CN107698680A - 抗pcsk9单克隆抗体 - Google Patents
抗pcsk9单克隆抗体 Download PDFInfo
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- CN107698680A CN107698680A CN201711161968.2A CN201711161968A CN107698680A CN 107698680 A CN107698680 A CN 107698680A CN 201711161968 A CN201711161968 A CN 201711161968A CN 107698680 A CN107698680 A CN 107698680A
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- ser
- dfsk9
- antibody
- seq
- thr
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Abstract
本发明涉及抗体工程技术领域，具体公开了抗PCSK9的单克隆抗体。本发明包含编码所述抗体可变区和CDR区的氨基酸序列，以及获得方法和单克隆抗体的用途。本发明从噬菌体抗体库中筛选出抗PCSK9的单克隆抗体，通过链置换构建噬菌体库的方法进行亲和力成熟，先对初筛获得的单克隆抗体的轻链CDR1、2、3区突变建库筛选后，选取亲和力较高的单克隆抗体，再对其重链CDR1、2、3区突变建库进行筛选，最终筛选到了高亲和力的抗PCSK9的单克隆抗体。本发明所得到的PCSK9抗体对PCSK9具有良好的亲和力并能够抑制PCSK9与其配体的结合，可用于血脂异常、心脑血管疾病和血栓闭塞性疾病的治疗。
Description
技术领域
本发明涉及抗体工程技术领域，具体涉及本发明涉及全人源的抗PCSK9的单克隆抗体、及其获得方法与应用。
背景技术
PCSK9(前蛋白转化酶枯草溶菌素9，Proprotein convertase subtilisin/kexintype 9)属于前蛋白转化酶家族蛋白酶K亚家族。人PCSK9基因定位于染色体1p32.3，长约22kb，共有12个外显子，编码含692个氨基酸残基的蛋白质。PCSK9蛋白由信号肽、前结构域、催化结构域和羧基末端结构域(V结构域)组成，它作为可溶性的74kDa的前体合成，在内质网中经过自身催化裂解产生14kDa的前肽和60kDa的成熟的蛋白酶。PCSK 9主要在肝脏、肠道和肾脏表达，皮肤和神经系统也有少量表达，但是只有肝脏中的PCSK9可以分泌到血液循环系统中。
研究表明，PCSK9能介导低密度脂蛋白受体(LDLR)降解从而调节血浆中LDL胆固醇(LDL-C)水平，而LDLR介导肝内的LDL胞吞过程是从循环系统中清除LDL的主要途径。LDLR是1个多结构域的蛋白，胞外域与表皮生长因子前体同源结构域EGF-A、EGF-B、EGF-C紧连。PCSK9介导LDLR降解首先需要与LDLR结合，LDLR上结合位点主要是EGF-A，形成PCSK9与EGF-A复合物。有研究表明，PCSK9也可通过极低密度脂蛋白受体、载脂蛋白B受体、载脂蛋白E受体2调节胆固醇代谢，但其中的分子机制并不明确。
基础研究和临床试验表明，通过外源性的干预措施抑制PCSK9活性，可以加速血浆中LDL的清除，从而达到降血脂的作用。目前PCSK9的抑制剂主要包括单克隆抗体、反义核苷酸、小分子干扰RNA、模拟肽和小分子抑制剂等。
单克隆抗体药物是今年生物医药领域的研发热点，具有靶向性强、特异性高和毒副作用低等特点，代表了药品治疗领域的最新发展方向。以PCSK9为靶标的单克隆抗体可以与PCSK9发生特异性结合，阻断PCSK9与LDLR的相互作用，减慢LDLR的降解过程从而发挥降低LDL-C水平的作用。临床实验数据显示了抗PCSK9单抗药物的安全性、有效性及独特的临床应用价值。
全人源抗体是治疗性抗体发展的主要方向。抗体库技术的出现为人源抗体的制备筛选提供了良好的技术平台。抗体库技术绕过了以往单抗研制过程中必须的杂交瘤过程，甚至不需要经过免疫过程即可获得各种抗体基因及抗体分子片段。噬菌体抗体库是最早出现也是目前应用最广泛的抗体库。噬菌体抗体库根据抗体基因的来源分为免疫库和非免疫库，非免疫库又包括天然库、半合成库和全合成库。噬菌体抗体库的筛选模拟了抗体亲和力成熟的过程，通常将抗原包被在固相介质上，加入待筛选的噬菌体抗体库，通过数轮“吸附-洗涤-洗脱-扩增”的过程(即淘选)直至筛选到高亲和力特异的抗体。
目前多家制药公司都在积极开发针对PCSK9的单抗药物。安进的Repatha(evolocumab)和赛诺菲/再生元的Praluent(alirocumab)均为全人源抗体，已于2015年先后被批准上市，用于原发性高胆固醇血症和家族性高胆固醇血症(杂合子和纯合子)的治疗。在他汀基础上加用Evolocumab可使原发性高胆固醇血症患者的LDL-C降低77％，杂合子家族性高胆固醇血症患者的LDL-C降低68％，纯合子家族性高胆固醇血症患者的LDL-C降低31％。evolocumab耐受性良好，目前无任何明显的安全性问题。辉瑞的人源化单抗bococizumab正在进行三期临床，诺华的人源化单抗lodelcizumab正在进行二期临床。罗氏及默沙东也在开展临床试验。
目前国内该领域依然缺乏自主研发的具有高亲和力的抗PCSK9的全人源抗体。
发明内容
本发明提供了抗PCSK9的单克隆抗体；本发明从全合成抗体库中筛选出抗PCSK9的单克隆抗体，然后通过计算机辅助设计分析，构建小容量合成噬菌体抗体轻链库的方法，对筛选获得的抗PCSK9单克隆抗体的轻链的CDR1、2、3区突变建库；筛选后，选取亲和力较高的单克隆抗体，再对其重链CDR1、2、3区突变建库进行筛选，最终筛选到了高亲和力的抗PCSK9的单克隆抗体。本发明抗PCSK9抗体具有全新的序列，该抗体在体外特别是细胞水平上功能良好，极具医学应用前景。
为实现上述目的，本发明一种抗PCSK9的单克隆抗体，包括：
轻链和重链；所述轻链的互补决定区CDR1、CDR2和CDR3分别用LCDR1、LCDR2和LCDR3表示；并且所述重链的互补决定区CDR1、CDR2和CDR3分别用HCDR1、HCDR2和HCDR3表示；LCDR1包含RASQSIDNRLT(SEQ NO.22)、RASQSVRNWLD(SEQ NO.23)、RASQGINSWLN(SEQNO.24)、RASQNVNNWLN(SEQ NO.25)、RASQNINSWLN(SEQ NO.26)、RASQNINNWLN(SEQ NO.27)、RASQGIHNWLN(SEQ NO.28)、RASQDVDSWLT(SEQ NO.29)、RASQSVRNWLN(SEQ NO.30)、RASQDVRNWLT(SEQ NO.31)或RASQSIRSYLN(SEQ NO.32)中的任一种；LCDR2包含DASSRQS(SEQNO.33)、GASTLES(SEQ NO.34)、AASTRET(SEQ NO.35)、GASSRQS(SEQ NO.36)、GASTRPT(SEQNO.37)、DASNRQS(SEQ NO.38)、GASNLAS(SEQ NO.39)、DASNLQS(SEQ NO.40)或DASSRPT(SEQNO.41)中的任一种；LCDR3包含QQPENDPTT(SEQ NO.42)、QQDNDIPLT(SEQ NO.43)、QQWNNTPNT(SEQ NO.44)、QQDNDMPLT(SEQ NO.45)、QQWFDVPTT(SEQ NO.46)、QQWDDTPNT(SEQ NO.47)、QQNSNIPLT(SEQ NO.48)、QQDSKIPLT(SEQ NO.49)、QQWTDTPLT(SEQ NO.50)、QQDDSTPPT(SEQNO.51)或QQGDSMPMT(SEQ NO.52)中的任一种；HCDR1包含GGTFTNNA(SEQ NO.53)、GYTVTSYG(SEQ NO.54)或GYSLTSYG(SEQ NO.55)中的任一种；HCDR2包含RIIPMFGMA(SEQ NO.56)、WLSFYNGNT(SEQ NO.57)、WVTFYNGNT(SEQ NO.58)、WVSFYQGNT(SEQ NO.59)、WVSFYNGQT(SEQNO.60)或WVSFYNGNS(SEQ NO.61)中的任一种；HCDR3包含AREGIPMI(SEQ NO.62)、ARGYSLDV(SEQ NO.63)、ARGYGMSI(SEQ NO.64)、ARGFGMDR(SEQ NO.65)、ARGYGMTV(SEQ NO.66)或ARGFGLSV(SEQ NO.67)。
