CN105348390B - 抗人pcsk9单克隆抗体 - Google Patents
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Abstract
本发明公开了一种利用噬菌体抗体库技术筛选并利用基因工程方法制备的新型抗人PCSK9的单克隆抗体或其片段，其特征在于对人PCSK9具有高亲和力。本发明抗体可以是全长抗体、基本上完整的抗体、Fab片段、F(ab')2片段或单链Fv片段。该单克隆抗体或其片段用于治疗由人PCSK9介导的疾病中的用途，所述的疾病包括但不局限于原发性高胆固醇血症、混合型高脂血症和家族性高胆固醇血症。
Description
说明书
技术领域
本发明为基因工程抗体药物领域，特别是抗人PCSK9的单克隆抗体领域。本发明涉及人源性功能性抗人PCSK9单克隆抗体，以及这类抗体在治疗人PCSK9介导的疾病中的用途。
背景技术
心血管疾病是目前我国首位的死亡原因，其中动脉粥样硬化引起的心脏病、脑卒中和外周动脉疾病是导致死亡的主要原因，而低密度脂蛋白胆固醇(LDL-C)水平升高是动脉粥样硬化性心血管疾病最大的危险因素。他汀类药物是目前降低LDL-C最有效的药物，但是仍有部分患者的血脂水平未能达标，以及有些患者不能耐受他汀类药物。因此，需要寻找他汀以外的强效降胆固醇药物，以降低动脉粥样硬化性心血管疾病的风险。
前蛋白枯草溶菌素转化酶9(proprotein convertase subtilisin kexin 9,PCSK9)，又称为神经凋亡调节转化酶1(neural apoptosis-regulated convertase 1,NARC-1)。PCSK9蛋白主要在肝脏中合成和分泌，PCSK9蛋白进入血液循环后，在肝脏与低密度脂蛋白受体(the low-density lipoprotein receptor,LDLR)结合，介导LDLR降解，从而降低肝脏对LDL-C的清除能力，升高血浆LDL-C水平。PCSK9基因敲除的小鼠表现出血浆胆固醇水平大约降低50％，而且使他汀类药物降低血浆胆固醇的灵敏度变高(Rashid S,et al2005,Proc Nath Acad Sci 102:5374-5379)。
抗PCSK9单克隆抗体可有效阻止PCSK9与LDL-R结合，从而发挥强效降胆固醇的作用，可用于他汀治疗未达标或他汀不耐受的高胆固醇血症的患者。
目前安进研发的PCSK9抑制剂Repatha(Evolocumab)和赛诺菲/再生元研发的PCSK9抑制剂Praluent(Alirocumab)都已经获得欧洲和美国FDA的批准上市，用于治疗原发性高胆固醇血症、混合型高脂血症和家族性高胆固醇血症。但是本领域仍然需要更多适于治疗患者的改善的抗PCSK9抗体。
发明内容
本发明的目的在于提供一种新型抗人PCSK9的单克隆抗体或其片段。由本发明涉及的抗体基因可变区的序列，可构建全长的抗体分子作为药物用于临床上由人PCSK9介导的疾病使用。这些适应症包括但不局限于下列：原发性高胆固醇血症、混合型高脂血症和家族性高胆固醇血症。
本发明从先前构建的大容量天然人源噬菌体抗体库(抗人IL-17单克隆抗体，专利申请号201510097117.0)中筛选并优化得到了全人源抗PCSK9单克隆抗体。
本发明的抗体为全人源的单克隆抗体，拮抗至少一种与PCSK9或其部分相关的体外或体内生物活性。
在一个实施例中，本发明抗体的特征在于能够有效抑制PCSK9结合重组人LDLR胞外区EGF-AB结构域。
在一个实施例中，本发明抗体的特征在于对人PCSK9具有强结合亲和力，亲和力优于(H7B12-IgG4)或与安进公司的同类抗体Evolocumab以及赛诺菲公司的同类抗体Alirocumab相似。
在一个实施例中，本发明抗PCSK9单克隆抗体含有重链可变区多肽，该多肽含有3个HCDR，其特征在于：所述HCDR1序列为GY X1X2X3NY；所述HCDR2序列为SFYNGN；所述HCDR3序列为GYVMD X4。其中X1X2X3序列为AIY、TLN或者ALY；X4为I或者F。而且其中HCDRs序列根据Chothia定义。优选地，本发明抗PCSK9单克隆抗体包含重链可变区多肽，该多肽具有选自SEQ ID NO:16-19的氨基酸序列。
在另一个实施例中，本发明抗PCSK9单克隆抗体含有轻链可变区多肽，该多肽含有3个LCDR序列，其特征在于：所述LCDR1序列为RASQSINNWLD；所述LCDR2序列为AASTRPS；所述LCDR3序列为QQDQDIPPT。而且其中LCDRs定义根据Chothia定义。优选地，本发明抗PCSK9单克隆抗体包含轻链可变区多肽，该多肽具有选自SEQ ID NO:20的氨基酸序列。
在优选的实施例中，本发明抗PCSK9单克隆抗体含有(i):含HCDR1、HCDR2、和HCDR3序列的重链可变区，其特征在于：所述HCDR1序列为GY X1X2X3NY；所述HCDR2序列为SFYNGN；所述HCDR3序列为GYVMD X4；其中X1X2X3序列为AIY、TLN或者ALY；X4为I或者F。而且其中HCDRs序列根据Chothia定义。(ii)：含LCDR1、LCDR2、和LCDR3序列的轻链可变区，其特征在于：所述LCDR1序列为RASQSINNWLD；所述LCDR2序列为AASTRPS；所述LCDR3序列为QQDQDIPPT。而且其中LCDRs定义根据Chothia定义。
在更优选的实施例中，本发明抗PCSK9单克隆抗体的重链可变区序列为SEQ IDNO：16，轻链可变区序列为SEQ ID NO：20；或重链可变区序列为SEQ ID NO：17，轻链可变区序列为SEQ ID NO：20；或重链可变区序列为SEQ ID NO：18，轻链可变区序列为SEQ ID NO：20；或重链可变区序列为SEQ ID NO：19，轻链可变区序列为SEQ ID NO：20。
本发明抗PCSK9单克隆抗体可包含或由完整的抗体(即全长)、基本上完整的抗体或其抗原结合部分，例如Fab片段、F(ab')2片段或单链Fv片段组成。
本发明抗PCSK9单克隆抗体包括可变区和恒定区，其中抗体重链恒定区可以是IgG1亚型(Seq ID 7)、IgG2(Seq ID 8)或者IgG4亚型(Seq ID 9)，轻链恒定区可以是kappa亚型(Seq ID 10)或者Lambda亚型(seq ID 11)。
本发明所述的单克隆抗体或其片段是全人源的。
本发明抗体的亲和力可以在标准测试方法中进行证明。简言之，测量使用仪器Biacore 3000，将抗人FC段的抗体偶联至芯片CM5的表面上，稀释抗体蛋白至合适浓度，保证200RU左右的抗体被抗人Fc的抗体捕获。将抗体设置一系列的浓度梯度(ex.100nm，50nm，25nm，12.5nm，6.25nm，3.125nm，1.5625nm，0nm)流经固定相表面，测定各单克隆抗体的亲和力。
在一个实施例中，分析了6种不同抗PCSK9单抗(包括4种本发明的单抗和2种对照单抗)抑制LDLR与PCSK9结合的能力，结果是均能有效抑制LDLR与PCSK9的结合。
本发明抗体在人血清中的稳定性可以在标准测试方法中进行证明。简言之，取过滤去菌的单抗样品，分别稀释于200ul无菌的正常人混合血清或PBS至终浓度30ug/ml，混匀后置37℃水浴放置12天(288小时)。12天后，利用ELISA分析血清样品(A:Ab/normal humanserum)，PBS样品(B:Ab/PBS)和冻存的单抗样品(C:Ab)与huPCSK9的结合，并分别比较各单抗样品结合huPCSK9能力的变化(A/C)。结果表明本发明中的四株抗huPCSK9单抗具有好的血清稳定性。
本发明抗体的抑制PCSK9诱导细胞表面LDLR下调的活性可以在标准测试方法中进行证明。简言之，将HEK293细胞密度调整至8*10E5cells/ml，接种于24孔板(500ul/孔)。然后在细胞中加入抗体和PCSK9，悬浮培养3h。然后分别收集各孔中细胞，用1％BSA-PBS洗涤2次，然后与FITC-anti-LDLR单抗(Sino Biological Inc.Cat#10231-R301-F)于冰上孵育45分钟，用PBS洗涤2次后，用BD公司的Accuri C6流式细胞仪分析FITC信号。以未处理的HEK293细胞为对照，比较各种处理后细胞表面LDLR的水平。结果显示本发明的抗PCSK9单抗(20ug/ml)能够明显抑制PCSK9诱导的LDLR水平下调。
本发明抗体抑制PCSK9诱导细胞摄取LDL-C能力的活性可以在标准测试方法中进行证明。简言之，将HEK293细胞密度调整至8*10E5cells/ml，接种于24孔板(500ul/孔)。然后在细胞中加入PCSK9和抗体，悬浮培养2.5h。然后再分别在每个孔中补加BODIPY FL LDL(Invitrogen，Cat#:L3483)至终浓度6ug/ml，继续悬浮培养2.5h。然后分别收集各孔中细胞，用PBS洗涤2次后，用BD公司的Accuri C6流式细胞仪分析BODIPY信号(激发波长488nM，发射波长520)。以未处理的HEK293细胞为对照，比较各种处理后细胞摄取LDL-C的水平。结果显示本发明的多种抗PCSK9单抗(20ug/ml)能够明显抑制PCSK9诱导的HEK293细胞摄取LDL-C水平的下调。
附图说明
图1：单抗抑制PCSK9结合人LDLR胞外区EGF-AB结构域。
图2：抗人PCSK9单克隆抗体竞争LDLR结合人PCSK9。
图3：不同抗huPCSK9单抗在在人血清中的稳定性分析。
