CN113527486A - 一种抗Nectin-4的抗体及其应用 - Google Patents

一种抗Nectin-4的抗体及其应用 Download PDF

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Publication number
CN113527486A
CN113527486A CN202010320420.3A CN202010320420A CN113527486A CN 113527486 A CN113527486 A CN 113527486A CN 202010320420 A CN202010320420 A CN 202010320420A CN 113527486 A CN113527486 A CN 113527486A
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China
Prior art keywords
cdr
seq
amino acid
acid sequence
ser
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CN202010320420.3A
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English (en)
Inventor
任红媛
朱戬
林鉴
王骊淳
徐晓红
邓小芳
毕建军
王晋
吴建
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Maiwei Shanghai Biotechnology Co ltd
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Maiwei Shanghai Biotechnology Co ltd
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Priority to CN202010320420.3A priority Critical patent/CN113527486A/zh
Priority to EP21791797.0A priority patent/EP4141029A1/en
Priority to KR1020227040582A priority patent/KR20230007406A/ko
Priority to PCT/CN2021/088661 priority patent/WO2021213434A1/zh
Priority to US17/996,403 priority patent/US20230265183A1/en
Priority to BR112022021322A priority patent/BR112022021322A2/pt
Priority to CA3176385A priority patent/CA3176385A1/en
Priority to CN202180030277.7A priority patent/CN115427452A/zh
Priority to AU2021260639A priority patent/AU2021260639A1/en
Priority to MX2022013163A priority patent/MX2022013163A/es
Priority to JP2022563917A priority patent/JP2023522229A/ja
Publication of CN113527486A publication Critical patent/CN113527486A/zh
Pending legal-status Critical Current

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Abstract

本发明提供一种结合人Nectin‑4的抗体分子或其片段及其用于预防或治疗癌症的应用。本发明提供的抗体通过杂交瘤筛选和人源化技术获得，为特异性结合Nectin‑4的高亲和力抗体，可作为有效的临床先导药物分子。

Description

一种抗Nectin-4的抗体及其应用
技术领域
本发明属于抗体药物领域，具体而言，本发明涉及针对人Nectin-4的抗体及其用于制备药物的用途。
背景技术
Nectin-4(又名PVRL4，脊髓灰质炎病毒受体样分子4)是一个大小为 66KD的一型跨膜糖蛋白，属于Nectin家族的IG超家族蛋白分子，其细胞外结构域由三个Ig样结构域(VCC型)组成，与钙粘蛋白一起参与粘附连接的形成和维持。
Nectin-4与多种肿瘤细胞的发生，发展有着密切联系。已发现Nectin-4 在多种实体瘤、尤其是膀胱癌中均有表达；在乳腺癌、卵巢癌和肺癌中，作为肿瘤相关抗原，分别占乳腺癌50％、卵巢癌49％和肺癌86％的组织表达检出率，并且在这些上皮恶性肿瘤的发生、侵袭和转移中起关键作用。因此， Nectin-4已成为许多实体瘤诊断和治疗的重要靶点。
目前针对Nectin-4的主要药物为Enfortumab vedotin，这是一种抗体偶联药物，由抗Nectin-4的单克隆抗体和细胞杀伤药物monomethyl auristatin E (MMAE)偶联而成，主治膀胱癌，尤其是尿路上皮癌，已在2018年3月获 FDA突破性疗法认定。此外另有研究显示，粘附因子Nnectin-4不仅可作为乳腺癌的有效预后因子，同时可作为三阴性乳腺癌(TNBC)患者的有效治疗靶点；体内外研究证实，抗Nectin-4的抗体偶联药物(ADC)对于局部、转移性TNBC具有较好疗效。
发明内容
本发明要解决的技术问题是，通过杂交瘤筛选和人源化技术，获得特异性结合Nectin-4的高亲和力抗体，其中通过人源化改造，获得全人抗体序列。
针对上述技术问题，本发明的目的是提供一种特异性结合Nectin-4、特别是人Nectin-4的抗体分子或其片段，并提供其用途。其中，本发明所述的抗体分子的片段涵盖抗体的各种功能性片段，例如其抗原结合部分，如Fab、 F(ab’)2或scFv片段。
本发明的技术方案如下：
一方面，本发明提供一种抗体分子或其片段，所述抗体分子或其片段包含重链可变区(VH)和轻链可变区(VL)，所述重链可变区和轻链可变区分别包含选自以下的重链CDR和轻链CDR的组合：
(1)依次示于SEQ ID NO：35、39、43的CDR-H1(GYTFTTY)、CDR-H2 (YPGNVN)、CDR-H3(GLYYFDY)；和，依.次示于SEQ ID NO：45、47、 49的CDR-L1(KASQSVSNDVA)、CDR-L2(YASNRYT)、CDR-L3 (QQDYSSPYT)；
(2)依次示于SEQ ID NO：36、40、43的CDR-H1(GYTFTTYYIH)、 CDR-H2(WIYPGNVNTK)、CDR-H3(GLYYFDY)；和，依次示于SEQ ID NO：45、47、49的CDR-L1(KASQSVSNDVA)、CDR-L2(YASNRYT)、 CDR-L3(QQDYSSPYT)；
(3)依次示于SEQ ID NO：37、41、43的CDR-H1(TYYIH)、CDR-H2(WIYPGNVNTKYNEKFKG)、CDR-H3(GLYYFDY)；和，依次示于SEQ ID NO：45、47、49的CDR-L1(KASQSVSNDVA)、CDR-L2(YASNRYT)、 CDR-L3(QQDYSSPYT)；
(4)依次示于SEQ ID NO：38、42、44的CDR-H1(TTYYIH)、CDR-H2 (WIGWIYPGNVNTK)、CDR-H3(ARGLYYFD)；和，依次示于SEQ ID NO： 46、48、50的CDR-L1(SNDVAWY)、CDR-L2(LLIYYASNRY)、CDR-L3 (QQDYSSPY)；
(5)依次示于SEQ ID NO：51、55、59的CDR-H1(GFSLIDY)、CDR-H2 (WGDGK)、CDR-H3(QGGLLFYAMDY)；和，依次示于SEQ ID NO：61、 63、65的CDR-L1(KSSQSLLNSYSQKNYLA)、CDR-L2(FASTRES)、CDR-L3 (QQHYNTPFT)；
(6)依次示于SEQ ID NO：52、56、59的CDR-H1(GFSLIDYGVS)、 CDR-H2(VIWGDGKIY)、CDR-H3(QGGLLFYAMDY)；和，依次示于SEQ ID NO：61、63、65的CDR-L1(KSSQSLLNSYSQKNYLA)、CDR-L2 (FASTRES)、CDR-L3(QQHYNTPFT)；
(7)依次示于SEQ ID NO：53、57、59的CDR-H1(DYGVS)、CDR-H2(VIWGDGKIYYNSVLKS)、CDR-H3(QGGLLFYAMDY)；和，依次示于SEQ ID NO：61、63、65的CDR-L1(KSSQSLLNSYSQKNYLA)、CDR-L2 (FASTRES)、CDR-L3(QQHYNTPFT)；