其中，所述轻链可变区氨基酸序列优选SEQ NO.11、SEQ NO.12、SEQ NO.13、SEQNO.14、SEQ NO.15、SEQ NO.16、SEQ NO.17、SEQ NO.18、SEQ NO.19、SEQ NO.20或SEQ NO.21中的任一种。
其中，所述重链可变区氨基酸序列优选SEQ NO.1、SEQ NO.2、SEQ NO.3、SEQ NO.4、SEQ NO.5、SEQ NO.6、SEQ NO.7、SEQ NO.8、SEQ NO.9或SEQ NO.10中的任一种。
其中，所述重链可变区HCDR1序列为选自GYTVTSYG(SEQ NO.54)或GYSLTSYG(SEQNO.55)的氨基酸序列；所述轻链可变区LCDR1序列选自RASQSVRNWLD(SEQ NO.23)、RASQNVNNWLN(SEQ NO.25)、RASQNINSWLN(SEQ NO.26)、RASQNINNWLN(SEQ NO.27)或RASQDVDSWLT(SEQ NO.29)中的任一种氨基酸序列；所述重链可变区HCDR2序列选自WVSFYQGNT(SEQ NO.59)、WVSFYNGQT(SEQ NO.60)或WVSFYNGNS(SEQ NO.61)中的任一种氨基酸序列；所述轻链可变区LCDR2序列选自GASTLES(SEQ NO.34)、AASTRET(SEQ NO.35)、GASSRQS(SEQ NO.36)、GASTRPT(SEQ NO.37)或GASNLAS(SEQ NO.39)中的任一种氨基酸序列；所述重链可变区HCDR3序列选自ARGYSLDV(SEQ NO.63)、ARGYGMSI(SEQ NO.64)、ARGFGMDR(SEQ NO.65)或ARGYGMTV(SEQ NO.66)中的任一种氨基酸序列；所述轻链可变区LCDR3序列选自QQDNDIPLT(SEQ NO.43)、QQDNDMPLT(SEQ NO.45)、QQWFDVPTT(SEQ NO.46)、QQWDDTPNT(SEQ NO.47)或QQDSKIPLT(SEQ NO.49)中的任一种氨基酸序列。
其中，本发明还提供了多种包含上述轻链或上述重链的抗体、多肽或蛋白。
其中，本发明还提供了多种包含上述轻链或上述重链的抗体，所述抗体能够特异性与PCSK9结合，阻断PCSK9与LDLR的结合，提高细胞表面LDLR的数量或血液循环系统中LDLR的水平，降低血液循环系统中LDL或LDL-C的水平。
其中，本发明还提供了多种包含上述轻链或上述重链的多核苷酸序列或组合。
其中，所述抗PCSK9的单克隆抗体的重链恒定区包含IgG1、IgG2、IgG3和IgG4；所述轻链恒定区包含Cκ或Cλ。
其中，所述重链恒定区优选IgG4或IgG2；所述重链的真核表达载体或原核生物表达载体。
其中，所述轻链恒定区优选Cκ；所述轻链的真核表达载体或原核生物表达载体。
其中，本发明还提供了一种包含上述多核苷酸序列或组合的重组DNA表达载体；所述重组DNA表达载体的DNA序列中包含编码抗PCSK9抗体的上述重链可变区、重链恒定区、轻链可变区和轻链恒定区的氨基酸序列。
其中，本发明还提供了一种转染上述重组DNA表达载体的宿主细胞，所述宿主细胞包含原核细胞、酵母、昆虫或哺乳动物细胞。
其中，所述原核细胞优选大肠杆菌。
其中，所述哺乳动物细胞优选HEK293E细胞、CHO细胞或NS0细胞。
其中，本发明还提供了多种含有上述轻链或上述重链的全长抗体、单链抗体、单域抗体、双特异抗体、抗体药物偶联物。
其中，本发明还提供了多种含有上述轻链或上述重链的单克隆抗体、人工载体、药物或药物组合物。
其中，所述单克隆抗体包含全长抗体和抗PCSK9单克隆抗体的片段，所述片段包含但不限于Fab、Fab’、F(ab’)2、Fv或ScFv。
其中，本发明还提供了一种含有上述轻链或上述重链的检测试剂或试剂盒。
本发明所述抗体可用于通过消除、抑制或降低PCSK9活性而被改善、缓解、抑制或预防的疾病；所述疾病包含血脂异常、心脑血管疾病和血栓闭塞性疾病。
其中，所述血脂异常包含胆固醇升高、甘油三脂升高、低密度脂蛋白升高和高密度脂蛋白降低。所述心血管疾病包含冠状动脉硬化性心脏病、急性心肌梗塞、动脉粥状硬化、中风和外周动脉闭塞性疾病。
本发明一种抗PCSK9的单克隆抗体的获得方法，包括如下步骤：
(1)、抗PCSK9单链抗体的生物淘选，通过三轮抗体库的富集筛选，从全合成的ScFv噬菌体库中获得了一种亲和力较高的抗体序列DFSK9-1，其重链DFSK9-H1具有SEQ NO.1的氨基酸序列，其轻链DFSK9-L1具有SEQ NO.11的氨基酸序列；
(2)、以DFSK9-1为基础，通过计算机三级结构模拟，设计并构建轻链互补决定区CDR1、CDR2、CDR3突变抗体库,并对这个突变抗体库进行生物淘选和阳性克隆的筛选及鉴定，获得了10种含有不同轻链的单链抗体序列，分别命名为DFSK9-2、DFSK9-3、DFSK9-4、DFSK9-5、DFSK9-6、DFSK9-7、DFSK9-8、DFSK9-9、DFSK9-10、DFSK9-11，对应的轻链可变区分别命名为DFSK9-L2、DFSK9-L3、DFSK9-L4、DFSK9-L5、DFSK9-L6、DFSK9-L7、DFSK9-L8、DFSK9-L9、DFSK9-L10和DFSK9-L11，其对应的氨基酸序列分别见SEQ NO.12、SEQ NO.13、SEQNO.14、SEQ NO.15、SEQ NO.16、SEQ NO.17、SEQ NO.18、SEQ NO.19、SEQ NO.20和SEQ NO.21；将上述单链抗体在噬菌体水平上进行亲和力比较；
(3)、选出5株亲和力较高的克隆DFSK9-2、DFSK9-4、DFSK9-5、DFSK9-6和DFSK9-8，设计并构建重链互补决定区CDR1、CDR2、CDR3突变抗体库，再对这个重链突变的抗体库进行生物淘选和阳性克隆的筛选及鉴定，获得了10种不同的单链抗体序列，分别命名为DFSK9-12、DFSK9-13、DFSK9-14、DFSK9-15、DFSK9-16、DFSK9-17、DFSK9-18、DFSK9-19、DFSK9-20和DFSK9-21；其中，DFSK9-12、DFSK9-13、DFSK9-14、DFSK9-15、DFSK9-16、DFSK9-17、DFSK9-18、DFSK9-19、DFSK9-20和DFSK9-21的轻链可变区序列分别是DFSK9-L2、DFSK9-L8、DFSK9-L5、DFSK9-L6、DFSK9-L5、DFSK9-L6、DFSK9-L5、DFSK9-L4、DFSK9-L6、DFSK9-L6；其对应的氨基酸序列分别见SEQ NO.12、SEQ NO.18、SEQ NO.15、SEQ NO.16、SEQ NO.15、SEQ NO.16、SEQNO.15、SEQ NO.14、SEQ NO.16、SEQ NO.16；DFSK9-12、DFSK9-13、DFSK9-14、DFSK9-15、DFSK9-16、DFSK9-17、DFSK9-18、DFSK9-19、DFSK9-20和DFSK9-21的重链可变区序列分别是DFSK9-H2、DFSK9-H8、DFSK9-H7、DFSK9-H3、DFSK9-H6、DFSK9-H4、DFSK9-H4、DFSK9-H9、DFSK9-H5、DFSK9-H10；其对应的氨基酸序列分别见SEQ NO.2、SEQ NO.8、SEQ NO.7、SEQNO.3、SEQ NO.6、SEQ NO.4、SEQ NO.4、SEQ NO.9、SEQ NO.5、SEQ NO.10；将上述单链抗体在噬菌体水平上进行亲和力比较；