图4：抗PCSK9单抗抑制PCSK9诱导的HEK293细胞表面的LDLR下调(其中anti-IL17为一株抗IL17的单抗；Aliro为抗PCSK9的对照单抗Alirocumab；Evolo为抗PCSK9的对照抗体Evolocumab；H4H4，H7B12，H8A7，H8F4为四株抗PCSK9的单抗)。
图5：抗PCSK9单抗抑制PCSK9诱导的HEK293细胞摄取LDL-C能力的下调(其中anti-IL17为一株抗IL17的单抗；Aliro为抗PCSK9的对照单抗Alirocumab；Evolo为抗PCSK9的对照抗体Evolocumab；H4H4，H7B12，H8A7，H8F4为四株抗PCSK9的单抗)。
具体实施方式
以下实施例仅仅对本发明进行进一步的说明，不应理解为对本发明的限制。
实施例1：各种重组蛋白的制备。
制备抗PCSK9单抗的过程中需要用到多种不同的重组蛋白，包括人PCSK9(huPCSK9，Seq ID No:1),小鼠PCSK9(moPCSK9,Seq ID No:2)和猕猴PCSK9(mmPCSK9,SeqID No:3)，人LDLR胞外区(EGF-AB，Seq ID No:4)，以及重组抗体。而这些蛋白都有大量的翻译后修饰(如：糖基化或二硫键等)，因而利用哺乳动物细胞表达系统将更有利于保持重组蛋白的结构和功能。此外，为了方便纯化，非抗体类的重组蛋白在C端添加了His-tag(SeqID No:5)或者鼠抗体的Fc段(mFc，Seq ID No:6)。重组抗体制备时，抗体重链恒定区可以是IgG1亚型(Seq ID No:7)，IgG2亚型(Seq ID No:8)或者IgG4亚型(Seq ID No:9)，轻链恒定区可以是kappa亚型(Seq ID No:10)或者Lambda亚型(Seq ID No:11)。
根据Uniprot数据库的各种目的重组蛋白的氨基酸序列，设计并合成上述各种重组蛋白的基因(包含His-tag或者mFc编码基因)。利用常规的分子生物学技术将合成的各种重组蛋白基因克隆至合适的真核表达载体(如invitrogen公司的pcDNA3.1等)，然后利用脂质体(如invitrogen公司的293fectin等)或其它转染试剂(如PEI等)将制备的重组蛋白表达质粒转染入HEK293细胞(如invitrogen公司的HEK293F)，在无血清悬浮培养条件下培养3-4天。然后通过离心等方式收获培养上清。
His-tag融合表达的重组蛋白利用金属螯合亲和层析柱(如GE公司的HisTrap FF等)对培养上清中的重组蛋白进行一步纯化。而mFc融合表达的重组蛋白和重组抗体用ProteinA/G亲和层析柱(如GE公司的Mabselect SURE等)进行一步纯化。然后利用脱盐柱(如GE公司的Hitrap desaulting等)将重组蛋白保存缓冲液置换为PBS(pH7.0)或者其它合适的缓冲液。必要时，可以对抗体样品进行过滤除菌，然后分装保存于-20℃。
实施例2：利用噬菌体呈现抗体库技术筛选和优化抗人PCSK9单抗。
以制备的重组huPCSK9-his(以下简写为PCSK9-His)为抗原，参照文献(抗人IL-17单克隆抗体，专利申请号201510097117.0)，利用固相筛选策略筛选呈现人单链抗体库的噬菌体,获得多株序列不同但特异性结合人PCSK9的人抗体，包括克隆：S2B8(Seq ID No:12)，S2F5(Seq ID No:13)，S2G1(Seq ID No:14)，S3A12(Seq ID No:15)。其中重组全抗体S3A12能够有效抑制PCSK9结合重组人LDLR胞外区EGF-AB结构域(图1)。
然后参照文献(抗人IL-17单克隆抗体，专利申请号201510097117.0)利用轻链置换和重链CDRs突变的策略对抗体进行体外亲和力成熟，最终获得四株亲和力提高的抗人PCSK9的单抗，具体序列信息见表1。
表1：亲和力成熟的抗人PCSK9的单抗。
| 单抗 | H4H4 | H7B12 | H8A7 | H8F4 |
| 重链(序列号) | H4H4(seqID 16) | H7B12(seqID 17) | H8A7(seqID 18) | H8F4(seqID 19) |
| 轻链(序列号) | L3A8(seqID 20) | L3A8(seqID 20) | L3A8(seqID 20) | L3A8(seqID 20) |
实施例3：BIacore 3000测定全抗体的亲和力。
Amine couling kit和human antibody capture kit以及CM5芯片和pH7.4的10×HBS-EP均购自GE Healthcare。
根据试剂盒中的说明书，将抗人FC段的抗体偶联至芯片CM5的表面上，稀释抗体蛋白至合适浓度，保证200RU左右的抗体被抗人Fc的抗体捕获。将huPCSK9设置一系列的浓度梯度(ex.100nm，50nm，25nm，12.5nm，6.25nm，3.125nm，1.5625nm，0nm)流经固定相表面，测定各单克隆抗体的亲和力，用同样的方法测定Evolocumab(Seq ID No:21，Seq ID No:22)和Alirocumab(Seq ID No:23，Seq ID No:24)的亲和力作为对照。结果如表2所示。
表2.anti-huPCSK9mAbs亲和力常数测定数值。
| Antibody | Kon(1/MS) | Koff(1/S) | KD |
| H4H4-IgG4 | 7.14E+05 | 4.70E-05 | 65.9pM |
| H7B12-IgG4 | 1.01E+06 | 3.26E-05 | 32.3pM |
| H8F4-IgG4 | 6.63E+05 | 4.48E-05 | 67.5pM |
| H8A7-IgG4 | 7.46E+05 | 4.83E-05 | 64.8pM |
| Evolocumab-IgG4 | 2.28E+05 | 1.93E-05 | 76.4pM |
| Alirocumab | 2.27E+06 | 1.54E-04 | 67.7pM |
实施例4：抗人PCSK9单克隆抗体竞争PCSK9受体LDLR结合PCSK9。
功能性的抗PCSK9单抗应该能在蛋白水平上阻断LDLR和PCSK9之间的结合，本实施例分析了6种不同抗PCSK9单抗(H4H4,H7B12，H8A7，H8F4，Evolocumab和Alirocumab)抑制LDLR与PCSK9结合的能力。
用5.6ug/ml的PCSK9-his对各单克隆抗体进行梯度稀释，共11个稀释浓度，前6个梯度1:1稀释，后5个梯度1:2稀释，各抗体起始浓度调整为30ug/ml。利用HRP-mouse-anti-his IgG单抗检测LDLR与PCSK9的结合信号，然后利用GraphPad Prism 6进行数据分析和作图(图2)
实施例5：抗PCSK9单抗在人血清中的稳定性分析。
为了初步分析不同抗PCSK9单抗分子的特异性及血清稳定性，进行了抗PCSK9单抗在人血清中的稳定性分析。此研究包括6种不同抗PCSK9单抗，分别为H4H4，H7B12，H8A7，H8F4，Evolocumab和Alirocumab。取过滤去菌的单抗样品，分别稀释于200ul无菌的正常人混合血清或PBS至终浓度30ug/ml，混匀后置37℃水浴放置12天(288小时)。12天后，利用ELISA分析血清样品(A:Ab/normal human serum)，PBS样品(B:Ab/PBS)和冻存的单抗样品(C:Ab)与huPCSK9的结合(图3)，并分别比较各单抗样品结合huPCSK9能力的变化(A/C)。结果(表3)表明本发明中的四株抗huPCSK9单抗具有好的血清稳定性。
表3：不同处理条件下单抗样品结合huPCSK9能力的变化。
实施例6：抗PCSK9单抗抑制PCSK9诱导HEK293细胞表面LDLR受体的下调。
本实施例研究用悬浮培养于293Freestyle培养基(Invitrogen)的HEK293细胞。将HEK293细胞密度调整至8*10E5cells/ml，接种于24孔板(500ul/孔)。然后按照表4所列方式处理细胞，悬浮培养3h。然后分别收集各孔中细胞，用1％BSA-PBS洗涤2次，然后与FITC-anti-LDLR单抗(Sino Biological Inc.Cat#10231-R301-F)于冰上孵育45分钟，用PBS洗涤2次后，用BD公司的Accuri C6流式细胞仪分析FITC信号。以未处理的HEK293细胞为对照，比较各种处理后细胞表面LDLR的水平。结果(图4)显示：10ug/ml的PCSK9处理HEK293细胞后导致细胞表明LDLR水平明显下降，而多种抗PCSK9单抗(20ug/ml)能够明显抑制PCSK9诱导的LDLR水平下调。
表4：HEK293细胞的不同处理方式。
实施例7：抗PCSK9单抗抑制PCSK9诱导HEK293细胞摄取LDL-C能力的下调。
本实施例研究用悬浮培养于293Freestyle培养基(Invitrogen)的HEK293细胞。将HEK293细胞密度调整至8*10E5cells/ml，接种于24孔板(500ul/孔)。然后按照表5所列方式处理细胞，悬浮培养2.5h。然后再分别在每个孔中补加BODIPY FL LDL(Invitrogen，Cat#:L3483)至终浓度6ug/ml，继续悬浮培养2.5h。然后分别收集各孔中细胞，用PBS洗涤2次后，用BD公司的Accuri C6流式细胞仪分析BODIPY信号(激发波长488nM，发射波长520)。以未处理的HEK293细胞为对照，比较各种处理后细胞摄取LDL-C的水平。结果(图5)显示：10ug/ml的PCSK9处理HEK293细胞后导致细胞摄取LDL-C量明显下降，而多种抗PCSK9单抗(20ug/ml)能够明显抑制PCSK9诱导的HEK293细胞摄取LDL-C水平的下调。