(8)依次示于SEQ ID NO：54、58、60的CDR-H1(IDYGVS)、CDR-H2 (WLGVIWGDGKIY)、CDR-H3(AKQGGLLFYAMD)；和，依次示于SEQ ID NO：62、64、66的CDR-L1(LNSYSQKNYLAWY)、CDR-L2 (LLIYFASTRE)、CDR-L3(QQHYNTPF)；
(9)依次示于SEQ ID NO：51、55、59的CDR-H1(GFSLIDY)、CDR-H2 (WGDGK)、CDR-H3(QGGLLFYAMDY)；和，依.次示于SEQ ID NO：67、 63、65的CDR-L1(KSSQSLLNTYSQKNYLA)、CDR-L2(FASTRES)、CDR-L3 (QQHYNTPFT)；
(10)依次示于SEQ ID NO：51、68、59的CDR-H1(GFSLIDY)、CDR-H2 (WGDAK)、CDR-H3(QGGLLFYAMDY)；和，依次示于SEQ ID NO：67、 63、65的CDR-L1(KSSQSLLNTYSQKNYLA)、CDR-L2(FASTRES)、CDR-L3 (QQHYNTPFT)；
(11)依次示于SEQ ID NO：51、69、59的CDR-H1(GFSLIDY)、CDR-H2 (WGGGK)、CDR-H3(QGGLLFYAMDY)；和，依次示于SEQ ID NO：67、 63、65的CDR-L1(KSSQSLLNTYSQKNYLA)、CDR-L2(FASTRES)、CDR-L3 (QQHYNTPFT)；
(12)依次示于SEQ ID NO：70、74、78的CDR-H1(GYTFTSY)、CDR-H2 (YPGNAN)、CDR-H3(SVYYFDY)；和，依次示于SEQ ID NO：45、80、 49的CDR-L1(KASQSVSNDVA)、CDR-L2(YASNRNT)、CDR-L3 (QQDYSSPYT)；
(13)依次示于SEQ ID NO：71、75、78的CDR-H1(GYTFTSYYIH)、 CDR-H2(WIYPGNANNK)、CDR-H3(SVYYFDY)；和，依次示于SEQ ID NO：45、80、49的CDR-L1(KASQSVSNDVA)、CDR-L2(YASNRNT)、 CDR-L3(QQDYSSPYT)；
(14)依次示于SEQ ID NO：72、76、78的CDR-H1(SYYIH)、CDR-H2(WIYPGNANNKYNENFKG)、CDR-H3(SVYYFDY)；和，依次示于SEQ ID NO：45、80、49的CDR-L1(KASQSVSNDVA)、CDR-L2(YASNRNT)、 CDR-L3(QQDYSSPYT)；
(15)依次示于SEQ ID NO：73、77、79的CDR-H1(TSYYIH)、CDR-H2(WIGWIYPGNANNK)、CDR-H3(ARSVYYFD)；和，依次示于SEQ ID NO：46、81、50的CDR-L1(SNDVAWY)、CDR-L2(LLIYYASNRN)、CDR-L3 (QQDYSSPY)；
(16)依次示于SEQ ID NO：82、86、90的CDR-H1(GYSFTDY)、CDR-H2 (NPNNGN)、CDR-H3(EDRYAFAY)；和，依次示于SEQ ID NO：92、94、 96的CDR-L1(RASQSVSTSSYTYMH)、CDR-L2(YASNLES)、CDR-L3 (QHTWEIPYT)；
(17)依次示于SEQ ID NO：83、87、90的CDR-H1(GYSFTDYYMH)、 CDR-H2(RVNPNNGNTL)、CDR-H3(EDRYAFAY)；和，依次示于SEQ ID NO：92、94、96的CDR-L1(RASQSVSTSSYTYMH)、CDR-L2(YASNLES)、 CDR-L3(QHTWEIPYT)；
(18)依次示于SEQ ID NO：84、88、90的CDR-H1(DYYMH)、CDR-H2(RVNPNNGNTLYNQKFRG)、CDR-H3(EDRYAFAY)；和，依次示于SEQ ID NO：92、94、96的CDR-L1(RASQSVSTSSYTYMH)、CDR-L2 (YASNLES)、CDR-L3(QHTWEIPYT)；
(19)依次示于SEQ ID NO：85、89、91的CDR-H1(TDYYMH)、CDR-H2(WIGRVNPNNGNTL)、CDR-H3(AREDRYAFA)；和，依次示于SEQ ID NO： 93、95、97的CDR-L1(STSSYTYMHWY)、CDR-L2(LLIKYASNLE)、 CDR-L3(QHTWEIPY)。
按照本领域公知的抗体中重链可变区、轻链可变区的结构域组成，本发明抗体分子或其片段中的重链可变区或轻链可变区以 FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4的顺序包含上述结构域组分，其中FR 为框架区。
优选地，在本发明提供的抗体分子或其片段中，所述重链可变区包含示于SEQ IDNO：7、9或10的氨基酸序列或与所述氨基酸序列具有至少75％同一性的氨基酸序列；和，所述轻链可变区包含示于SEQ ID NO：8、11或 12的氨基酸序列或与所述氨基酸序列具有至少75％同一性的氨基酸序列；或者，
所述重链可变区包含示于SEQ ID NO：13、15、16、18、19、20或21 的氨基酸序列或与所述氨基酸序列具有至少75％同一性的氨基酸序列；和，所述轻链可变区包含示于SEQID NO：14、22、23或25的氨基酸序列或与所述氨基酸序列具有至少75％同一性的氨基酸序列；或者
所述重链可变区包含示于SEQ ID NO：27、29或30的氨基酸序列或与所述氨基酸序列具有至少75％同一性的氨基酸序列；和，所述轻链可变区包含示于SEQ ID NO：28、31或32的氨基酸序列或与所述氨基酸序列具有至少75％同一性的氨基酸序列；或者，
所述重链可变区包含示于SEQ ID NO：33的氨基酸序列或与所述氨基酸序列具有至少75％同一性的氨基酸序列；和，所述轻链可变区包含示于SEQ ID NO：34的氨基酸序列或与所述氨基酸序列具有至少75％同一性的氨基酸序列。
根据本发明的具体实施方式，所述抗体分子或其片段中的重链可变区和轻链可变区选自以下氨基酸序列的组合：
(1)如SEQ ID NO：7所示的氨基酸序列或与如SEQ ID NO：7所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO：8所示的氨基酸序列或与如SEQ ID NO：8所示的氨基酸序列具有至少75％同一性的氨基酸序列；
(2)如SEQ ID NO：9所示的氨基酸序列或与如SEQ ID NO：9所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO：11所示的氨基酸序列或与如SEQ ID NO：11所示的氨基酸序列具有至少75％同一性的氨基酸序列；
(3)如SEQ ID NO：9所示的氨基酸序列或与如SEQ ID NO：9所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO：12所示的氨基酸序列或与如SEQ ID NO：12所示的氨基酸序列具有至少75％同一性的氨基酸序列；
(4)如SEQ ID NO：10所示的氨基酸序列或与如SEQ ID NO：10所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO：11所示的氨基酸序列或与如SEQ IDNO：11所示的氨基酸序列具有至少75％同一性的氨基酸序列；
(5)如SEQ ID NO：10所示的氨基酸序列或与如SEQ ID NO：10所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO：12所示的氨基酸序列或与如SEQ IDNO：12所示的氨基酸序列具有至少75％同一性的氨基酸序列；
(6)如SEQ ID NO：13所示的氨基酸序列或与如SEQ ID NO：13所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO：14所示的氨基酸序列或与如SEQ IDNO：14所示的氨基酸序列具有至少75％同一性的氨基酸序列；
(7)如SEQ ID NO：16所示的氨基酸序列或与如SEQ ID NO：16所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO：22所示的氨基酸序列或与如SEQ IDNO：22所示的氨基酸序列具有至少75％同一性的氨基酸序列；
(8)如SEQ ID NO：16所示的氨基酸序列或与如SEQ ID NO：16所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO：23所示的氨基酸序列或与如SEQ IDNO：23所示的氨基酸序列具有至少75％同一性的氨基酸序列；