(4)、将(3)中所述单克隆重链可变区基因和轻链可变基因克隆到真核表达载体，转染宿主细胞，获得抗PCSK9单克隆抗体的全抗。
其中，所述CDR为互补决定区(complementarity-determining region)；所述ScFv为单链抗体(single-chain fragment variable)；所述ADCs为抗体药物偶联物(antibody-drug conjugates)；所述LDLR为低密度脂蛋白受体(Low Density LipoproteinReceptor)；所述LDL-C为低密度脂蛋白胆固醇(Low Density Lipoprotein-Cholesterol)；所述HEK293E细胞为人胚肾293E细胞(human embryonic kidney 293E cell)；CHO细胞为中国仓鼠卵巢细胞(chinese hamster ovary cell)；NS0细胞为小鼠NS0胸腺瘤细胞。
本发明相对于现有技术而言，具备的有益效果是：
本发明提供的是多种全新的抗PCSK9抗体，与底物结合的亲和力高，能够很好的阻断PCSK9与LDLR的结合，并且影响细胞表面的LDLR的分布和表达，从而提高细胞表面的LDL-R与LDL结合能力，增加细胞对LDL摄取和降解，达到降低胞外的LDL和LDL-C的含量，达到降低LDL、LDL-C和总胆固醇目的。
本发明提供的单克隆抗体可以用于通过消除、抑制或降低PCSK9活性而改善、缓解、抑制或预防的疾病；所述疾病包含血脂异常、心脑血管疾病和血栓闭塞性疾病；血脂异常包含胆固醇升高、甘油三脂升高、低密度脂蛋白升高和高密度脂蛋白降低等；心血管疾病包含冠状动脉硬化性心脏病、急性心肌梗塞、动脉粥状硬化、中风和外周动脉闭塞性疾病等。
说明书附图
图1、pScFvDisb-S1质粒图谱；
图2、轻链突变抗体库阳性克隆噬菌体单克隆ELISA鉴定单链抗体的相对亲和力；
图3、轻链突变抗体库阳性克隆噬菌体梯度ELISA比较单链抗体的相对亲和力；
图4、重链突变抗体库阳性克隆噬菌体单克隆ELISA鉴定单链抗体的相对亲和力；
图5、重链突变抗体库阳性克隆噬菌体梯度ELISA比较单链抗体的相对亲和力；
图6、pTSEG4质粒图谱；
图7、pTSEK质粒图谱
图8、抗PCSK9全抗体与PCSK9在分子水平上结合试验；
图9、抗PCSK9全抗体竞争抑制LDLR与PCSK9结合实验；
图10、抗PCSK9全抗体生物学活性实验。
具体实施方式
本发明详细的实施方法参见实施例，实施例中所述的实验方法和试剂，若无特殊说明均为常规实验方法和试剂。以下实施例仅用于说明和解释本发明，而不是以任何方式限制本发明。
本发明提供了一种特异性结合PCSK9的单克隆抗体，所述重链可变区序列包含SEQNO.1、2、3、4、5、6、7、8、9和10中的任一种,所述轻链可变区序列包含SEQ NO.11、12、13、14、15、16、17、18、19、20和21中的任一种。
优选地，所述特异性结合PCSK9的单克隆抗体的重链可变区序列选自SEQ NO.2、3、4、5、6、7、8、9和10中的任一种，所述轻链可变区序列选自SEQ NO.12、14、15、16和18中的任一种。
通过轻链噬菌体库筛选，所述抗体轻链或其功能性片段的轻链互补决定区LCDR1、LCDR2和LCDR3的氨基酸序列选自以下氨基酸序列中的任一组合(如表1所示)：
表1、轻链各个CDR区的氨基酸序列
通过重链噬菌体库筛选，所述抗体重链或其功能性片段重链的互补决定区CDR1、CDR2和CDR3分别用HCDR1、HCDR2和HCDR3表示：HCDR1为GGTFTNNA(SEQ NO.53)、GYTVTSYG(SEQ NO.54)或GYSLTSYG(SEQ NO.55)中的任一种；HCDR2为RIIPMFGMA(SEQ NO.56)、WLSFYNGNT(SEQ NO.57)、WVTFYNGNT(SEQ NO.58)、WVSFYQGNT(SEQ NO.59)、WVSFYNGQT(SEQNO.60)或WVSFYNGNS(SEQ NO.61)中的任一种；HCDR3为AREGIPMI(SEQ NO.62)、ARGYSLDV(SEQ NO.63)、ARGYGMSI(SEQ NO.64)、ARGFGMDR(SEQ NO.65)、ARGYGMTV(SEQ NO.66)或ARGFGLSV(SEQ NO.67)中的任一种。
优选地，通过重链噬菌体库筛选，所述特异性结合PCSK9的单克隆抗体包含HCDR1、HCDR2和HCDR3序列的重链可变区及含LCDR1、LCDR2和LCDR3序列的轻链可变区；其中,所述重链可变区HCDR1序列为选自GYTVTSYG(SEQ NO.54)或GYSLTSYG(SEQ NO.55)的氨基酸序列；所述轻链可变区LCDR1序列选自RASQSVRNWLD(SEQ NO.23)、RASQNVNNWLN(SEQ NO.25)、RASQNINSWLN(SEQ NO.26)、RASQNINNWLN(SEQ NO.27)或RASQDVDSWLT(SEQ NO.29)中的任一种氨基酸序列；所述重链可变区HCDR2序列选自WVSFYQGNT(SEQ NO.59)、WVSFYNGQT(SEQNO.60)或WVSFYNGNS(SEQ NO.61)中的任一种氨基酸序列；所述轻链可变区LCDR2序列选自GASTLES(SEQ NO.34)、AASTRET(SEQ NO.35)、GASSRQS(SEQ NO.36)、GASTRPT(SEQ NO.37)或GASNLAS(SEQ NO.39)中的一种氨基酸序列；所述重链可变区HCDR3序列选自ARGYSLDV(SEQNO.63)、ARGYGMSI(SEQ NO.64)、ARGFGMDR(SEQ NO.65)或ARGYGMTV(SEQ NO.66)中的任一种氨基酸序列；所述轻链可变区LCDR3序列选自QQDNDIPLT(SEQ NO.43)、QQDNDMPLT(SEQNO.45)、QQWFDVPTT(SEQ NO.46)、QQWDDTPNT(SEQ NO.47)或QQDSKIPLT(SEQ NO.49)中的任一种氨基酸序列。
本发明用全合成ScFv单链噬菌抗体库获得特异性抗体的方法，所述全人源的特异性结合PCSK9的单克隆抗体是用噬菌体抗体库技术筛选而得，其步骤在于：
(1)、抗PCSK9单链抗体的生物淘选，通过三轮抗体库的富集筛选，获得了一种亲和力较高的抗体序列DFSK9-1；
(2)、以DFSK9-1为基础，通过计算机辅助设计，构建轻链CDR123突变库并对该抗体库进行生物淘选和阳性克隆的筛选及鉴定，获得了10种不同轻链的抗体序列的克隆DFSK9-2、DFSK9-3、DFSK9-4、DFSK9-5、DFSK9-6、DFSK9-7、DFSK9-8、DFSK9-9、DFSK9-10、DFSK9-11。将上述10种单链抗体在噬菌体水平进行亲和力比较；