表5：HEK293细胞的不同处理方式。
序列表
<110>北京智仁美博生物科技有限公司
<120>抗人PCSK9单克隆抗体
<160>24
<210>1
<211>639
<212>PRT
<213>智人（Homo sapiens）
<220>
<223>huPCSK9
<400>1
Glu Ala Pro Glu His Gly Thr Thr Ala Thr Phe His Arg Cys Ala Lys
1 5 10 15
Asp Pro Trp Arg Leu Pro Gly Thr Tyr Val Val Val Leu Lys Glu Glu
20 25 30
Thr His Leu Ser Gln Ser Glu Arg Thr Ala Arg Arg Leu Gln Ala Gln
35 40 45
Ala Ala Arg Arg Gly Tyr Leu Thr Lys Ile Leu His Val Phe His Gly
50 55 60
Leu Leu Pro Gly Phe Leu Val Lys Met Ser Gly Asp Leu Leu Glu Leu
65 70 75 80
Ala Leu Lys Leu Pro His Val Asp Tyr Ile Glu Glu Asp Ser Ser Val
85 90 95
Phe Ala Gln Ser Ile Pro Trp Asn Leu Glu Arg Ile Thr Pro Pro Arg
100 105 110
Tyr Arg Ala Asp Glu Tyr Gln Pro Pro Asp Gly Gly Ser Leu Val Glu
115 120 125
Val Tyr Leu Leu Asp Thr Ser Ile Gln Ser Asp His Arg Glu Ile Glu
130 135 140
Gly Arg Val Met Val Thr Asp Phe Glu Asn Val Pro Glu Glu Asp Gly
145 150 155 160
Thr Arg Phe His Arg Gln Ala Ser Lys Cys Asp Ser His Gly Thr His
165 170 175
Leu Ala Gly Val Val Ser Gly Arg Asp Ala Gly Val Ala Lys Gly Ala
180 185 190
Ser Met Arg Ser Leu Arg Val Leu Asn Cys Gln Gly Lys Gly Thr Val
195 200 205
Ser Gly Thr Leu Ile Gly Leu Glu Phe Ile Arg Lys Ser Gln Leu Val
210 215 220
Gln Pro Val Gly Pro Leu Val Val Leu Leu Pro Leu Ala Gly Gly Tyr
225 230 235 240
Ser Arg Val Leu Asn Ala Ala Cys Gln Arg Leu Ala Arg Ala Gly Val
245 250 255
Val Leu Val Thr Ala Ala Gly Asn Phe Arg Asp Asp Ala Cys Leu Tyr
260 265 270
Ser Pro Ala Ser Ala Pro Glu Val Ile Thr Val Gly Ala Thr Asn Ala
275 280 285
Gln Asp Gln Pro Val Thr Leu Gly Thr Leu Gly Thr Asn Phe Gly Arg
290 295 300
Cys Val Asp Leu Phe Ala Pro Gly Glu Asp Ile Ile Gly Ala Ser Ser
305 310 315 320
Asp Cys Ser Thr Cys Phe Val Ser Gln Ser Gly Thr Ser Gln Ala Ala
325 330 335
Ala His Val Ala Gly Ile Ala Ala Met Met Leu Ser Ala Glu Pro Glu
340 345 350
Leu Thr Leu Ala Glu Leu Arg Gln Arg Leu Ile His Phe Ser Ala Lys
355 360 365
Asp Val Ile Asn Glu Ala Trp Phe Pro Glu Asp Gln Arg Val Leu Thr
370 375 380
Pro Asn Leu Val Ala Ala Leu Pro Pro Ser Thr His Gly Ala Gly Trp
385 390 395 400
Gln Leu Phe Cys Arg Thr Val Trp Ser Ala His Ser Gly Pro Thr Arg
405 410 415
Met Ala Thr Ala Ile Ala Arg Cys Ala Pro Asp Glu Glu Leu Leu Ser
420 425 430
Cys Ser Ser Phe Ser Arg Ser Gly Lys Arg Arg Gly Glu Arg Met Glu
435 440 445
Ala Gln Gly Gly Lys Leu Val Cys Arg Ala His Asn Ala Phe Gly Gly
450 455 460
Glu Gly Val Tyr Ala Ile Ala Arg Cys Cys Leu Leu Pro Gln Ala Asn
465 470 475 480
Cys Ser Val His Thr Ala Pro Pro Ala Glu Ala Ser Met Gly Thr Arg
485 490 495
Val His Cys His Gln Gln Gly His Val Leu Thr Gly Cys Ser Ser His
500 505 510
Trp Glu Val Glu Asp Leu Gly Thr His Lys Pro Pro Val Leu Arg Pro
515 520 525
Arg Gly Gln Pro Asn Gln Cys Val Gly His Arg Glu Ala Ser Ile His
530 535 540
Ala Ser Cys Cys His Ala Pro Gly Leu Glu Cys Lys Val Lys Glu His
545 550 555 560
Gly Ile Pro Ala Pro Gln Glu Gln Val Thr Val Ala Cys Glu Glu Gly
565 570 575
Trp Thr Leu Thr Gly Cys Ser Ala Leu Pro Gly Thr Ser His Val Leu
580 585 590
Gly Ala Tyr Ala Val Asp Asn Thr Cys Val Val Arg Ser Arg Asp Val
595 600 605
Ser Thr Thr Gly Ser Thr Ser Glu Glu Ala Val Thr Ala Val Ala Ile
610 615 620
Cys Cys Arg Ser Arg His Leu Ala Gln Ala Ser Gln Glu Leu Gln
625 630 635
<210>2
<211>660
<212>PRT
<213>小鼠（Mus musculus）
<220>
<223>mopcsk9
<400>2
Gln Asp Glu Asp Gly Asp Tyr Glu Glu Leu Met Leu Ala Leu Pro Ser
1 5 10 15
Gln Glu Asp Gly Leu Ala Asp Glu Ala Ala His Val Ala Thr Ala Thr
20 25 30
Phe Arg Arg Cys Ser Lys Glu Ala Trp Arg Leu Pro Gly Thr Tyr Ile
35 40 45
Val Val Leu Met Glu Glu Thr Gln Arg Leu Gln Ile Glu Gln Thr Ala
50 55 60
His Arg Leu Gln Thr Arg Ala Ala Arg Arg Gly Tyr Val Ile Lys Val
65 70 75 80
Leu His Ile Phe Tyr Asp Leu Phe Pro Gly Phe Leu Val Lys Met Ser
85 90 95
Ser Asp Leu Leu Gly Leu Ala Leu Lys Leu Pro His Val Glu Tyr Ile
100 105 110
Glu Glu Asp Ser Phe Val Phe Ala Gln Ser Ile Pro Trp Asn Leu Glu
115 120 125
Arg Ile Ile Pro Ala Trp His Gln Thr Glu Glu Asp Arg Ser Pro Asp
130 135 140
Gly Ser Ser Gln Val Glu Val Tyr Leu Leu Asp Thr Ser Ile Gln Gly
145 150 155 160
Ala His Arg Glu Ile Glu Gly Arg Val Thr Ile Thr Asp Phe Asn Ser