(9)如SEQ ID NO：16所示的氨基酸序列或与如SEQ ID NO：16所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO：25所示的氨基酸序列或与如SEQ IDNO：25所示的氨基酸序列具有至少75％同一性的氨基酸序列；
(10)如SEQ ID NO：19所示的氨基酸序列或与如SEQ ID NO：19所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO：25所示的氨基酸序列或与如SEQ IDNO：25所示的氨基酸序列具有至少75％同一性的氨基酸序列；
(11)如SEQ ID NO：21所示的氨基酸序列或与如SEQ ID NO：21所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO：25所示的氨基酸序列或与如SEQ IDNO：25所示的氨基酸序列具有至少75％同一性的氨基酸序列；
(12)如SEQ ID NO：27所示的氨基酸序列或与如SEQ ID NO：27所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO：28所示的氨基酸序列或与如SEQ IDNO：28所示的氨基酸序列具有至少75％同一性的氨基酸序列；
(13)如SEQ ID NO：29所示的氨基酸序列或与如SEQ ID NO：29所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO：32所示的氨基酸序列或与如SEQ IDNO：32所示的氨基酸序列具有至少75％同一性的氨基酸序列；
(14)如SEQ ID NO：30所示的氨基酸序列或与如SEQ ID NO：30所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO：32所示的氨基酸序列或与如SEQ IDNO：32所示的氨基酸序列具有至少75％同一性的氨基酸序列；或者
(15)如SEQ ID NO：33所示的氨基酸序列或与如SEQ ID NO：33所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO：34所示的氨基酸序列或与如SEQ IDNO：34所示的氨基酸序列具有至少75％同一性的氨基酸序列。
基于本发明提供的上述重链可变区或轻链可变区的具体氨基酸序列，本领域技术人员可以常规地确定其中包含的重链CDR和轻链CDR的氨基酸序列，以本领域其他已知方法确定得到的重轻链CDR及其组合也被涵盖在本发明的范围内。
本发明提供的所述抗体分子或其片段结合脊髓灰质炎病毒受体样分子4(Nectin-4)，优选哺乳动物Nectin-4，更优选灵长类动物Nectin-4，进一步优选人或猴Nectin-4，特别是人Nectin-4。
优选地，所述抗体分子为鼠源抗体、嵌合抗体或者完全或部分人源化抗体；所述片段为所述抗体分子的能够特异性结合Nectin-4的任何片段，例如 scFv、dsFv、(dsFv)2、Fab、Fab′、F(ab′)2或Fv片段。
优选地，所述抗体分子为单克隆抗体或单链抗体。
优选地，所述抗体分子或其片段还包含人或鼠的恒定区，优选包含鼠或人的重链恒定区(CH)和/或轻链恒定区(CL)；优选地，所述抗体分子或其片段包含重链和轻链，例如两条重链和轻链。更优选地，所述抗体分子或其片段包含IgG、IgA、IgM、IgD或IgE的重链恒定区和/或κ或λ型轻链恒定区。
根据本发明的具体实施方式，本发明提供的抗体分子为单克隆抗体，优选为人源化的单克隆抗体；优选地，所述单克隆抗体的重链恒定区为IgG1 型，轻链恒定区为κ型。例如，所述单克隆抗体的重链恒定区包含示于SEQ ID NO：4的氨基酸序列或者与所述氨基酸序列具有至少75％同一性的氨基酸序列；所述单克隆抗体的轻链恒定区包含示于SEQ IDNO：5的氨基酸序列或者与所述氨基酸序列具有至少75％同一性的氨基酸序列。
在本发明的上下文中，“至少75％同一性”为75％至100％之间的任何百分比数字的同一性，例如75％、80％、85％、90％，甚至91％、92％、93％、 94％、95％、96％、97％、98％或99％同一性。
另一方面，本发明提供一种核酸分子，其包含编码本发明所述的抗体分子或其片段中包含的轻链可变区、重链可变区、重链或轻链的核苷酸序列。
本发明的核酸分子可以被克隆到载体中，进而转化或转染宿主细胞。因此，还一方面，本发明提供一种载体，其包含本发明的核酸分子。所述载体可以为真核表达载体、原核表达载体、人工染色体及噬菌体载体等。
本发明的载体或核酸分子可以用于转化或转染宿主细胞或者以任何方式进入宿主细胞内，用于保存或表达抗体等目的。因此，又一方面，本发明提供一种宿主细胞，所述宿主细胞包含本发明的核酸分子和/或载体，或者所述宿主细胞被本发明的核酸分子和/或载体转化或转染。宿主细胞可以是任何原核或真核细胞，例如细菌或昆虫、真菌、植物或动物细胞。
本发明提供的抗体分子可以利用本领域已知的任何方法获得。例如，可以先由本发明提供的核酸分子获得所述抗体的重链可变区和/或轻链可变区，或者获得所述抗体分子的重链和/或轻链，然后与所述抗体分子的任选其他结构域组装成抗体；或者，在允许本发明提供的宿主细胞表达所述抗体分子的重链可变区和/或轻链可变区或者所述抗体分子的重链和/或轻链以组装成所述抗体的情况下，培养所述宿主细胞。任选地，所述方法还包括回收产生的抗体分子的步骤。
本发明提供的抗体分子或其片段、核酸分子、载体、宿主细胞或融合蛋白可以被包含在组合物中，更特别地被包含在药物制剂中，从而根据实际需要用于各种目的。因此，在又一方面，本发明还提供一种组合物，所述组合物包含本发明提供的抗体分子或其片段、核酸分子、载体和/或宿主细胞。优选地，所述组合物为药物组合物，其可选地还包含药学上可接受的载体、辅料或赋形剂。
再一方面，本发明还提供所述抗体分子或其片段、核酸分子、载体、宿主细胞和/或组合物在制备预防或治疗癌症的药物中的用途。优选地，所述癌症为膀胱癌、乳腺癌、卵巢癌或肺癌。
另一方面，本发明还提供一种预防或治疗癌症的方法，所述方法包括给有此需要的受试者施用本发明的抗体分子或其片段、核酸分子、载体、宿主细胞和/或组合物。优选地，所述癌症为膀胱癌、乳腺癌、卵巢癌或肺癌。其中，所述受试者为哺乳类动物，更优选人。
再一方面，本发明还提供所述抗体分子或其片段、核酸分子、载体、宿主细胞和/或组合物在制备用于诊断癌症的试剂中的用途。优选地，所述癌症为膀胱癌、乳腺癌、卵巢癌或肺癌。
由此相应地，本发明还提供一种诊断癌症的方法，所述方法包括使本发明的抗体分子或其片段、核酸分子、载体、宿主细胞和/或组合物与来自受试者的样品相接触。优选地，所述癌症为膀胱癌、乳腺癌、卵巢癌或肺癌。其中，所述受试者为哺乳类动物，更优选人。
由此相应地，又一方面，本发明提供一种试剂盒，所述试剂盒包括本发明的抗体分子或其片段、核酸分子、载体、宿主细胞和/或组合物。所述试剂盒可以用于治疗或诊断用途。
再一方面，本发明还提供所述抗体分子或其片段在制备抗体药物偶联物中的用途。
由此相应地，本发明提供一种抗体药物偶联物，其由本发明的抗体分子或其片段与细胞毒性部分偶联形成。
优选地，所述细胞毒性部分为微管蛋白抑制剂、拓扑异构酶抑制剂或 DNA结合剂。优选地，所述微管蛋白抑制剂选自美登素衍生物、Monomethyl auristatin E(MMAE)、Monomethylauristatin F(MMAF)、Monomethyl Dolastatin 10、Tubulysin类衍生物、Cryptophycin类衍生物和Taltobulin。优选地，所述拓扑异构酶抑制剂选自阿霉素(Doxorubicin)代谢产物PNU-159682 衍生物和伊立替康(irinotecan，CPT-11)代谢产物SN38衍生物。优选地，所述DNA结合剂选自PBD类衍生物和Duocarmycin类衍生物。
相对于现有技术，本发明通过杂交瘤筛选和人源化技术，获得特异性结合Nectin-4的高亲和力抗体，其中通过人源化改造，获得全人抗体序列。并且，通过对该分子进行了理化性质和细胞学活性研究，确认得到了有效的临床先导药物分子序列。
附图说明
以下，结合附图来详细说明本发明的实施方案，其中：
图1显示了抗原表达细胞株的流式细胞术鉴定结果，其中小图1A： HT-1376膀胱癌细胞，小图1B：CHO-huNectin4 S8细胞。
图2显示了杂交瘤细胞培养上清的FACS结合活性测定结果，其中小图 2A：第一轮筛选，小图2B：第二轮筛选，小图2C：第三轮筛选。
图3显示了抗体与表达BT474细胞的FACS结合实验结果。
图4显示了抗体的BT474细胞内吞活性实验结果。
图5显示了抗体与表达huNectin4、muNectin4、cynoNectin4的不同细胞的FACS结合实验结果。