(3)、选出两株亲和力较高的克隆，构建重链CDR123库并对该库进行生物淘选和阳性克隆的筛选，获得了种10不同序列的单链抗体DFSK9-12、DFSK9-13、DFSK9-14、DFSK9-15、DFSK9-16、DFSK9-17、DFSK9-18、DFSK9-19、DFSK9-20、DFSK9-21；将上述单链抗体在噬菌体水平上进行亲和力比较；
(4)、将(3)中所述单克隆重链可变区基因和轻链可变基因克隆到真核表达载体，转染宿主细胞，获得抗PCSK9单克隆抗体的全抗。
优选地，对(4)的抗PCSK9单克隆抗体的全抗再进行亲和力和生物活性试验。
具体实施例
以下结合附图和实施例详述本发明。
实施例1、抗PCSK9单链抗体的生物淘选
采用基因克隆的方法改造pCom3载体，改造后的载体命名为pScFvDisb-S1(图1)。以该载体为基础构建全合成噬菌体抗体库。
免疫管中包被抗原PCSK9-His 10μg/1ml/管，4℃包被过夜。用PBST-4％milk分别封闭免疫管和噬菌体抗体库(噬菌体投入量约为109-1012)，37℃封闭1h。封闭后的噬菌体抗体库加入免疫管中进行抗原抗体结合，37℃反应1h。PBST-PBS洗去未结合的噬菌体，0.1MpH2.2的Glycine-HCl洗脱，用1.5M pH8.8的Tris-HCl中和洗脱液至pH7.0左右。洗脱液感染10ml生长至OD值约0.5-0.8的XL1-Blue菌液，37℃先静置30min后150rpm摇床振荡培养1h。取出1％菌液进行梯度稀释，涂布于2YTATG小平皿上，用于计算噬菌体产出量。剩余的菌液离心后涂布于2YTATG大平皿，37℃过夜培养。将过夜培养菌转接至2YTATG液体培养基，摇至对数期后加入M13K07辅助噬菌体感染，28℃培养过夜扩增噬菌体，PEG6000-NaCl沉降纯化噬菌体用于下一轮筛选。共进行3轮噬菌体库富集筛选。
实施例2、抗PCSK9单链抗体阳性克隆的筛选
经过三轮筛选后，挑取分隔良好的单克隆菌落，接种于加有2YTATG液体培养基的96孔深孔板，37℃，220rpm培养约5h至其对数生长期，每孔加入约1010的辅助噬菌体M13KO7，37℃静置30min后150rpm振荡培养1h。4000rpm，离心15min，沉淀重悬于2YTATKA液体培养基中，28℃，220rpm培养过夜。4000rpm，4℃离心15min，吸取含噬菌体上清进行单克隆ELISA鉴定。筛选得到亲和力较高的单链抗体DFSK9-1，其重链可变区命名为DFSK9-H1,其氨基酸序列见SEQ NO.1；其轻链可变区命名为DFSK9-L1,氨基酸序列见SEQ NO.11；
SEQ NO.1(DFSK9-H1重链可变区序列)：QVQLVQSGAEVKRPGASVKVSCKASGGTFTNNAISWVRQAPGQGLEWMGRIIPMFGMANYAQKLQGRGTMTTDPSTSTAYMELRSLRSDDTAVYYCAREGIPMIWGQGTTVTVSS
SEQ NO.11(DFSK9-L1轻链可变区序列)：DIQMTQSPSSLSASVGDRVTITCRASQSIDNRLTWYQQKPGKAPKLLIYDASSRQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQPENDPTTFGQGTKVEIK
实施例3、对单链抗体DFSK9-1进行体外亲和力成熟
3.1.构建DFSK9-1轻链CDR123突变库
用NheI和NotI对pScFvDisb-DFSK9-1质粒进行双酶切，酶切产物琼脂糖凝胶电泳后，切胶回收大小5.5kb的条带；用NheI和NotI对合成的轻链突变库基因VLCDR123M进行双酶切，通用产物回收试剂盒回收产物。突变库基因与载体按摩尔比3：1比例经T4DNA连接酶16℃连接4h。将连接产物电击法转化至XL1-Blue电转感受态中。37℃，150rpm振动培养1h复苏。取1％菌液，稀释后涂小平皿，计算库容量。其余菌液4000rpm离心15min后，沉淀涂布于2YTATG大平皿上，37℃倒置培养过夜。建成的抗体库库容大约为108，随机挑取20个克隆进行序列分析，序列正确率和多样性都大于90％。
3.2.噬菌体抗体库的生物淘选和阳性克隆的筛选
在按照实施例1和实施例2的方法进行生物淘选和阳性克隆筛选，对亲和力较高的克隆进行测序，共得到10种不同的单链抗体序列，分别命名为DFSK9-2、DFSK9-3、DFSK9-4、DFSK9-5、DFSK9-6、DFSK9-7、DFSK9-8、DFSK9-9、DFSK9-10、DFSK9-11，对应的轻链可变区命名为DFSK9-L2、DFSK9-L3、DFSK9-L4、DFSK9-L5、DFSK9-L6、DFSK9-L7、DFSK9-L8、DFSK9-L9、DFSK9-L10、DFSK9-L11，它们对应的氨基酸序列分别见SEQ NO.12、SEQ NO.13、SEQ NO.14、SEQ NO.15、SEQ NO.16、SEQ NO.17、SEQ NO.18、SEQ NO.19、SEQ NO.20和SEQ NO.21；轻链可变区序列SEQ NO.12-SEQ NO.21如下：
SEQ NO.12(DFSK9-L2轻链可变区序列)：DIQMTQSPSSLSASVGDRVTITCRASQSVRNWLDWYQQKPGKAPKLLIYGASTLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQDNDIPLTFGQGTKVEIK
SEQ NO.13(DFSK9-L3轻链可变区序列)：DIQMTQSPSSLSASVGDRVTITCRASQGINSWLNWYQQKPGKAPKLLIYAASTRETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWNNTPNTFGQGTKVEIK
SEQ NO.14(DFSK9-L4轻链可变区序列)：DIQMTQSPSSLSASVGDRVTITCRASQNVNNWLNWYQQKPGKAPKLLIYAASTRETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQDNDMPLTFGQGTKVEIK
SEQ NO.15(DFSK9-L5轻链可变区序列)：DIQMTQSPSSLSASVGDRVTITCRASQNINSWLNWYQQKPGKAPKLLIYGASSRQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWFDVPTTFGQGTKVEIK
SEQ NO.16(DFSK9-L6轻链可变区序列)：DIQMTQSPSSLSASVGDRVTITCRASQNINNWLNWYQQKPGKAPKLLIYGASTRPTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWDDTPNTFGQGTKVEIK
SEQ NO.17(DFSK9-L7轻链可变区序列)：DIQMTQSPSSLSASVGDRVTITCRASQGIHNWLNWYQQKPGKAPKLLIYDASNRQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNSNIPLTFGQGTKVEIK
SEQ NO.18(DFSK9-L8轻链可变区序列)：DIQMTQSPSSLSASVGDRVTITCRASQDVDSWLTWYQQKPGKAPKLLIYGASNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQDSKIPLTFGQGTKVEIK
SEQ NO.19(DFSK9-L9轻链可变区序列)：DIQMTQSPSSLSASVGDRVTITCRASQSVRNWLNWYQQKPGKAPKLLIYDASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWTDTPLTFGQGTKVEIK
SEQ NO.20(DFSK9-L10轻链可变区序列)：DIQMTQSPSSLSASVGDRVTITCRASQDVRNWLTWYQQKPGKAPKLLIYGASNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQDDSTPPTFGQGTKVEIK
SEQ NO.21(DFSK9-L11轻链可变区序列)：DIQMTQSPSSLSASVGDRVTITCRASQSIRSYLNWYQQKPGKAPKLLIYDASSRPTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGDSMPMTFGQGTKVEIK
单克隆噬菌体ELISA鉴定结果如图2所示；
3.3.噬菌体水平梯度稀释ELISA鉴定抗PCSK9单链抗体的相对亲和力
将本实施例3.2中获得的克隆进行单克隆噬菌体的展示和纯化，进行噬菌体水平的梯度稀释ELISA鉴定单链抗体的相对亲和力。
用pH7.2的0.01M PBS缓冲液包被PCSK9-His(300ng/孔/100μl)，4℃包被过夜。PBST洗涤3次，PBST-4％milk 37℃封闭1h。加入PBST-4％milk五倍梯度稀释的纯化噬菌体样品(100μl/孔)，37℃静置1h。用PBST洗涤5次，加入PBST-4％milk 1:5000稀释的anti-M13-HRP单克隆抗体(100μl/孔)，37℃放置1h。TMB显色试剂盒显色(100μl/孔)，室温显色10min，用2M H2SO4(50μl/孔)终止显色,。酶标仪波长450nm和630nm读数。使用软件GraphPadPrism5Demo分析数据并作图，结果如图3所示：结果显示筛选出的噬菌体单链抗体均能与PCSK9进行结合，而且DFSK9-2、DFSK9-4、DFSK9-5、DFSK9-6和DFSK9-8的亲和力明显高于其它克隆，选取上述5个单链抗体进行下一步试验。
实施例4、对筛选出的抗PCSK9的单链抗体再次进行体外亲和力成熟
4.1链置换法构建重链CDR123突变库
用NcoI-HF和KpnI对实施例3.3中DFSK9-2、DFSK9-4、DFSK9-5、DFSK9-6和DFSK9-8等5种单链抗体的混合质粒进行双酶切，切胶回收大小5.5kb的条带；用NcoI-HF和KpnI对合成的重链突变库基因VHCDR123M进行双酶切，通用回收试剂盒回收酶切产物。突变库基因与载体按摩尔比3：1比例经T4DNA连接酶16℃连接4h。将连接产物电击法转化至XL1电转感受态中。37℃，150rpm培养1h复苏。取1％菌液，稀释后涂小平皿，计算库容量。其余菌液4000rpm，离心15min后，沉淀涂布于2YTATG大平皿上，37℃倒置培养过夜。构建成的抗体库库容大约为5*108，随机挑取20个克隆进行序列分析，正确率和多样性均大于90％。
4.2噬菌体抗体库的生物淘选和阳性克隆的筛选
将实施例4.1中的抗体库进行噬菌体展示和纯化回收。从该库中淘选抗PCSK9的单链抗体。噬菌体抗体库的生物淘选方法和阳性克隆的筛选同实施案例1和实施例2。测序后发现共筛选到10种不同的抗PCSK9抗体序列，分别命名为DFSK9-12、DFSK9-13、DFSK9-14、DFSK9-15、DFSK9-16、DFSK9-17、DFSK9-18、DFSK9-19、DFSK9-20、DFSK9-21。其中DFSK9-12的轻链可变区序列是DFSK9-L2，对应的氨基酸序列见SEQ NO.12；DFSK9-13的轻链可变区序列是DFSK9-L8，对应的氨基酸序列见SEQ NO.18；DFSK9-14、DFSK9-16和DFSK9-18的轻链可变区序列是DFSK9-L5，对应的氨基酸序列见SEQ NO.15；DFSK9-15、DFSK9-17、DFSK9-20和DFSK9-21的轻链可变区序列是DFSK9-L6，对应的氨基酸序列见SEQ NO.16；DFSK9-19的轻链可变区序列是DFSK9-L4，对应的氨基酸序列见SEQ NO.14。DFSK9-12的重链可变区序列是DFSK9-H2，对应的氨基酸序列见SEQ NO.2；DFSK9-13的重链可变区序列是DFSK9-H8，对应的氨基酸序列见SEQ NO.8；DFSK9-14的重链可变区序列是DFSK9-H7，对应的氨基酸序列见SEQNO.7；DFSK9-15的重链可变区序列是DFSK9-H3，对应的氨基酸序列见SEQ NO.3；DFSK9-16的重链可变区序列是DFSK9-H6，对应的氨基酸序列见SEQ NO.6；DFSK9-17和DFSK9-18的重链可变区序列是DFSK9-H4，，对应的氨基酸序列见SEQ NO.4；DFSK9-19的重链可变区是DFSK9-H9，对应的氨基酸序列见SEQ NO.9；DFSK9-20的重链可变区是DFSK9-H5，对应的氨基酸序列见SEQ NO.5；DFSK9-21的重链可变区序列是DFSK9-H10，对应的氨基酸序列见SEQ NO.10。轻链可变区序列参照实施例3.2中氨基酸序列SEQ NO.12、SEQ NO.14、SEQ NO.15、SEQ NO.16和SEQ NO.18，重链可变区氨基酸序列SEQ NO.2-SEQ NO.10如下：
SEQ NO.2(DFSK9-H2重链可变区序列)：QVQLVQSGAEVKKPGASVKVSCKASGYTVTSYGISWVRQAPGQGLEWMGWLSFYNGNTNYAQKLQGRGTMTTDPSTSTAYMELRSLRSDDTAVYYCARGYSLDVWGQGTTVTVSS
SEQ NO.3(DFSK9-H3重链可变区序列)：QVQLVQSGAEVKKPGASVKVSCKASGYTVTSYGISWVRQAPGQGLEWMGWVSFYNGQTNYAQKLQGRGTMTTDPSTSTAYMELRSLRSDDTAVYYCARGYSLDVWGQGTTVTVSS
SEQ NO.4(DFSK9-H4重链可变区序列)：QVQLVQSGAEVKKPGASVKVSCKASGYTVTSYGISWVRQAPGQGLEWMGWVSFYNGNSNYAQKLQGRGTMTTDPSTSTAYMELRSLRSDDTAVYYCARGYSLDVWGQGTTVTVSS
SEQ NO.5(DFSK9-H5重链可变区序列)：QVQLVQSGAEVKKPGASVKVSCKASGYSLTSYGISWVRQAPGQGLEWMGWVSFYNGNSNYAQKLQGRGTMTTDPSTSTAYMELRSLRSDDTAVYYCARGFGMDRWGQGTTVTVSS
SEQ NO.6(DFSK9-H6重链可变区序列)：QVQLVQSGAEVKKPGASVKVSCKASGYTVTSYGISWVRQAPGQGLEWMGWVSFYQGNTNYAQKLQGRGTMTTDPSTSTAYMELRSLRSDDTAVYYCARGFGMDRWGQGTTVTVSS
SEQ NO.7(DFSK9-H7重链可变区序列)：QVQLVQSGAEVKKPGASVKVSCKASGYSLTSYGISWVRQAPGQGLEWMGWVSFYNGQTNYAQKLQGRGTMTTDPSTSTAYMELRSLRSDDTAVYYCARGYGMSIWGQGTTVTVSS
SEQ NO.8(DFSK9-H8重链可变区序列)：QVQLVQSGAEVKKPGASVKVSCKASGYSLTSYGISWVRQAPGQGLEWMGWVSFYQGNTNYAQKLQGRGTMTTDPSTSTAYMELRSLRSDDTAVYYCARGYGMTVWGQGTTVTVSS