165 170 175
Val Pro Glu Glu Asp Gly Thr Arg Phe His Arg Gln Ala Ser Lys Cys
180 185 190
Asp Ser His Gly Thr His Leu Ala Gly Val Val Ser Gly Arg Asp Ala
195 200 205
Gly Val Ala Lys Gly Thr Ser Leu His Ser Leu Arg Val Leu Asn Cys
210 215 220
Gln Gly Lys Gly Thr Val Ser Gly Thr Leu Ile Gly Leu Glu Phe Ile
225 230 235 240
Arg Lys Ser Gln Leu Ile Gln Pro Ser Gly Pro Leu Val Val Leu Leu
245 250 255
Pro Leu Ala Gly Gly Tyr Ser Arg Ile Leu Asn Ala Ala Cys Arg His
260 265 270
Leu Ala Arg Thr Gly Val Val Leu Val Ala Ala Ala Gly Asn Phe Arg
275 280 285
Asp Asp Ala Cys Leu Tyr Ser Pro Ala Ser Ala Pro Glu Val Ile Thr
290 295 300
Val Gly Ala Thr Asn Ala Gln Asp Gln Pro Val Thr Leu Gly Thr Leu
305 310 315 320
Gly Thr Asn Phe Gly Arg Cys Val Asp Leu Phe Ala Pro Gly Lys Asp
325 330 335
Ile Ile Gly Ala Ser Ser Asp Cys Ser Thr Cys Phe Met Ser Gln Ser
340 345 350
Gly Thr Ser Gln Ala Ala Ala His Val Ala Gly Ile Val Ala Arg Met
355 360 365
Leu Ser Arg Glu Pro Thr Leu Thr Leu Ala Glu Leu Arg Gln Arg Leu
370 375 380
Ile His Phe Ser Thr Lys Asp Val Ile Asn Met Ala Trp Phe Pro Glu
385 390 395 400
Asp Gln Gln Val Leu Thr Pro Asn Leu Val Ala Thr Leu Pro Pro Ser
405 410 415
Thr His Glu Thr Gly Gly Gln Leu Leu Cys Arg Thr Val Trp Ser Ala
420 425 430
His Ser Gly Pro Thr Arg Thr Ala Thr Ala Thr Ala Arg Cys Ala Pro
435 440 445
Glu Glu Glu Leu Leu Ser Cys Ser Ser Phe Ser Arg Ser Gly Arg Arg
450 455 460
Arg Gly Asp Trp Ile Glu Ala Ile Gly Gly Gln Gln Val Cys Lys Ala
465 470 475 480
Leu Asn Ala Phe Gly Gly Glu Gly Val Tyr Ala Val Ala Arg Cys Cys
485 490 495
Leu Val Pro Arg Ala Asn Cys Ser Ile His Asn Thr Pro Ala Ala Arg
500 505 510
Ala Gly Leu Glu Thr His Val His Cys His Gln Lys Asp His Val Leu
515 520 525
Thr Gly Cys Ser Phe His Trp Glu Val Glu Asp Leu Ser Val Arg Arg
530 535 540
Gln Pro Ala Leu Arg Ser Arg Arg Gln Pro Gly Gln Cys Val Gly His
545 550 555 560
Gln Ala Ala Ser Val Tyr Ala Ser Cys Cys His Ala Pro Gly Leu Glu
565 570 575
Cys Lys Ile Lys Glu His Gly Ile Ser Gly Pro Ser Glu Gln Val Thr
580 585 590
Val Ala Cys Glu Ala Gly Trp Thr Leu Thr Gly Cys Asn Val Leu Pro
595 600 605
Gly Ala Ser Leu Thr Leu Gly Ala Tyr Ser Val Asp Asn Leu Cys Val
610 615 620
Ala Arg Val His Asp Thr Ala Arg Ala Asp Arg Thr Ser Gly Glu Ala
625 630 635 640
Thr Val Ala Ala Ala Ile Cys Cys Arg Ser Arg Pro Ser Ala Lys Ala
645 650 655
Ser Trp Val Gln
660
<210>3
<211>639
<212>PRT
<213>猕猴（Macaca mulatta）
<220>
<223>mmPCSK9
<400>3
Asp Ala Pro Glu His Gly Ala Thr Ala Thr Phe His Arg Cys Ala Lys
1 5 10 15
Asp Pro Trp Arg Leu Pro Gly Thr Tyr Val Val Val Leu Lys Glu Glu
20 25 30
Thr His Arg Ser Gln Ser Glu Arg Thr Ala Arg Arg Leu Gln Ala Gln
35 40 45
Ala Ala Arg Arg Gly Tyr Leu Thr Lys Ile Leu His Val Phe His His
50 55 60
Leu Leu Pro Gly Phe Leu Val Lys Met Ser Gly Asp Leu Leu Glu Leu
65 70 75 80
Ala Leu Lys Leu Pro His Val Asp Tyr Ile Glu Glu Asp Ser Ser Val
85 90 95
Phe Ala Gln Ser Ile Pro Trp Asn Leu Glu Arg Ile Thr Pro Ala Arg
100 105 110
Tyr Arg Ala Asp Glu Tyr Gln Pro Pro Lys Gly Gly Ser Leu Val Glu
115 120 125
Val Tyr Leu Leu Asp Thr Ser Ile Gln Ser Asp His Arg Glu Ile Glu
130 135 140
Gly Arg Val Met Val Thr Asp Phe Glu Ser Val Pro Glu Glu Asp Gly
145 150 155 160
Thr Arg Phe His Arg Gln Ala Ser Lys Cys Asp Ser His Gly Thr His
165 170 175
Leu Ala Gly Val Val Ser Gly Arg Asp Ala Gly Val Ala Lys Gly Ala
180 185 190
Gly Leu Arg Ser Leu Arg Val Leu Asn Cys Gln Gly Lys Gly Thr Val
195 200 205
Ser Gly Thr Leu Ile Gly Leu Glu Phe Ile Arg Lys Ser Gln Leu Val
210 215 220
Gln Pro Val Gly Pro Leu Val Val Leu Leu Pro Leu Ala Gly Gly Tyr
225 230 235 240
Ser Arg Val Phe Asn Ala Ala Cys Gln Arg Leu Ala Arg Ala Gly Val
245 250 255
Val Leu Val Thr Ala Ala Gly Asn Phe Arg Asp Asp Ala Cys Leu Tyr
260 265 270
Ser Pro Ala Ser Ala Pro Glu Val Ile Thr Val Gly Ala Thr Asn Ala
275 280 285
Gln Asp Gln Pro Val Thr Leu Gly Thr Leu Gly Thr Asn Phe Gly Arg
290 295 300
Cys Val Asp Leu Phe Ala Pro Gly Glu Asp Ile Ile Gly Ala Ser Ser
305 310 315 320
Asp Cys Ser Thr Cys Phe Val Ser Arg Ser Gly Thr Ser Gln Ala Ala
325 330 335
Ala His Val Ala Gly Ile Ala Ala Met Met Leu Ser Ala Glu Pro Glu
340 345 350
Leu Thr Leu Ala Glu Leu Arg Gln Arg Leu Ile His Phe Ser Ala Lys