图6显示了抗体与Nectin蛋白质家族成员的结合实验结果，其中小图6A：Nectin-1，小图6B：Nectin-2，小图6C：Nectin-3，小图6D：Nectin-4。
图7显示了抗体的猴血清稳定性实验结果，其中小图7A：对照抗体 Enfortumab，小图7B：42D20 hz10。
图8显示了抗体的小鼠体内代谢情况，其中小图8A：对照抗体 Enfortumab，小图8B：42D20 hz10。
具体实施方式
以下参照具体的实施例来说明本发明。本领域技术人员能够理解，这些实施例仅用于说明本发明，其不以任何方式限制本发明的范围。
下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的药材原料、试剂材料等，如无特殊说明，均为市售购买产品。其中：
对照抗体Enfortumab的重链氨基酸序列示于SEQ ID NO：1，轻链氨基酸序列示于SEQ ID NO：2。
本发明的抗体序列见附表I至附表IV。
抗原NECTIN4重组蛋白(序列号：NP_002178.2，32aa-349aa)，示于 SEQ ID NO：3。
实施例1对照抗体的合成与表达
将全合成的Enfortumab抗体轻链可变区和重链可变区基因分别克隆至装有人-kappa轻链恒定区和人IgG1重链恒定区编码基因上游的真核表达载体pCDNA3.1中，获得Enfortumab轻、重链表达质粒，转入大肠杆菌扩增，分离获得大量含Enfortumab抗体轻链(SEQ ID NO：2)和重链(SEQ ID NO： 1)的质粒，将二者与聚乙烯亚胺(PEI)混合后共转染入HEK293细胞中。细胞转染5-6天，取培养上清，利用Mabselect亲和层析柱对表达上清进行纯化，获得Enfortumab抗体。
实施例2Nectin-4抗原表达细胞株的制备
通过PCR的方法，从含有Nectin-4cDNA的载体(北京义翘神州，Cat： HG19771-UT)中克隆出Nectin-4基因的阅读框，并将Nectin-4基因的阅读框通过酶切的方法克隆入含有谷氨酰氨合成酶(GS)筛选基因的稳定表达载体中，电转染(Nucleofector IIb，Lonza)悬浮培养的CHO-K1细胞，将转染后的细胞置于含有50μM MSX(Sigma，Cat：M5379)的CD CHOAGTTM 培养基(Gibco，Cat：12490-025)中，接种于96孔细胞培养板，37℃，5％CO2静置培养2-3周，通过MSX加压筛选，从镜下预筛获得9个细胞生长孔，并将其放大培养至24孔细胞培养板，最终通过流式细胞分析(FACS)挑选出抗原高表达克隆S8，进行放大培养和冻存。
将该高表达克隆S8命名为CHO-huNectin4 S8，该细胞与内源表达 Nectin-4的HT-1376膀胱癌细胞的比较鉴定结果见图1。
实施例3杂交瘤细胞的筛选和鉴定
1.小鼠免疫
10只8周龄Balb/c小鼠分成两组，分别采用传统免疫和快速免疫两个方法，其中传统免疫用CHO-huNectin4 S8工程细胞株为免疫剂，快速免疫用重组蛋白人Nectin4(购自近岸蛋白，Cat：CJ19)为免疫剂。
小鼠免疫前采血作为阴性对照。两种免疫剂分别腹腔注射，间隔2周进行第二、三次免疫，第三次免疫后一周采血测效价，选取效价高的小鼠在融合前3天进行冲刺免疫。
2.融合筛选
将SP20骨髓瘤细胞复苏、扩大培养至一定数量级。融合前一天给细胞换液，以保证融合时细胞生长状态良好。融合当天收集骨髓瘤细胞，离心后用基础培养基悬浮，计数待用。
分别无菌取小鼠的脾脏、淋巴结，研磨制备细胞悬液，细胞过滤器过滤，然后进行红细胞裂解，裂解后合并、计数。按B细胞与SP20骨髓瘤细胞的数量比为1∶2进行混合，离心后用电融合液洗两遍混合的细胞，再重悬细胞并将密度调整至1-2×107/ml左右。将细胞悬液加入电击杯进行融合，融合后加入到完整培养基中，放入到37℃、8％CO2培养箱中恢复30-240分钟，然后加入HAT培养基铺至384孔板中培养，第5天补液，第7天换成HT培养基，第8-10天进行如下的阳性杂交瘤筛选。
取杂交瘤细胞培养上清用FACS进行分析，筛选得到能够结合细胞表面稳定表达人Nectin4抗原的CHO-huNECTIN4 S8细胞，同时不结合空白 CHOK1细胞的阳性孔。通过有限稀释法将筛选到的阳性孔单细胞化，待连续两次亚克隆的阳性都为100％时结束亚克隆，得到的杂交瘤细胞株只分泌一个抗体。
杂交瘤细胞培养上清的FACS结合活性测定结果见图2和表1至表3。
表1.第一轮筛选出的克隆
ID FACS FACS(*10<sup>5</sup>)
5M21 1731598.512 173.1599
7I18 900458.7143 90.04587
26G13 493254.2857 49.32543
29B12 685057.4296 68.50574
30J3 440992.4 44.09924
表2.第二轮筛选出的克隆
ID FACS FACS(*10<sup>5</sup>)
4A15 221093.1035 22.1
21N4 264347.4081 26.4
28J3 208131.2025 20.8
32A13 677419.4598 67.7
38D17 266536.8786 26.7
41O24 1749182.695 174.9
42D20 533378.824 53.3
45F2 393898.7755 39.4
49C23 1510386.33 151.0
50I15 1299424.85 129.9
表3.第三轮筛选出的克隆
ID FACS FACS(*10<sup>5</sup>)
4E9 180199.602 180.199
9M13 448161.147 44.816
12C7 639255.386 63.926
16E10 515026.703 51.503
20M12 783128.784 78.313
30L7 505457.216 50.546
30L18 284554.941 28.455
得到的鼠源单克隆抗体以杂交瘤细胞株ID命名。
实施例4鼠源单克隆抗体的可变区序列鉴定
将分泌抗人Nectin-4抗体的杂交瘤细胞扩大培养后，按照RNAfast200 试剂盒(上海飞捷生物技术有限公司)说明书步骤提取细胞总RNA；利用 5×PrimeScript RT MasterMix(Takara)将杂交瘤细胞总RNA反转录成cDNA；使用简并引物(Anke Krebber.1997)和Extaq PCR试剂(Takara)扩增抗体轻链可变区IgVL(κ)和重链可变区VH序列；利用PCRclean-up Gel extraction 试剂盒(Macherey-Nagel公司)纯化PCR扩增产物；按照pClone007 Simple Vector Kit试剂盒(擎科生物科技有限公司)说明书将扩增PCR产物连接至 T载体并转化大肠杆菌感受态细胞，菌株扩增、抽提质粒后进行DNA测序获得单克隆抗体可变区序列。
实施例5嵌合抗体的制备
将鼠源抗人Nectin-4单克隆抗体的重链可变区序列和公开发表的人单克隆抗体IgG1亚类的重链恒定区序列(SEQ ID NO：4)拼接在一起，构建到哺乳动物细胞表达载体中；将鼠源抗人Nectin-4单克隆抗体的轻链可变区序列和公开发表的人单克隆抗体κ亚类的轻链恒定区序列(SEQ ID NO：5)拼接在一起，构建到哺乳动物细胞表达载体中。构建好的抗人Nectin-4嵌合抗体的重链载体和轻链载体配对混合，使用PEI转染HEK293细胞，约7天后收集细胞上清，使用Mabselect纯化得到抗人NECTIN4嵌合抗体蛋白。
得到的嵌合抗体在本文以“鼠源抗体命名xiIgG”表示。
实施例6鼠源抗体的人源化与人源化抗体制备
综合抗体编码方案，确定鼠源抗体的重链和轻链的6个抗原互补决定簇 (CDR)的氨基酸序列区域及支撑抗体保守三维构象的框架区域(framework region)。随后通过分析搜索已知人源抗体序列，选择与鼠源抗体最为相似接近的人源抗体重链可变区序列，如IGHV1|IGHJ4*01，选择其抗体框架区序列作为模板，将鼠源抗体重链CDR与人源抗体框架区结合，最终生成人源化抗体重链可变区序列。同样过程，生成人源化抗体轻链可变区序列。
鼠源抗体CDR直接移植至人框架区的抗体常出现结合活性急剧下降，因此需要将框架区个别氨基酸从人源的改回鼠源的。确定回复突变位点，一是对照设计好的人源化抗体序列和原始的鼠源抗体序列，检查有哪些氨基酸的不同；二是检查这些氨基酸是否对支持抗体结构起重要作用或者对与抗原的结合起重要作用。人源化设计后的序列的同时需要检查是否有一些潜在的翻译后修饰位点，如N(天冬酰胺)糖基化位点、N脱酰胺化位点、D(天冬氨酸)异构化位点等。
将人源化的重链可变区和轻链可变区两两组合，参考实施例5所述的嵌合抗体的制备，获得人源化抗体。将人源化抗体命名为“鼠源抗体命名 hzmn”，其中m和n分别为鼠源抗体VH和VL的人源化(hz)改造序列(VH_hz 和VL_hz)编号。
实施例7抗体药物缀合物(ADC)的制备