SEQ NO.9(DFSK9-H9重链可变区序列)：QVQLVQSGAEVKKPGASVKVSCKASGYSLTSYGISWVRQAPGQGLEWMGWVTFYNGNTNYAQKLQGRGTMTTDPSTSTAYMELRSLRSDDTAVYYCARGFGLSVWGQGTTVTVSS
SEQ NO.10(DFSK9-H10重链可变区序列)：QVQLVQSGAEVKKPGASVKVSCKASGYSLTSYGISWVRQAPGQGLEWMGWVSFYNGQTNYAQKLQGRGTMTTDPSTSTAYMELRSLRSDDTAVYYCARGYGMDRWGQGTTVTVSS
单克隆phage ELISA鉴定phage-Abs的相对亲和力如图4所示。
4.3.梯度稀释噬菌体ELISA鉴定抗PCSK9单链抗体的亲和力
将本实施例4.2中获得的克隆进行单克隆噬菌体的展示和纯化，进行噬菌体梯度稀释ELISA实验鉴定单链抗体的亲和力，方法同实施例3中3.3。结果如图5所示：筛选出的10种不同的单链抗体均能与PCSK9进行很好的结合，且亲和力均高于最初的单链DFSK9-1。
实施例5、抗PCSK9全抗体的亲和力鉴定
5.1抗PCSK9全抗体的制备
将实施例4中筛到抗体的重链VH克隆至含有重链恒定区基因(γ4)的载体pTSEG4(图6)上，轻链VK基因克隆到含有轻链恒定区基因(κ链)的载体pTSEK(图7)上，载体pTSEG4和pTSEK均以PTT载体为基础改造获得。PTT载体的制备过程在参考文献(Yves.Durocher,Sylvie.Perret and Amine.Kamen Nucleic Acids Research，2002Vol.30，No.2e9)中有详细描述。瞬时转染HEK293E细胞，进行全抗体表达。使用AKTA仪器protein A亲和柱纯化获得全抗体蛋白。
5.2全抗体与PCSK9的结合实验
用pH7.2的0.01M PBS缓冲液包被PCSK9-His(300ng/孔/100μl)，4℃包被过夜。用300μl/孔PBST(1‰Tween 20)洗3次，再加入PBST-4％milk 37℃封闭1h。加入不同稀释度的全抗体DFSK9-1，DFSK9-12、DFSK9-13、DFSK9-14、DFSK9-15、DFSK9-16、DFSK9-17、DFSK9-18、DFSK9-19、DFSK9-20和DFSK9-21。11种全抗体最高浓度是50ug/ml，5倍梯度稀释，每个全抗体做8个梯度，37℃孵育1h。用300μl/孔PBST洗5次，再加入用PBST-4％milk 1:5000稀释的山羊抗人IgG-HRP二抗37℃孵育1h。用300μl/孔PBST洗5次，TMB显色试剂盒显色(100μl/孔)，室温显色10min，然后用2M H2SO4(50μl/孔)终止显色。酶标仪波长450nm和630nm读数。使用软件GraphPad Prism 5Demo分析数据作图，实验结果如图8和表2所示，所有抗体均能很好的与PCSK9分子结合,其中DFSK9-14、DFSK9-15、DFSK9-16、DFSK9-17、DFSK9-18、DFSK9-20和DFSK9-21具有相对较高的亲和力。
表2、全抗体亲和力EC50值
5.3全抗体竞争抑制LDLR与PCSK9结合实验
用pH7.2的0.01M PBS包被LDLR-Fc(100ng/孔/100μl)，4℃包被过夜。PBST洗涤3次，再加入PBST-4％milk 37℃封闭1h。加入PBST-4％milk稀释的浓度为2μg/ml的PCSK9-His(100μl/孔)，37℃孵育1h。加入PBST-4％milk稀释不同稀释度的全抗体DFSK9-1，DFSK9-12、DFSK9-13、DFSK9-14、DFSK9-15、DFSK9-16、DFSK9-17、DFSK9-18、DFSK9-19、DFSK9-20和DFSK9-21。11种全抗体最高浓度是100ug/ml，五倍梯度稀释，每个全抗体做8个梯度，37℃孵育2h。PBST洗5次，加入用PBST-4％milk稀释的小鼠抗His IgG-HRP二抗，37℃孵育1h。TMB显色试剂盒显色(100μl/孔)，室温显色10min，用2M H2SO4(50μl/孔)终止显色。酶标仪波长450nm和630nm读数。使用软件GraphPad Prism 5Demo作图，实验结果如图9和表3所示，所有抗体均能有效的抑制PCSK9与LDLR的结合，其中DFSK9-14、DFSK9-15、DFSK9-16、DFSK9-17、DFSK9-18、DFSK9-20和DFSK9-21抑制能力稍强一些。
表3、全抗体竞争实验IC50值
5.4BIAcore X100测定全抗体的亲和力
采用捕获法测定全抗体的亲和力。将山羊抗人IgG偶联到CM5芯片表面，分别稀释DFSK9-1、DFSK9-12、DFSK9-13、DFSK9-14、DFSK9-15、DFSK9-16、DFSK9-17、DFSK9-18、DFSK9-19、DFSK9-20和DFSK9-21保证大约200RU左右的抗体被山羊抗人IgG捕获。将PCSK9设置一系列的浓度梯度(200nM、100nM、50nM、25nM、12.5nM、6.25nM、3.125nM、1.5625nM、0.78125nM)流经固定相表面，测定抗体的亲和力。结果发现筛出的抗体都具有较高的亲和力(见表4)，选取亲和力最高的8种全抗体进行生物学活性实验。
表4、抗PCSK9全抗体亲和力常数测定数值
实例6、抗PCSK9全抗体生物学活性实验
将HepG2细胞以2.5X105cells/ml接种到96孔中。次日，将10％FBS的EMEM生长培养基换成80μl含5％除脂蛋白FBS的分析培养基，37℃培养24h。次日，将分析培养基稀释的不同稀释度的全抗体(10μl/孔)加入到已接种HepG2细胞的培养板中。8种全抗体样品初始浓度为900nmol/L，3倍梯度稀释，每个全抗体做8个梯度。然后再加入30nmol/L的PCSK9溶液(10μl/孔)，混匀后37℃，5％CO2条件下培养4h。每孔加入10ul 0.1mg/m L BODIPY标记的LDL溶液，混匀后37℃，5％CO2条件下继续培养15-20h。洗涤每孔加入200μl PBS洗涤2次。每孔加入100μl PBS，酶标仪波长490nm和520nm读取相对荧光单位RFU数值。GraphPadPrism5Demo软件分析数据作图，结果如图10所示，以同型IgG为阴性对照，由图上可以看出8个抗体均能以剂量依赖性方式阻断PCSK9与低密度脂蛋白受体LDLR结合，增加低密度脂蛋白LDL在HepG2细胞内摄取率，各抗体之间的生物学活性相差不多。
表5、生物学活性实验EC50值
| No. | Sample | EC50(nmol/L) | No. | Sample | EC50(nmol/L) |
| 1 | DFSK9-13 | 29.61 | 7 | DFSK9-17 | 18.51 |
| 2 | DFSK9-14 | 16.59 | 8 | DFSK9-18 | 23.91 |
| 3 | DFSK9-15 | 15.31 | 9 | DFSK9-20 | 17.95 |
| 4 | DFSK9-16 | 23.80 | 10 | DFSK9-21 | 20.33 |
对于本领域的普通技术人员而言，具体实施例只是对本发明进行了示例性描述，显然本发明具体实现并不受上述方式的限制，只要采用了本发明的方法构思和技术方案进行的各种非实质性的改进，或未经改进将本发明的构思和技术方案直接应用于其它场合的，均在本发明的保护范围之内。
序列表
<110> 北京东方百泰生物科技有限公司.
<120> 抗PCSK9单克隆抗体.