355 360 365
Asp Val Ile Asn Glu Ala Trp Phe Pro Glu Asp Gln Arg Val Leu Thr
370 375 380
Pro Asn Leu Val Ala Ala Leu Pro Pro Ser Thr His Arg Ala Gly Trp
385 390 395 400
Gln Leu Phe Cys Arg Thr Val Trp Ser Ala His Ser Gly Pro Thr Arg
405 410 415
Met Ala Thr Ala Val Ala Arg Cys Ala Gln Asp Glu Glu Leu Leu Ser
420 425 430
Cys Ser Ser Phe Ser Arg Ser Gly Lys Arg Arg Gly Glu Arg Ile Glu
435 440 445
Ala Gln Gly Gly Lys Arg Val Cys Arg Ala His Asn Ala Phe Gly Gly
450 455 460
Glu Gly Val Tyr Ala Ile Ala Arg Cys Cys Leu Leu Pro Gln Val Asn
465 470 475 480
Cys Ser Val His Thr Ala Pro Pro Ala Gly Ala Ser Met Gly Thr Arg
485 490 495
Val His Cys His Gln Gln Gly His Val Leu Thr Gly Cys Ser Ser His
500 505 510
Trp Glu Val Glu Asp Leu Gly Thr His Lys Pro Pro Val Leu Arg Pro
515 520 525
Arg Gly Gln Pro Asn Gln Cys Val Gly His Arg Glu Ala Ser Ile His
530 535 540
Ala Ser Cys Cys His Ala Pro Gly Leu Glu Cys Lys Val Lys Glu His
545 550 555 560
Gly Ile Pro Ala Pro Gln Glu Gln Val Ile Val Ala Cys Glu Asp Gly
565 570 575
Trp Thr Leu Thr Gly Cys Ser Pro Leu Pro Gly Thr Ser His Val Leu
580 585 590
Gly Ala Tyr Ala Val Asp Asn Thr Cys Val Val Arg Ser Arg Asp Val
595 600 605
Ser Thr Thr Gly Ser Thr Ser Lys Glu Ala Val Ala Ala Val Ala Ile
610 615 620
Cys Cys Arg Ser Arg His Leu Val Gln Ala Ser Gln Glu Leu Gln
625 630 635
<210>4
<211>120
<212>PRT
<213>智人（Homo sapiens）
<220>
<223>LDLR-EGFAB
<400>4
Thr Leu Cys Glu Gly Pro Asn Lys Phe Lys Cys His Ser Gly Glu Cys
1 5 10 15
Ile Thr Leu Asp Lys Val Cys Asn Met Ala Arg Asp Cys Arg Asp Trp
20 25 30
Ser Asp Glu Pro Ile Lys Glu Cys Gly Thr Asn Glu Cys Leu Asp Asn
35 40 45
Asn Gly Gly Cys Ser His Val Cys Asn Asp Leu Lys Ile Gly Tyr Glu
50 55 60
Cys Leu Cys Pro Asp Gly Phe Gln Leu Val Ala Gln Arg Arg Cys Glu
65 70 75 80
Asp Ile Asp Glu Cys Gln Asp Pro Asp Thr Cys Ser Gln Leu Cys Val
85 90 95
Asn Leu Glu Gly Gly Tyr Lys Cys Gln Cys Glu Glu Gly Phe Gln Leu
100 105 110
Asp Pro His Thr Lys Ala Cys Lys
115 120
<210>5
<211>11
<212>PRT
<213>人工序列
<220>
<223>his标签
<400>5
Ala Ser Gly Ala Ala His His His His His His
1 5 10
<210>6
<211>234
<212>PRT
<213>小鼠（Mus musculus）
<220>
<223>mFc标签
<400>6
Ala Ser Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys
1 5 10 15
Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro
20 25 30
Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys
35 40 45
Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp
50 55 60
Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg
65 70 75 80
Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln
85 90 95
His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn
100 105 110
Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly
115 120 125
Ser Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu Glu Glu
130 135 140
Met Thr Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp Phe Met
145 150 155 160
Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu
165 170 175
Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe
180 185 190
Met Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn
195 200 205
Ser Tyr Ser Cys Ser Val Val His Glu Gly Leu His Asn His His Thr
210 215 220
Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys
225 230
<210>7
<211>330
<212>PRT
<213>智人（Homo sapiens）
<220>
<223>IgG-C1
<400>7
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
225 230 235 240
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
325 330
<210>8
<211>326
<212>PRT
<213>智人（Homo sapiens）
<220>
<223>IgG2-C
<400>8
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg
1 5 10 15
Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr
65 70 75 80
Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro
100 105 110
Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
115 120 125
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
130 135 140
Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly
145 150 155 160
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn
165 170 175
Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp
180 185 190
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro
195 200 205
Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu
210 215 220
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn
225 230 235 240
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
245 250 255
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
260 265 270
Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
275 280 285
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
290 295 300
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
305 310 315 320
Ser Leu Ser Pro Gly Lys
325
<210>9
<211>327
<212>PRT
<213>智人（Homo sapiens）
<220>
<223>IgG4-C
<400>9
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg
1 5 10 15
Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr
65 70 75 80
Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro
100 105 110
Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
115 120 125
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
130 135 140
Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp
145 150 155 160
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe
165 170 175
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
180 185 190
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu
195 200 205
Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
210 215 220
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys
225 230 235 240
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
245 250 255
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
260 265 270
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
275 280 285
Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser
290 295 300
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
305 310 315 320
Leu Ser Leu Ser Leu Gly Lys
325
<210>10
<211>107
<212>PRT
<213>智人（Homo sapiens）
<220>
<223>kappa亚型轻链恒定区
<400>10
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
1 5 10 15
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
20 25 30
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
35 40 45
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
50 55 60
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
65 70 75 80
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
85 90 95
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 105
<210>11
<211>105
<212>PRT
<213>智人（Homo sapiens）
<220>
<223>lambda亚型轻链恒定区
<400>11
Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu
1 5 10 15
Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe
20 25 30
Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val
35 40 45
Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys
50 55 60
Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser
65 70 75 80
His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu
85 90 95
Lys Thr Val Ala Pro Thr Glu Cys Ser
100 105
<210>12
<211>245
<212>PRT
<213>人工序列
<220>
<223>S2B8
<400>12
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Ser Arg His
20 25 30
Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ser Ile Ser Ser Gly Ser Ser Asn Ile Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Gly Leu Gly Pro Glu Tyr Leu Gln His Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Ser Ser Glu Leu Thr Gln Pro Ala Ser Val
130 135 140
Ser Gly Ser Pro Gly Gln Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser
145 150 155 160
Ser Asp Ile Gly Asp Asn Asn Phe Val Ser Trp Tyr Gln Gln Tyr Pro
165 170 175
Gly Lys Ala Pro Lys Leu Met Ile Tyr Asp Val Ser Asn Arg Pro Ser
180 185 190
Gly Val Ser Asn Arg Phe Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser
195 200 205
Leu Thr Ile Ser Gly Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys
210 215 220
Ser Ser Tyr Thr Ser Ser Ser Thr Leu Val Phe Gly Gly Gly Thr Lys
225 230 235 240
Val Thr Val Leu Gly
245
<210>13
<211>242
<212>PRT
<213>人工序列
<220>
<223>S2F5
<400>13
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Thr Gly Tyr
20 25 30
Ser Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Asn Pro Ile Ser Gly Ser Ala Asn Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Thr Gln Asn Asn Thr Gly Tyr Arg Gly Thr Asp Tyr Trp Gly
100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly
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Gly Gly Ser Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser
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Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala
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180 185 190
Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
195 200 205
Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln
210 215 220
Gln His Ala Asp Thr Pro Thr Thr Phe Gly Gln Gly Thr Lys Val Glu
225 230 235 240
Ile Lys
<210>14
<211>237
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<223>S2G1
<400>14
Gln Met Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Gly Ser Ser
20 25 30
Ala Ile His Trp Val Arg Gln Ala Pro Arg Gln Gly Leu Glu Trp Met
35 40 45
Gly Arg Ile Asn Pro Val Leu Gly Thr Thr Asn Tyr Thr Gln Lys Phe
50 55 60
His Asp Arg Leu Thr Ile Thr Ala Asp Glu Ser Thr Thr Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Gly Ile Pro Met Ile Trp Gly Gln Gly Thr Met Val Thr
100 105 110
Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
115 120 125
Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
130 135 140
Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile His
145 150 155 160
Asn Trp Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
165 170 175
Leu Ile Tyr Ala Ala Ser Asn Arg Ala Ser Gly Val Pro Ser Arg Phe
180 185 190
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
195 200 205
Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Gly Lys Gln
210 215 220
Pro Val Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
225 230 235
<210>15
<211>235
<212>PRT
<213>人工序列
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Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Ile Tyr Asn Tyr
20 25 30
Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Val Ser Tyr Phe Gly Gly Ser Thr Thr Tyr Ala Gln Lys Leu
50 55 60
Gln Gly Arg Gly Thr Met Thr Thr Asp Pro Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Lys Gly Tyr Gly Met Glu Ile Trp Gly Gln Gly Thr Thr Val Ser
100 105 110
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
115 120 125
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
130 135 140
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asn Val Asn Asn Trp
145 150 155 160
Leu Asp Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
165 170 175
Tyr Ala Ala Ser Thr Arg Ala Ser Gly Val Pro Ser Arg Phe Ser Gly
180 185 190
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
195 200 205
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210 215 220
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
225 230 235
<210>16
<211>115
<212>PRT
<213>人工序列
<220>
<223>H4H4
<400>16
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Ile Tyr Asn Tyr
20 25 30
Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Val Ser Phe Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu
50 55 60
Gln Gly Arg Gly Thr Met Thr Thr Asp Pro Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Tyr Val Met Asp Ile Trp Gly Gln Gly Thr Thr Val Thr
100 105 110
Val Ser Ser
115
<210>17
<211>115
<212>PRT
<213>人工序列
<220>
<223>H7B12
<400>17
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Leu Asn Asn Tyr
20 25 30
Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Val Ser Phe Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu
50 55 60
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65 70 75 80
Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Tyr Val Met Asp Ile Trp Gly Gln Gly Thr Thr Val Thr
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Val Ser Ser
115
<210>18
<211>115
<212>PRT
<213>人工序列
<220>
<223>H8H7
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Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
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20 25 30
Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Val Ser Phe Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu
50 55 60
Gln Gly Arg Gly Thr Met Thr Thr Asp Pro Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
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115
<210>19
<211>115
<212>PRT
<213>人工序列
<220>
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<400>19
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20 25 30