在pH为7.4的PBS中，2.0-2.6当量TECP还原抗体2小时后，将vcMMAE 的DMA溶液加入到还原后的抗体溶液中(vcMMAE和抗体的摩尔比为6： 1)，2-8℃搅拌1小时后超滤换液除去DMA和小分子残留。紫外分光光度计测定偶联物在248nm～280nm处的吸光值，计算得到偶联物的浓度。样品分装到冻存管中-80℃保存。样品的DAR值(4.0±1)由HPLC-HIC测定。
将抗体对应的ADC以“抗体命名-E”来命名。
实施例8体外细胞结合实验
将抗人Nectin-4对照抗体Enfortumumab、本发明的抗体或ADC从 100nM的起始浓度开始做2倍的梯度倍比稀释，共16个浓度点，各个浓度点的抗体取10ul加入384孔板。
100g室温离心5分钟收集细胞表面表达Nectin-4的BT474细胞(乳腺癌细胞)，用含0.5％BSA的PBS洗涤细胞一次，100g室温离心5分钟，重悬细胞为密度约2x106个细胞/ml，取10ul加入到已加抗体的384孔板的孔中。4℃孵育1小时后，加入荧光标记的羊抗人IgG二抗。继续于4℃孵育1 小时后，用流式细胞仪分析细胞群的平均荧光读值。
本发明的鼠抗分子与BT474细胞的FACS结合实验结果见图3中的图3A 和表4、表5。
表4.鼠源单克隆抗体与BT474细胞的结合
Figure BDA0002460638400000141
Figure BDA0002460638400000151
表5.鼠源单克隆抗体与BT474细胞的结合
Figure BDA0002460638400000152
本发明的人源化改造分子与BT474细胞的FACS结合实验结果见图3中的图3B、图3C和表6、表7。
表6.人源化抗体与BT474细胞的结合
Figure BDA0002460638400000153
表7.人源化抗体与BT474细胞的结合
Figure BDA0002460638400000154
Figure BDA0002460638400000161
本发明的ADC与BT474细胞的FACS结合实验结果见图3中的图3D和表 8。
表8.抗体药物缀合物与BT474细胞的结合
Figure BDA0002460638400000162
实施例9抗体的体外细胞学实验
9.1BT474细胞内吞实验
1.收集BT474细胞，1200rpm离心8分钟，DPBS(Gibco Cat.：14190-136) 洗两次。
2.1E5每孔种细胞，抗体从10ug/ml浓度起，按照1∶2浓度梯度稀释做7 个点，最后一点为空白孔，将混合物冰上孵育1h。
3.冰PBS洗两次，1200rpm离心8分钟，用补充L-glutamine和HEPES 的RPMI 1640培养基重悬细胞，根据时间点等量分成相应的份数，置于37℃孵育不同的时间段，其中一份一直置于冰上，做0点无内吞对照，设未加抗体组做NC对照。
4.用pH2.7的柠檬酸洗3.5分钟，用1M pH9.5的Tris-HCl溶液中和，用PBS洗两次，用适量的1％BSA-PBS重悬，上IQplus仪器检测。
5.用GraphPad Prism软件分析处理数据。
本发明的鼠抗分子的BT474细胞内吞活性实验结果见图4中的图4A和表 9。
表9.鼠源单克隆抗体的BT474细胞内吞活性
内吞效率(％)
Enfortumab 63
mIgG 5M21 69
mIgG 26G13 37
mIgG 29B12 57
mIgG 30J3 56
mIgG 7118 66
同型对照 16
本发明的人源化改造分子的BT474细胞内吞活性实验结果见图4中的图 4B、4C和表10、表11。
表10.人源化抗体的BT474细胞内吞活性
内吞效率(％)
Enfortumab 62.4
5M21 xiIgG 56.9
5M21 hz00 54.8
5M21 hz01 62.2
5M21 hz10 64.8
5M21 hz11 64.4
表11.人源化抗体的BT474细胞内吞活性
Figure BDA0002460638400000171
Figure BDA0002460638400000181
本发明的ADC的BT474细胞内吞活性实验结果见图4中的图4D和表 12。
表12.抗体药物缀合物的BT474细胞内吞活性
内吞效率(％)
Enfortumab-E 58.9
42D20hz10-E 74
42D20hz43-E 68.9
42D20hz44-E 71.1
42D20hz63-E 67.9
42D20hz64-E 66.6
20M12xiIgG-E 67
20M12hz01-E 56.6
20M12hz11-E 58.4
同型对照 --
9.2 BT474细胞增殖抑制试验
培养表达Nectin4的乳腺癌细胞BT474，胰蛋白酶消化收集细胞，400g 离心5分钟，弃上清。按照4000细胞每孔的密度种板，37℃，5％CO2培养 24小时。待测抗体溶于含1％BSA的培养基，起始浓度为200ug/ml，3倍稀释，9个稀释浓度，包含0浓度，加100ul每个浓度的抗体至96孔板。将100 ul稀释好的抗体样品加100ul细胞混合至96孔板中，37℃，5％CO2孵育120 小时。使用含1％BSA的培养基配制浓度为5uM的CCK-8，加20ul CCK-8 至96孔板中，37℃，5％CO2孵育4小时。最后取出细胞板室温静置15min 后，将细胞板充分振荡混匀。以450nm为检测波长读板。以裸抗和ADC样品工作浓度(ng/ml)为X轴，测得吸光度值为Y轴，用SoftMax Pro进行四参数拟合，得到裸抗样品与ADC样品的EC50值。
结果见表13、表14和表15。
表13.鼠抗的BT474细胞增殖抑制活性
EC50(ng/ml) 浓度最高点细胞杀伤效果百分比
Enfortumab 3.367 43.3％
mIgG 5M21 42.34 33.5％
mIgG 29B12 387.0 33.2％
mIgG 30J3 16.88 52.8％
同型对照 N/A N/A
表14.ADC的BT474细胞增殖抑制活性
Figure BDA0002460638400000191
表15.ADC的BT474细胞增殖抑制活性
Figure BDA0002460638400000192
Figure BDA0002460638400000201
实施例10抗体交叉抗原活性分析-Facs
细胞：
CHO-huNectin4 S8；
表达鼠Nectin4(NP_082169)的HEK293细胞：HEK-muNectin4；和
表达Cyno Nectin4(SEQ ID NO：6)的HEK293细胞：HEK-cynoNectin4。
参照实施例8所述进行实验，结果分别见图5的图5A、5B和5C，以及表 16、表17和表18。
表16.人源化抗体与CHO-huNectin4 S8细胞的结合
Figure BDA0002460638400000202
表17.人源化抗体与HEK-muNectin4细胞的结合
Figure BDA0002460638400000203
表18.人源化抗体与HEK-cynoNectin4细胞的结合
Figure BDA0002460638400000204
实施例11抗体的体外结合亲和力和动力学实验
采用GE公司BIAcore仪器S200测定抗体抗原相互作用力。参考GE公司BiotinCAPture Kit操作说明，首先在传感芯片CAP分析通道和对照样品通道都偶联His标签的人NECTIN4抗原，然后在分析通道和对照样品通道一起流过梯度稀释的抗体样品(起始浓度20nM，1∶3稀释8个浓度点，并且设定0.741nm浓度点重复)，测定抗体抗原结合后发生的光反应值。经仪器软件拟合分析(1∶1结合模式)，最终得到抗体的结合常数Kon和解离常数Koff，以及亲和力常数KD。
结果见表19。
表19.抗体的结合亲和力和动力学结果
ka(1/Ms) kd(1/s) KD(M)
Enfortumab 1.12E+06 5.52E-03 4.94E-09
42D20xiIgG 7.90E+05 7.98E-04 1.01E-09
42D20hz10 8.31E+05 9.46E-04 1.14E-09
42D20hz11 1.19E+06 1.15E-03 9.60E-10
42D20hz13 1.19E+06 1.08E-03 9.04E-10
42D20hz63 8.73E+05 1.57E-03 1.80E-09
实施例12抗体与同家族抗原蛋白的交叉结合活性验证
抗原：
human Nectin-1(C-6His)Novoprotein Cat#C492
human Nectin-2(C-6His)Novoprotein Cat#C440
human Nectin-3(C-6His)Novoprotein Cat#C630
human Nectin-4(C-6His)Novoprotein Cat#CJ19
抗体(一抗)：本发明的抗体；
对照抗体；
CD111/Nectin-1/PVRL1 Antibody，Rabbit Fab，80244-RP01-100，Sinobiologics.
