。3.6显色：弃上清，每孔用200μlpbst清洗三次。加入tmb100μl。颜色变化后，加入2m硫酸终止反应。3.7读数：酶标仪450nm、620nm处读取吸光度，选择读数大于0.2的孔为阳性克隆。4)阳性克隆的筛选结果见表2。表2克隆号数值2c60.66377d50.65738g20.793916d20.225117e80.401119b60.250322g90.293529b60.247632d121.000434g100.372861c70.771362e110.665763f120.607864a50.86545)阳性克隆与猴il-5蛋白交叉实验筛选的阳性结果见表3。表3克隆号数值17e80.367419b60.214822g90.415332d120.579534g100.265762e110.337864a50.4896将表3中筛选出的7株阳性克隆转移到24孔板培养，计数铺亚克隆，十天后检测。亚克隆检测采用同上述类似的elisa方法进行细胞培养上清的筛选。筛选结果如下表4。表4实施例2阳性克隆体外生物学功能测定按照以下步骤对杂交瘤elisa阳性克隆的表达上清进行体外生物学功能测定：1)离心收获稳转il5rαβ-ctll-2的细胞系，用il5rαβ-ctll-2细胞培养基(rpmi1640+10％fbs+2mml-glutamine+1mmsodiumpyruvate)重悬细胞，调整细胞密度至2e5cells/ml。2)在96孔细胞培养板中按照每孔50μl体积培养1中获得的重悬细胞。3)用细胞培养基稀释il-5蛋白至4×20ng/ml，之后用阳性对照抗体或者杂交瘤上清按照1:1比例混合稀释后的il-5蛋白。4)将3中混合后的待测样品加入2中的96孔板中，37℃，5％co2培养48h。5)加入50μlμlcelltiterglo试剂反应15min，用septramaxm5仪器测定样品的荧光值。细胞功能实验测定数据如下表5。表5上表中，pc为本实验中的阳性对照抗体，来源于序列seqidno.121和seqidno.122的表达组合，经过proteina纯化获得。通过biocare仪器测定鼠源抗体亲和力，同事考虑到有些亚克隆来自于同一株母克隆，我们最终选择32d12c12和64a5e1进行进一步的抗体结合特性测定。亚克隆32d12c12与64a5e1与il-5的亲和力测试结果如图1和图2。实施例3抗体基因克隆本实施例中分子生物学实验步骤所涉及的技术细节或者具体方案可以参照奥斯伯等人的著作(奥斯伯等主编，金由辛等译校著，2018年，《精编分子生物学实验指南(第五版)》，科学出版社)进行完成，在此不再赘述。本实施例中具体的按照如下的步骤对杂交瘤细胞株进行鼠源抗体基因的克隆。(1)总mrna的提取1)细胞离心好去上清，加1mlrnaisoplus,混匀后室温静置5min。2)加200μl氯仿，充分混匀后室温静置15min。3)12000rpm，4℃，离心15分钟，将上层液体移入新的1.5mlep管中。4)加入等体积的异丙醇，混匀后室温静置10min。5)12000rpm，4℃，离心10分钟，去上清。6)加入1ml75％的乙醇，12000rpm，4℃，离心5分钟，去上清。7)重复步骤6。8)离心后取出残留液体，带乙醇完全挥发后用50μldepc水溶。(2)从cdna中获得抗体基因片段按照常规的技术方案我们从mrna反转录后获得了对应的cdna模板，接下来使用以下的技术方法从cdna中获得抗体基因片段。pcr反应体系和反应条件如表6。表6pcr反应条件如表7。表7参照试剂盒的说明进行实验，具体步骤如下：1)向pcr反应液(或其它酶促反应液)中加入3倍量的bufferdc(如果需加入的bufferdc量不足100μl时应加入100μl)，然后均匀混合。2)将试剂盒中的spincolumn安置于collectiontube上。3)将上述操作1)的溶液转移至spincolumn中，室温12,000rpm离心1分钟，弃滤液。注)如将滤液再加入spincolumn中离心一次，可以提高dna的回收率。4)将700μl的bufferwb加入spincolumn中，室温12,000rpm离心30秒钟，弃滤液。5)重复操作步骤4。6)将spincolumn安置于collectiontube上，室温12,000rpm离心1分钟。