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35 40 45
Gly Trp Val Ser Phe Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu
50 55 60
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65 70 75 80
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115
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<212>PRT
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<220>
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35 40 45
Tyr Ala Ala Ser Thr Arg Pro Ser Gly Val Pro Ser Arg Phe Ser Gly
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Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Val Ser Phe Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu
50 55 60
Gln Gly Arg Gly Thr Met Thr Thr Asp Pro Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
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Val Ser Ser
115
<210>22
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<213>人工序列
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Asn Ser Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu
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Met Ile Tyr Glu Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe
50 55 60
Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu
65 70 75 80
Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Tyr Thr Ser Thr
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Ser Met Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105
<210>23
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<213>人工序列
<220>
<223>AliroVH
<400>23
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asn Tyr
20 25 30
Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Asp Trp Val
35 40 45
Ser Thr Ile Ser Gly Ser Gly Gly Thr Thr Asn Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Ile Ile Ser Arg Asp Ser Ser Lys His Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Lys Asp Ser Asn Trp Gly Asn Phe Asp Leu Trp Gly Arg Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
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50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln
85 90 95
Tyr Tyr Thr Thr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile
100 105 110
Lys
Claims (7)
1.一种特异性结合PCSK9的单克隆抗体，其包含含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，其特征在于：
所述HCDR1序列为GYAIYNY，所述HCDR2序列为SFYNGN，所述HCDR3序列为GYVMDI；或
所述HCDR1序列为GYTLNNY，所述HCDR2序列为SFYNGN，所述HCDR3序列为GYVMDI；或
所述HCDR1序列为GYAIYNY，所述HCDR2序列为SFYNGN，所述HCDR3序列为GYVMDF；或
所述HCDR1序列为GYALYNY，所述HCDR2序列为SFYNGN，所述HCDR3序列为GYVMDI；
所述LCDR1序列为RASQSINNWLD，所述LCDR2序列为AASTRPS，所述LCDR3序列为QQDQDIPPT；
而且其中HCDR和LCDR序列根据Chothia定义。
2.根据权利要求1所述的单克隆抗体，所述抗体的重链可变区序列为SEQ ID NO：16，轻链可变区序列为SEQ ID NO：20；或重链可变区序列为SEQ ID NO：17，轻链可变区序列为SEQID NO：20；或重链可变区序列为SEQ ID NO：18，轻链可变区序列为SEQ ID NO：20；或重链可变区序列为SEQ ID NO：19，轻链可变区序列为SEQ ID NO：20。
3.根据权利要求1或2所述的单克隆抗体，其中所述抗体为全长抗体、Fab片段、F(ab')2片段或单链Fv片段。
4.根据权利要求3所述的单克隆抗体，其特征在于，所述抗体是全人源抗体。
5.根据权利要求1或2所述的单克隆抗体，其中所述抗体还包含选自IgG1、IgG2或IgG4亚型的重链恒定区和包含选自kappa或Lambda亚型的轻链恒定区。
6.权利要求1-5中任一项所述的单克隆抗体在制备用于治疗由人PCSK9介导的高胆固醇血症的药物中的用途。
7.根据权利要求6所述的用途，其特征在于，所述高胆固醇血症为原发性高胆固醇血症、混合型高脂血症或家族性高胆固醇血症。
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| CN105348390B (zh) * | 2015-10-26 | 2018-08-28 | 北京智仁美博生物科技有限公司 | 抗人pcsk9单克隆抗体 |
| CN107474140B (zh) * | 2016-06-08 | 2022-06-03 | 常州博嘉生物医药科技有限公司 | Pcsk9特异性的结合蛋白mv072及其应用 |
| EP3515950A4 (en) * | 2016-09-20 | 2020-10-28 | Wuxi Biologics Ireland Limited. | NEW ANTI-PCSK9 ANTIBODIES |
| CN108239150A (zh) * | 2016-12-24 | 2018-07-03 | 信达生物制药(苏州)有限公司 | 抗pcsk9抗体及其用途 |
| CN107698680B (zh) * | 2017-01-22 | 2019-03-01 | 北京东方百泰生物科技有限公司 | 抗pcsk9单克隆抗体 |
| CN107957494B (zh) * | 2017-08-23 | 2020-06-02 | 武汉菲思特生物科技有限公司 | 一种人前蛋白转化酶枯草溶菌素9化学发光检测试剂和检测试剂盒与应用 |
| EP4063506A4 (en) * | 2019-09-19 | 2023-09-06 | Innovent Biologics (Suzhou) Co., Ltd. | METHOD FOR PREVENTING OR TREATING CHOLESTEROL-RELATED DISEASES USING AN ANTI-PCSK9 ANTIBODY |
| 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 |
| 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 |
| 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 |
| 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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