Anti-Nectin 2antibody(ab233085)，Rabbit antibody，Abcam.
Anti-Nectin 3antibody(ab137961)，Rabbit antibody，Abcam.
二抗：
Goat anti-Rabbit IgG-Fc Secondary antibody(HRP)Cat#SSA003，JacksonImmuno.
用1ug/ml抗原包板，4℃过夜孵育，然后依次加入梯度稀释的抗体，最后加入HRP标记的二抗来检测A450的吸收值。结果见图6的图6A、6B、6C 和6D。
从结果可知，本发明的抗体和对照抗体表现的性质相一致，都能特异性识别Nectin-4抗原，有剂量依赖的结合效应，而且并不与Nectin-4同家族的其他蛋白有交叉结合活性。
实施例13抗体体外猴血清稳定性研究
实验材料：待测抗体，FBS，待测抗体结合抗原，anti-huIgG Fab单克隆抗体(Sigma，I5260-1ML)，HRP标记山羊抗人IgG二抗(Jackson， code：109-035-098)。
实验仪器：37℃培养箱，酶标仪。
实验步骤：
样品制备：
1)待测抗体调浓度至20ug/ml，过滤除菌，分装为250ul/管备用；
2)猴血清等体积加入分装好的待测抗体，即终浓度为50％血清浓度， 10ug/ml抗体浓度；
3)共制备7个样品，封口膜封口，放置于37℃，全程需保持无菌；
4)分别于第0天、3天、7天、10天、14天、21天取样放置于4℃保存待测，其中第21天取样应至少在4℃放置一天。
检测方法：
1)分别PBS包被结合抗原、anti-IgG Fab单克隆抗体于96孔酶联板， 0.2ug/ml，100ul/孔，4℃过夜；
2)配置所需试剂：
封闭液5％BSA+PBS
抗体稀释液5％BSA+PBS+50％FBS
酶联板洗涤液0.1％Tween+PBS
3)包被好的酶联板PBS清洗三遍，300ul/孔，洗去游离的未包被抗原；
4)加入封闭液，200ul/孔，37℃封闭1h；
5)待测抗体使用抗体稀释液稀释至2ug/ml，并3倍稀释共8个梯度；
6)倒掉封闭液，两种包被方法的酶联板分别加入稀释好的抗体，100ul/ 孔，于37℃孵育1h；
7)PBST洗板3遍；
8)1∶5000稀释二抗，加入洗涤好的酶联板中，100ul/孔，37℃孵育40min；
9)PBST洗板3遍；
10)TMB显色，100ul/孔，避光10min；
11)加入50ul 2M HCL终止，450nm读数。
结果处理：
根据ELISA显色值制定结合曲线，观察放置不同时间结合曲线的变化，评估放置后抗体结合活性稳定性。结果见图7的图7A、7B。
结果证明本发明抗体在37℃孵育21天以后，有效抗体含量没有任何变化，即能够在37℃条件下稳定存在21天以上。
实施例14抗体小鼠体内药物代谢分析
实验材料：待测抗体，小鼠不同时间点采集血清，待测抗体的结合抗原人Nectin-4，anti-huIgG Fab单克隆抗体(Sigma，I5260-1ML)，HRP标记山羊抗人IgG二抗(Jackson，code：109-035-098)。
实验方法：
血清收集：
1)雌性Balb/C小鼠，3只/组，分别于尾静脉或者腹腔给药，200ug/只；
2)按照实验设计时间点尾静脉采血，收集血样，室温放置30min以上， 4000rpm，15min收集血清，-20℃保存，为防止血清蒸发，最后血清收集体积尽量大于20ul；
3)最后一次收集血清至少于-20℃冻存24h。
检测方法：
1)分别PBS包被结合抗原、anti-IgG Fab单克隆抗体于96孔酶联板， 0.2ug/ml，100ul/孔，4℃过夜；
2)配制所需试剂：
封闭液5％BSA+PBS
抗体稀释液5％BSA+PBS+20％空白小鼠血清
酶联板洗涤液0.1％Tween+PBS
3)包被好的酶联板PBS清洗三遍，300ul/孔；
4)加入封闭液，200ul/孔，37℃封闭1h；
5)起始血清使用封闭液稀释至合适浓度，用抗体稀释液稀释至合适的浓度范围，具体浓度的稀释倍数需根据预实验来调整，原则上使检测的血清最终显色值在标准品显色值范围内；
6)抗体稀释液稀释抗体标准品，标准品的稀释仍然根据预实验来调整，使标准品拟合出线性曲线(如有合适软件，也可拟合出S形曲线)。
7)倒掉酶联板中的封闭液，两种包被方法的酶联板分别加入稀释好的抗体标准品与待测血清，100ul/孔，于37℃孵育1h；
8)PBST洗板3遍；
9)1∶5000稀释二抗，加入洗涤好的酶联板中，100ul/孔，37℃孵育40min；
10)PBST洗板3遍；
11)TMB显色，100ul/孔，避光10min；
12)加入50ul 2M HCL终止，450nm读数。
结果见图8的图8A、8B和表20。
表20.抗体的小鼠体内代谢结果
Figure BDA0002460638400000241
浓度：ug/ml
NA：低于检测下限，检测下限为9.77ng/ml。
以上对本发明具体实施方式的描述并不限制本发明，本领域技术人员可以根据本发明作出各种改变或变形，只要不脱离本发明的精神，均应属于本发明所附权利要求的范围。
Figure BDA0002460638400000251
Figure BDA0002460638400000261
Figure BDA0002460638400000271
Figure BDA0002460638400000281
Figure BDA0002460638400000291
Figure BDA0002460638400000301
Figure BDA0002460638400000311
Figure BDA0002460638400000321
Figure BDA0002460638400000331
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100 105
<210> 9
<211> 116
<212> PRT
<213> 人工序列(artificial sequence)
<400> 9
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 Phe Thr Thr Tyr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met
35 40 45
Gly Trp Ile Tyr Pro Gly Asn Val Asn Thr Lys Tyr Asn Glu Lys Phe
50 55 60
Lys Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala 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 Gly Leu Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser
115
<210> 10
<211> 116
<212> PRT
<213> 人工序列(artificial sequence)
<400> 10
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 Phe Thr Thr Tyr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met
35 40 45
Gly Trp Ile Tyr Pro Gly Asn Val Asn Thr Lys Tyr Asn Glu Lys Phe
50 55 60
Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Ala 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 Gly Leu Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser
115
<210> 11
<211> 107
<212> PRT
<213> 人工序列(artificial sequence)
<400> 11
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 Lys Ala Ser Gln Ser Val Ser Asn Asp
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile
35 40 45
Tyr Tyr Ala Ser Asn Arg Tyr 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 Val Ala Thr Tyr Tyr Cys Gln Gln Asp Tyr Ser Ser Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105
<210> 12
<211> 107
<212> PRT
<213> 人工序列(artificial sequence)
<400> 12
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 Lys Ala Ser Gln Ser Val Ser Asn Asp
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile
35 40 45
Tyr Tyr Ala Ser Asn Arg Tyr 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 Leu Ala Thr Tyr Phe Cys Gln Gln Asp Tyr Ser Ser Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105
<210> 13
<211> 119
<212> PRT
<213> 人工序列(artificial sequence)
<400> 13
Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln
1 5 10 15
Ser Leu Ser Ile Ser Cys Thr Val Ser Gly Phe Ser Leu Ile Asp Tyr
20 25 30
Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Gly Val Ile Trp Gly Asp Gly Lys Ile Tyr Tyr Asn Ser Val Leu Lys
50 55 60
Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu
65 70 75 80
Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Met Tyr Tyr Cys Ala
85 90 95
Lys Gln Gly Gly Leu Leu Phe Tyr Ala Met Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Ser Val Thr Val Ser Ser
115
<210> 14
<211> 113
<212> PRT
<213> 人工序列(artificial sequence)
<400> 14
Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ala Met Ser Val Gly
1 5 10 15
Gln Arg Val Thr Met Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser
20 25 30
Tyr Ser Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Ser Pro Lys Leu Leu Ile Tyr Phe Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Ile Gly Ser Gly Ser Glu Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln
85 90 95
His Tyr Asn Thr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile
100 105 110
Lys
<210> 15
<211> 119
<212> PRT
<213> 人工序列(artificial sequence)
<400> 15
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ile Asp Tyr
20 25 30
Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Gly Val Ile Trp Gly Asp Gly Lys Ile Tyr Tyr Asn Ser Val Leu Lys
50 55 60
Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu
65 70 75 80
Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Gln Gly Gly Leu Leu Phe Tyr Ala Met Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
<210> 16
<211> 119
<212> PRT