<141> 2017-11-17
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Tyr Ala Ala Ser Thr Arg Glu Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Asn Asn Thr Pro Asn
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 14
<211> 107
<212> PRT
<213> 人工序列(Homo sapiens)
<400> 14
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asn Val Asn Asn Trp
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ala Ala Ser Thr Arg Glu Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Asp Asn Asp Met Pro Leu
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 15
<211> 107
<212> PRT
<213> 人工序列(Homo sapiens)
<400> 15
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asn Ile Asn Ser Trp
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Gly Ala Ser Ser Arg Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Phe Asp Val Pro Thr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 16
<211> 107
<212> PRT
<213> 人工序列(Homo sapiens)
<400> 16
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asn Ile Asn Asn Trp
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Gly Ala Ser Thr Arg Pro Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Asp Asp Thr Pro Asn
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 17
<211> 107
<212> PRT
<213> 人工序列(Homo sapiens)
<400> 17
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile His Asn Trp
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Asp Ala Ser Asn Arg Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Asn Ser Asn Ile Pro Leu
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 18
<211> 107
<212> PRT
<213> 人工序列(Homo sapiens)
<400> 18
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asp Ser Trp
20 25 30
Leu Thr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Gly Ala Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Asp Ser Lys Ile Pro Leu
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 19
<211> 107
<212> PRT
<213> 人工序列(Homo sapiens)
<400> 19
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Arg Asn Trp
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Asp Ala Ser Asn Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Asp Thr Pro Leu
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 20
<211> 107
<212> PRT
<213> 人工序列(Homo sapiens)
<400> 20
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Arg Asn Trp
20 25 30
Leu Thr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Gly Ala Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Asp Asp Ser Thr Pro Pro
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 21
<211> 107
<212> PRT
<213> 人工序列(Homo sapiens)
<400> 21
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Arg Ser Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Asp Ala Ser Ser Arg Pro Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asp Ser Met Pro Met
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 22
<211> 11
<212> PRT
<213> 人工序列(Homo sapiens)
<400> 22
Arg Ala Ser Gln Ser Ile Asp Asn Arg Leu Thr
1 5 10
<210> 23
<211> 11
<212> PRT
<213> 人工序列(Homo sapiens)
<400> 23
Arg Ala Ser Gln Ser Val Arg Asn Trp Leu Asp
1 5 10
<210> 24
<211> 11
<212> PRT
<213> 人工序列(Homo sapiens)
<400> 24
Arg Ala Ser Gln Gly Ile Asn Ser Trp Leu Asn
1 5 10
<210> 25
<211> 11
<212> PRT
<213> 人工序列(Homo sapiens)
<400> 25
Arg Ala Ser Gln Asn Val Asn Asn Trp Leu Asn
1 5 10
<210> 26
<211> 11
<212> PRT
<213> 人工序列(Homo sapiens)
<400> 26
Arg Ala Ser Gln Asn Ile Asn Ser Trp Leu Asn
1 5 10
<210> 27
<211> 11
<212> PRT
<213> 人工序列(Homo sapiens)
<400> 27
Arg Ala Ser Gln Asn Ile Asn Asn Trp Leu Asn
1 5 10
<210> 28
<211> 11
<212> PRT
<213> 人工序列(Homo sapiens)
<400> 28
Arg Ala Ser Gln Gly Ile His Asn Trp Leu Asn
1 5 10
<210> 29
<211> 11
<212> PRT
<213> 人工序列(Homo sapiens)
<400> 29
Arg Ala Ser Gln Asp Val Asp Ser Trp Leu Thr
1 5 10
<210> 30
<211> 11
<212> PRT
<213> 人工序列(Homo sapiens)
<400> 30
Arg Ala Ser Gln Ser Val Arg Asn Trp Leu Asn
1 5 10
<210> 31
<211> 11
<212> PRT
<213> 人工序列(Homo sapiens)
<400> 31
Arg Ala Ser Gln Asp Val Arg Asn Trp Leu Thr
1 5 10
<210> 32
<211> 11
<212> PRT
<213> 人工序列(Homo sapiens)
<400> 32
Arg Ala Ser Gln Ser Ile Arg Ser Tyr Leu Asn
1 5 10
<210> 33
<211> 7
<212> PRT
<213> 人工序列(Homo sapiens)
<400> 33
Asp Ala Ser Ser Arg Gln Ser
1 5
<210> 34
<211> 7
<212> PRT
<213> 人工序列(Homo sapiens)
<400> 34
Gly Ala Ser Thr Leu Glu Ser
1 5
<210> 35
<211> 7
<212> PRT
<213> 人工序列(Homo sapiens)
<400> 35
Ala Ala Ser Thr Arg Glu Thr
1 5
<210> 36
<211> 7
<212> PRT
<213> 人工序列(Homo sapiens)
<400> 36
Gly Ala Ser Ser Arg Gln Ser
1 5
<210> 37
<211> 7
<212> PRT
<213> 人工序列(Homo sapiens)
<400> 37
Gly Ala Ser Thr Arg Pro Thr
1 5
<210> 38
<211> 7
<212> PRT
<213> 人工序列(Homo sapiens)
<400> 38
Asp Ala Ser Asn Arg Gln Ser
1 5
<210> 39
<211> 7
<212> PRT
<213> 人工序列(Homo sapiens)
<400> 39
Gly Ala Ser Asn Leu Ala Ser
1 5
<210> 40
<211> 7
<212> PRT
<213> 人工序列(Homo sapiens)
<400> 40
Asp Ala Ser Asn Leu Gln Ser
1 5
<210> 41
<211> 7
<212> PRT
<213> 人工序列(Homo sapiens)
<400> 41
Asp Ala Ser Ser Arg Pro Thr
1 5
<210> 42
<211> 9
<212> PRT
<213> 人工序列(Homo sapiens)
<400> 42
Gln Gln Pro Glu Asn Asp Pro Thr Thr
1 5
<210> 43
<211> 9
<212> PRT
<213> 人工序列(Homo sapiens)
<400> 43
Gln Gln Asp Asn Asp Ile Pro Leu Thr
1 5
<210> 44
<211> 9
<212> PRT
<213> 人工序列(Homo sapiens)
<400> 44
Gln Gln Trp Asn Asn Thr Pro Asn Thr
1 5
<210> 45
<211> 9
<212> PRT
<213> 人工序列(Homo sapiens)
<400> 45
Gln Gln Asp Asn Asp Met Pro Leu Thr
1 5
<210> 46
<211> 9
<212> PRT
<213> 人工序列(Homo sapiens)
<400> 46
Gln Gln Trp Phe Asp Val Pro Thr Thr
1 5
<210> 47
<211> 9
<212> PRT
<213> 人工序列(Homo sapiens)
<400> 47
Gln Gln Trp Asp Asp Thr Pro Asn Thr
1 5
<210> 48
<211> 9
<212> PRT
<213> 人工序列(Homo sapiens)
<400> 48
Gln Gln Asn Ser Asn Ile Pro Leu Thr
1 5
<210> 49
<211> 9
<212> PRT
<213> 人工序列(Homo sapiens)
<400> 49