7)将spincolumn安置于新的1.5ml的离心管上，在spincolumn膜的中央处加入25～30μl的灭菌水或elutionbuffer，室温静置1分钟。注)将灭菌水或elutionbuffer加热至60℃使用时有利于提高洗脱效率。8)室温12,000rpm离心1分钟洗脱dna。(3)ta载体的连接及克隆转化按照下表8的载体连接体系进行dna片段与ta克隆载体连接。表8试剂名称添加体积t4dnaligase1μl10×buffer1μlt-vector0.5μl目的dna7.5μl之后将连接后的载体进行转化：1)将连接产物加入感受态细胞中，轻轻混匀，冰浴20分钟。2)42℃热激90s，立即取出冰浴2分钟。3)在医用型洁净台内加入lb培养基800μl，37℃振荡培养1小时。4)8000rpm，1min离心后，弃上清，将细胞加入配置的固体lb平板上，进行涂板，倒置放入37℃培养箱中过夜培养。5)第二天挑单克隆，送菌液测序。经过测序结果比对分析后，我们获得了64a5e1的重轻链可变区序列seqidno.104和seqidno.108；以及32d12c12的重轻链可变区序列seqidno.112和seqidno.116。选取32d12c12的可变区序列进行工程化改造，我们筛选到一组高亲和力的人源化32d12c12抗体可变区的组合序列seqidno.117和seqidno.118，亲和力测定结果如图3所示。实施例4单克隆抗体阻断增殖作用的检测在另一个实验中，我们获得了rb0003-30e6d5和rb0003-35c8f7两个阳性克隆，将这两个抗体的克隆进行表达纯化后，对这两个克隆也做进一步的抗体阻断增殖实验的测定，具体步骤如下：1、用外加10ng/ml的il-5的完全培养基培养“il-5敏感型tf-1细胞”。2、将培养数代的“il-5敏感型tf-1细胞”转到无任何添加的rpmi1640培养基中饥饿过夜。3、使用不同浓度的待测抗体溶液、il-5ra溶液(25μl，完全培养基稀释)与setup中确立的终浓度为1ng/ml的rb0003样品(25μl，完全培养基稀释)室温孵育1小时，并设立相应的对照组，详见platemap。4、350g离心收集tf-1细胞，用完全培养基重悬到4e5cells/ml。以2e4cells/well(50μl)的细胞密度接种于96孔通底白色板，随后将rb0003-30e6d5和rb0003-35c8f7抗体的混合溶液(共50μl)加入细胞孔，rb0003的终浓度为1ng/ml。总体系为100μl(blank组只加100μl的完全培养基)。5、37℃/5％co2培养箱孵育96h后，将板室温平衡30min。6、加入100μl的已平衡至室温的ctg反应液(celltiter-gloluminescencecellviabilityassaykit)，水平摇床上裂解2min，室温平衡10min后在96孔板底部贴膜，检测细胞增殖信号，检测结果如表9。表9rb0003阻断增殖作用的检测结果见表10。表10检测数据如下表11所示。表11经过软件处理上表结果后，可以得出上述两个抗体的半数抑制浓度(见表12和图4)。表12antibodyic50(ng/ml)30e6d516.3235c8f7682.45对30e6d5鼠源的抗体可变区序列seqidno.62和seqidno.66进行人源化改造，我们获得了高亲和力的人源化可变区序列组合seqidno.67和seqidno.68。通过将重链可变区拼接至seqidno.119，轻链可变区拼接至seqidno.120后进行基因合成、克隆至ptt5载体进行载体构建，对30e6d5人源化序列进行表达纯化，参照实施例2的方法对30e6d5人源化抗体的ic50进行测定(测定结果见图5，重链氨基酸序列如seqidno.121，轻链氨基酸序列如seqidno.122)，测定结果见表对30e6d5人源化抗体的亲和性进行测定(测定结果见图6)。从检测结果可以看出，人源化后的抗体30e6d5不仅具有良好的抗体亲和力，同时还显示出和阳性对照抗体同一水平的中和活性。序列表<110>瑞阳（苏州）生物科技有限公司<120>结合人il-5的单克隆抗体及其应用<160>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