<213> 人工序列(artificial sequence)
<400> 16
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ile Asp Tyr
20 25 30
Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Gly Val Ile Trp Gly Asp Gly Lys Ile Tyr Tyr Asn Ser Val Leu Lys
50 55 60
Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Ser Leu
65 70 75 80
Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Lys Gln Gly Gly Leu Leu Phe Tyr Ala Met Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
<210> 17
<211> 119
<212> PRT
<213> 人工序列(artificial sequence)
<400> 17
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ile Asp Tyr
20 25 30
Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Gly Val Ile Trp Gly Glu Gly Lys Ile Tyr Tyr Asn Ser Val Leu Lys
50 55 60
Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Ser Leu
65 70 75 80
Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Lys Gln Gly Gly Leu Leu Phe Tyr Ala Met Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
<210> 18
<211> 119
<212> PRT
<213> 人工序列(artificial sequence)
<400> 18
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ile Asp Tyr
20 25 30
Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Gly Val Ile Trp Gly Asp Gly Lys Ile Tyr Tyr Asn Ser Val Leu Lys
50 55 60
Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Ser Leu
65 70 75 80
Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Gln Gly Gly Leu Leu Phe Tyr Ala Met Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
<210> 19
<211> 119
<212> PRT
<213> 人工序列(artificial sequence)
<400> 19
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ile Asp Tyr
20 25 30
Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Gly Val Ile Trp Gly Asp Ala Lys Ile Tyr Tyr Asn Ser Val Leu Lys
50 55 60
Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Ser Leu
65 70 75 80
Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Lys Gln Gly Gly Leu Leu Phe Tyr Ala Met Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
<210> 20
<211> 119
<212> PRT
<213> 人工序列(artificial sequence)
<400> 20
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ile Asp Tyr
20 25 30
Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Gly Val Ile Trp Gly Gly Asp Lys Ile Tyr Tyr Asn Ser Val Leu Lys
50 55 60
Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Ser Leu
65 70 75 80
Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Lys Gln Gly Gly Leu Leu Phe Tyr Ala Met Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
<210> 21
<211> 119
<212> PRT
<213> 人工序列(artificial sequence)
<400> 21
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ile Asp Tyr
20 25 30
Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Gly Val Ile Trp Gly Gly Gly Lys Ile Tyr Tyr Asn Ser Val Leu Lys
50 55 60
Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Ser Leu
65 70 75 80
Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Lys Gln Gly Gly Leu Leu Phe Tyr Ala Met Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
<210> 22
<211> 114
<212> PRT
<213> 人工序列(artificial sequence)
<400> 22
Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser
20 25 30
Tyr Ser Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Pro Pro Lys Leu Leu Ile Tyr Phe Ala Ser Thr Arg Glu Ser Gly Val
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
His Tyr Asn Thr Pro Phe Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu
100 105 110
Lys Arg
<210> 23
<211> 114
<212> PRT
<213> 人工序列(artificial sequence)
<400> 23
Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser
20 25 30
Tyr Ser Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Ser Pro Lys Leu Leu Ile Tyr Phe Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Ser Glu Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Ala Glu Asp Leu Ala Val Tyr Phe Cys Gln Gln
85 90 95
His Tyr Asn Thr Pro Phe Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu
100 105 110
Lys Arg
<210> 24
<211> 114
<212> PRT
<213> 人工序列(artificial sequence)
<400> 24
Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser
20 25 30
Tyr Ser Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Ser Pro Lys Leu Leu Ile Tyr Phe Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Ser Glu Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Ala Glu Asp Leu Ala Val Tyr Phe Cys Gln Gln
85 90 95
His Tyr Asn Thr Pro Phe Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu
100 105 110
Lys Arg
<210> 25
<211> 114
<212> PRT
<213> 人工序列(artificial sequence)
<400> 25
Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Thr
20 25 30
Tyr Ser Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Ser Pro Lys Leu Leu Ile Tyr Phe Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Ser Glu Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Ala Glu Asp Leu Ala Val Tyr Phe Cys Gln Gln
85 90 95
His Tyr Asn Thr Pro Phe Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu
100 105 110
Lys Arg
<210> 26
<211> 114
<212> PRT
<213> 人工序列(artificial sequence)
<400> 26
Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Lys
20 25 30
Tyr Ser Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Pro Pro Lys Leu Leu Ile Tyr Phe Ala Ser Thr Arg Glu Ser Gly Val
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
His Tyr Asn Thr Pro Phe Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu
100 105 110
Lys Arg
<210> 27
<211> 116
<212> PRT
<213> 人工序列(artificial sequence)
<400> 27
Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Arg Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Tyr Ile His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Trp Ile Tyr Pro Gly Asn Ala Asn Asn Lys Tyr Asn Glu Asn Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys
85 90 95
Ala Arg Ser Val Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu
100 105 110
Thr Val Ser Ser
115
<210> 28
<211> 107
<212> PRT
<213> 人工序列(artificial sequence)
<400> 28
Ser Val Val Met Thr Gln Thr Pro Lys Phe Leu Leu Val Ser Ala Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Ser Asn Asp
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile
35 40 45
Tyr Tyr Ala Ser Asn Arg Asn Thr Gly Val Pro Asp Arg Phe Thr Gly