Gln Gln Asp Ser Lys Ile Pro Leu Thr
1 5
<210> 50
<211> 9
<212> PRT
<213> 人工序列(Homo sapiens)
<400> 50
Gln Gln Trp Thr Asp Thr Pro Leu Thr
1 5
<210> 51
<211> 9
<212> PRT
<213> 人工序列(Homo sapiens)
<400> 51
Gln Gln Asp Asp Ser Thr Pro Pro Thr
1 5
<210> 52
<211> 9
<212> PRT
<213> 人工序列(Homo sapiens)
<400> 52
Gln Gln Gly Asp Ser Met Pro Met Thr
1 5
<210> 53
<211> 8
<212> PRT
<213> 人工序列(Homo sapiens)
<400> 53
Gly Gly Thr Phe Thr Asn Asn Ala
1 5
<210> 54
<211> 8
<212> PRT
<213> 人工序列(Homo sapiens)
<400> 54
Gly Tyr Thr Val Thr Ser Tyr Gly
1 5
<210> 55
<211> 8
<212> PRT
<213> 人工序列(Homo sapiens)
<400> 55
Gly Tyr Ser Leu Thr Ser Tyr Gly
1 5
<210> 56
<211> 9
<212> PRT
<213> 人工序列(Homo sapiens)
<400> 56
Arg Ile Ile Pro Met Phe Gly Met Ala
1 5
<210> 57
<211> 9
<212> PRT
<213> 人工序列(Homo sapiens)
<400> 57
Trp Leu Ser Phe Tyr Asn Gly Asn Thr
1 5
<210> 58
<211> 9
<212> PRT
<213> 人工序列(Homo sapiens)
<400> 58
Trp Val Thr Phe Tyr Asn Gly Asn Thr
1 5
<210> 59
<211> 9
<212> PRT
<213> 人工序列(Homo sapiens)
<400> 59
Trp Val Ser Phe Tyr Gln Gly Asn Thr
1 5
<210> 60
<211> 9
<212> PRT
<213> 人工序列(Homo sapiens)
<400> 60
Trp Val Ser Phe Tyr Asn Gly Gln Thr
1 5
<210> 61
<211> 9
<212> PRT
<213> 人工序列(Homo sapiens)
<400> 61
Trp Val Ser Phe Tyr Asn Gly Asn Ser
1 5
<210> 62
<211> 8
<212> PRT
<213> 人工序列(Homo sapiens)
<400> 62
Ala Arg Glu Gly Ile Pro Met Ile
1 5
<210> 63
<211> 8
<212> PRT
<213> 人工序列(Homo sapiens)
<400> 63
Ala Arg Gly Tyr Ser Leu Asp Val
1 5
<210> 64
<211> 8
<212> PRT
<213> 人工序列(Homo sapiens)
<400> 64
Ala Arg Gly Tyr Gly Met Ser Ile
1 5
<210> 65
<211> 8
<212> PRT
<213> 人工序列(Homo sapiens)
<400> 65
Ala Arg Gly Phe Gly Met Asp Arg
1 5
<210> 66
<211> 8
<212> PRT
<213> 人工序列(Homo sapiens)
<400> 66
Ala Arg Gly Tyr Gly Met Thr Val
1 5
<210> 67
<211> 8
<212> PRT
<213> 人工序列(Homo sapiens)
<400> 67
Ala Arg Gly Phe Gly Leu Ser Val
1 5
Claims (17)
1.一种抗PCSK9的单克隆抗体，所述抗体包括重链和轻链，其特征在于，所述抗体包含如下轻链互补决定区和重链互补决定区；所述轻链互补决定区CDR1、CDR2和CDR3分别用LCDR1、LCDR2和LCDR3表示；所述重链互补决定区CDR1，CDR2和CDR3分别用HCDR1、HCDR2和HCDR3表示；
第一组：LCDR1、LCDR2和LCDR3分别选自RASQNINSWLN、GASSRQS和QQWFDVPTT；HCDR1、HCDR2和HCDR3分别选自GYSLTSYG、WVSFYNGQT和ARGYGMSI；
第二组：LCDR1、LCDR2和LCDR3分别选自RASQNINSWLN、GASSRQS和QQWFDVPTT；HCDR1、HCDR2和HCDR3分别选自GYTVTSYG、WVSFYQGNT和ARGFGMDR；
第三组：LCDR1、LCDR2和LCDR3分别选自RASQNINNWLN、GASTRPT和QQWDDTPNT；HCDR1、HCDR2和HCDR3分别选自GYSLTSYG、WVSFYNGNS和ARGFGMDR。
2.根据权利要求1所述的抗PCSK9的单克隆抗体，所述抗体包括重链和轻链，其特征在于，所述抗体包含如下轻链可变区和重链可变区；
第一组：所述轻链可变区和所述重链可变区分别选自序列SEQ NO.15和SEQNO.7；
第二组：所述轻链可变区和所述重链可变区分别选自序列SEQ NO.15和SEQ NO.6；
第三组：所述轻链可变区和所述重链可变区分别选自序列SEQ NO.16和SEQ NO.5。
3.一种包含上述权利要求1或2所述的轻链和重链的抗体、多肽或蛋白。
4.一种包含如权利要求1或2所述的轻链和重链的氨基酸序列用于全长抗体、单链抗体、单域抗体、双特异抗体和抗体药物偶联物。
5.一种包含如权利要求1或2所述的轻链和重链的抗体，所述抗体能够特异性与PCSK9结合。
6.一种编码如权利要求1或2所述的重链和轻链的多核苷酸序列或组合。
7.一种包含如权利要求6所述的多核苷酸序列或组合的重组DNA表达载体。
8.一种转染如权利要求7所述的重组DNA表达载体的宿主细胞，所述宿主细胞包含原核细胞、酵母、昆虫或哺乳动物细胞。
9.根据权利要求8所述的宿主细胞，其特征在于，所述原核细胞为大肠杆菌；所述哺乳动物细胞为HEK293E细胞、CHO细胞或NS0细胞。
10.根据权利要求1或2所述的抗PCSK9的单克隆抗体，其特征在于，所述抗PCSK9的单克隆抗体的重链恒定区包含IgG1、IgG2、IgG3或IgG4；所述轻链恒定区包含Cκ或Cλ。
11.根据权利要求10所述的抗PCSK9的单克隆抗体，其特征在于，所述重链恒定区为IgG4或IgG2；所述轻链恒定区为Cκ。
12.一种包含如权利要求1或2所述的轻链和重链的单克隆抗体、人工载体、药物或药物组合物。
13.根据权利要求12所述的单克隆抗体，其特征在于，所述单克隆抗体包含全长抗体或抗PCSK9单克隆抗体的片段，所述片段包含Fab、Fab’、F(ab’)2、Fv和ScFv中的一种或几种的组合。
14.根据权利要求1或2所述的单克隆抗体，其特征在于，所述单克隆抗体是全人源的。
15.一种包含如权利要求1或2所述的轻链和重链的检测试剂或试剂盒。
16.一种包含如权利要求1或2所述的轻链和重链的抗体，用于制备消除、抑制或降低PCSK9活性及其改善、缓解、抑制或预防相关疾病的药物用途。
17.根据权利要求16所述的抗体，其特征在于，所述抗体用于制备改善、缓解、抑制或预防血脂异常、心脑血管疾病和血栓闭塞性疾病的药物用途。
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110981962A (zh) * | 2019-12-19 | 2020-04-10 | 中国药科大学 | Pcsk9抗体、其抗原结合片段及其应用 |
| CN111620950A (zh) * | 2020-06-16 | 2020-09-04 | 中国药科大学 | 全人源抗pcsk9抗体、其抗原结合片段及其应用 |
| CN111690065A (zh) * | 2020-06-19 | 2020-09-22 | 北京东方百泰生物科技有限公司 | 一种抗il-17ra单克隆抗体的纯化方法 |
| CN114525258A (zh) * | 2020-10-30 | 2022-05-24 | 未来智人再生医学研究院(广州)有限公司 | 一种表达pcsk9阻断物的多能干细胞或其衍生物及应用 |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106749670B (zh) * | 2017-01-22 | 2018-06-12 | 北京东方百泰生物科技有限公司 | 抗pcsk9单克隆抗体 |
| WO2018228406A1 (zh) * | 2017-06-14 | 2018-12-20 | 江苏恒瑞医药股份有限公司 | Pcsk9抗体、其抗原结合片段及其医药用途 |
| CN109897110B (zh) * | 2017-12-08 | 2022-05-17 | 深圳华大生命科学研究院 | 纳米抗体及其制备方法 |
| CN110872353B (zh) * | 2018-09-03 | 2021-05-04 | 深圳华大基因科技有限公司 | 特异性结合pcsk9抗原的纳米抗体及其制备方法和应用 |
| CN111171152B (zh) * | 2020-01-15 | 2023-04-18 | 吉林医药学院 | Pcsk9抗体及其制备方法和应用 |
| CN117343171B (zh) * | 2022-11-01 | 2024-03-19 | 上海百英生物科技股份有限公司 | 一种抗bsa的兔单克隆抗体及其应用 |
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| CN110981962A (zh) * | 2019-12-19 | 2020-04-10 | 中国药科大学 | Pcsk9抗体、其抗原结合片段及其应用 |
| CN110981962B (zh) * | 2019-12-19 | 2022-07-12 | 中国药科大学 | Pcsk9抗体、其抗原结合片段及其应用 |
| CN111620950A (zh) * | 2020-06-16 | 2020-09-04 | 中国药科大学 | 全人源抗pcsk9抗体、其抗原结合片段及其应用 |
| CN111620950B (zh) * | 2020-06-16 | 2023-01-31 | 中国药科大学 | 全人源抗pcsk9抗体、其抗原结合片段及其应用 |
| CN111690065A (zh) * | 2020-06-19 | 2020-09-22 | 北京东方百泰生物科技有限公司 | 一种抗il-17ra单克隆抗体的纯化方法 |
| CN114525258A (zh) * | 2020-10-30 | 2022-05-24 | 未来智人再生医学研究院(广州)有限公司 | 一种表达pcsk9阻断物的多能干细胞或其衍生物及应用 |
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| CN107698680B (zh) | 2019-03-01 |
| CN106749670A (zh) | 2017-05-31 |
| AU2017394444A1 (en) | 2019-02-21 |
| EA039191B1 (ru) | 2021-12-15 |
| EP3480216A1 (en) | 2019-05-08 |
| CN107698679B (zh) | 2019-03-01 |
| KR20190025980A (ko) | 2019-03-12 |
| CA3032506C (en) | 2023-08-22 |
| CN107698679A (zh) | 2018-02-16 |
| CN106749670B (zh) | 2018-06-12 |
| AU2017394444B2 (en) | 2020-04-09 |
| US11390688B2 (en) | 2022-07-19 |
| EA201990170A1 (ru) | 2019-07-31 |
| JP6734467B2 (ja) | 2020-08-05 |
| CA3032506A1 (en) | 2018-07-26 |
| JP2019531057A (ja) | 2019-10-31 |
| US20210277146A1 (en) | 2021-09-09 |
| EP3480216A4 (en) | 2020-07-01 |
| WO2018133649A1 (zh) | 2018-07-26 |
| KR102257462B1 (ko) | 2021-05-31 |
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