50 55 60
Ser Gly Tyr Gly Thr Asp Phe Thr Phe Asn Ile Ser Thr Val Gln Ala
65 70 75 80
Glu Asp Leu Ala Val Tyr Phe Cys Gln Gln Asp Tyr Ser Ser Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 29
<211> 116
<212> PRT
<213> 人工序列(artificial sequence)
<400> 29
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 Phe Thr Ser Tyr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met
35 40 45
Gly Trp Ile Tyr Pro Gly Asn Ala Asn Asn Lys Tyr Asn Glu Asn Phe
50 55 60
Lys Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala 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 Ser Val Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser
115
<210> 30
<211> 116
<212> PRT
<213> 人工序列(artificial sequence)
<400> 30
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 Phe Thr Ser Tyr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met
35 40 45
Gly Trp Ile Tyr Pro Gly Asn Ala Asn Asn Lys Tyr Asn Glu Asn Phe
50 55 60
Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Ala 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 Ser Val Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser
115
<210> 31
<211> 107
<212> PRT
<213> 人工序列(artificial sequence)
<400> 31
Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Lys Ala Ser Gln Ser Val Ser Asn Asp
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Tyr Ala Ser Asn Arg Asn Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Asp Tyr Ser Ser Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105
<210> 32
<211> 107
<212> PRT
<213> 人工序列(artificial sequence)
<400> 32
Glu Val Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Lys Ala Ser Gln Ser Val Ser Asn Asp
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Tyr Ala Ser Asn Arg Asn Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser
65 70 75 80
Glu Asp Leu Ala Val Tyr Phe Cys Gln Gln Asp Tyr Ser Ser Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105
<210> 33
<211> 117
<212> PRT
<213> 人工序列(artificial sequence)
<400> 33
Glu Val Gln Leu Gln Gln Ser Gly Pro Asp Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr
20 25 30
Tyr Met His Trp Val Lys Gln Ser Arg Gly Lys Gly Leu Glu Trp Ile
35 40 45
Gly Arg Val Asn Pro Asn Asn Gly Asn Thr Leu Tyr Asn Gln Lys Phe
50 55 60
Arg Gly Lys Ala Ile Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Thr Ser Asp Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Asp Arg Tyr Ala Phe Ala Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ala
115
<210> 34
<211> 111
<212> PRT
<213> 人工序列(artificial sequence)
<400> 34
Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Ser Val Ser Thr Ser
20 25 30
Ser Tyr Thr Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Lys Tyr Ala Ser Asn Leu Glu Ser Gly Val Pro Ala
50 55 60
Arg Phe Ile Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His
65 70 75 80
Pro Val Glu Glu Glu Asp Thr Ala Thr Tyr Tyr Cys Gln His Thr Trp
85 90 95
Glu Ile Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 35
<211> 7
<212> PRT
<213> 人工序列(artificial sequence)
<400> 35
Gly Tyr Thr Phe Thr Thr Tyr
1 5
<210> 36
<211> 10
<212> PRT
<213> 人工序列(artificial sequence)
<400> 36
Gly Tyr Thr Phe Thr Thr Tyr Tyr Ile His
1 5 10
<210> 37
<211> 5
<212> PRT
<213> 人工序列(artificial sequence)
<400> 37
Thr Tyr Tyr Ile His
1 5
<210> 38
<211> 6
<212> PRT
<213> 人工序列(artificial sequence)
<400> 38
Thr Thr Tyr Tyr Ile His
1 5
<210> 39
<211> 6
<212> PRT
<213> 人工序列(artificial sequence)
<400> 39
Tyr Pro Gly Asn Val Asn
1 5
<210> 40
<211> 10
<212> PRT
<213> 人工序列(artificial sequence)
<400> 40
Trp Ile Tyr Pro Gly Asn Val Asn Thr Lys
1 5 10
<210> 41
<211> 17
<212> PRT
<213> 人工序列(artificial sequence)
<400> 41
Trp Ile Tyr Pro Gly Asn Val Asn Thr Lys Tyr Asn Glu Lys Phe Lys
1 5 10 15
Gly
<210> 42
<211> 13
<212> PRT
<213> 人工序列(artificial sequence)
<400> 42
Trp Ile Gly Trp Ile Tyr Pro Gly Asn Val Asn Thr Lys
1 5 10
<210> 43
<211> 7
<212> PRT
<213> 人工序列(artificial sequence)
<400> 43
Gly Leu Tyr Tyr Phe Asp Tyr
1 5
<210> 44
<211> 8
<212> PRT
<213> 人工序列(artificial sequence)
<400> 44
Ala Arg Gly Leu Tyr Tyr Phe Asp
1 5
<210> 45
<211> 11
<212> PRT
<213> 人工序列(artificial sequence)
<400> 45
Lys Ala Ser Gln Ser Val Ser Asn Asp Val Ala
1 5 10
<210> 46
<211> 7
<212> PRT
<213> 人工序列(artificial sequence)
<400> 46
Ser Asn Asp Val Ala Trp Tyr
1 5
<210> 47
<211> 7
<212> PRT
<213> 人工序列(artificial sequence)
<400> 47
Tyr Ala Ser Asn Arg Tyr Thr
1 5
<210> 48
<211> 10
<212> PRT
<213> 人工序列(artificial sequence)
<400> 48
Leu Leu Ile Tyr Tyr Ala Ser Asn Arg Tyr
1 5 10
<210> 49
<211> 9
<212> PRT
<213> 人工序列(artificial sequence)
<400> 49
Gln Gln Asp Tyr Ser Ser Pro Tyr Thr
1 5
<210> 50
<211> 8
<212> PRT
<213> 人工序列(artificial sequence)
<400> 50
Gln Gln Asp Tyr Ser Ser Pro Tyr
1 5
<210> 51
<211> 7
<212> PRT
<213> 人工序列(artificial sequence)
<400> 51
Gly Phe Ser Leu Ile Asp Tyr
1 5
<210> 52
<211> 10
<212> PRT
<213> 人工序列(artificial sequence)
<400> 52
Gly Phe Ser Leu Ile Asp Tyr Gly Val Ser
1 5 10
<210> 53
<211> 5
<212> PRT
<213> 人工序列(artificial sequence)
<400> 53
Asp Tyr Gly Val Ser
1 5
<210> 54
<211> 6
<212> PRT
<213> 人工序列(artificial sequence)
<400> 54
Ile Asp Tyr Gly Val Ser
1 5
<210> 55
<211> 5
<212> PRT
<213> 人工序列(artificial sequence)
<400> 55
Trp Gly Asp Gly Lys
1 5
<210> 56
<211> 9
<212> PRT
<213> 人工序列(artificial sequence)
<400> 56
Val Ile Trp Gly Asp Gly Lys Ile Tyr
1 5
<210> 57
<211> 16
<212> PRT
<213> 人工序列(artificial sequence)
<400> 57
Val Ile Trp Gly Asp Gly Lys Ile Tyr Tyr Asn Ser Val Leu Lys Ser
1 5 10 15
<210> 58
<211> 12
<212> PRT
<213> 人工序列(artificial sequence)
<400> 58
Trp Leu Gly Val Ile Trp Gly Asp Gly Lys Ile Tyr
1 5 10
<210> 59
<211> 11
<212> PRT
<213> 人工序列(artificial sequence)
<400> 59
Gln Gly Gly Leu Leu Phe Tyr Ala Met Asp Tyr
1 5 10
<210> 60
<211> 12
<212> PRT
<213> 人工序列(artificial sequence)
<400> 60
Ala Lys Gln Gly Gly Leu Leu Phe Tyr Ala Met Asp
1 5 10
<210> 61
<211> 17
<212> PRT
<213> 人工序列(artificial sequence)
<400> 61
Lys Ser Ser Gln Ser Leu Leu Asn Ser Tyr Ser Gln Lys Asn Tyr Leu
1 5 10 15
Ala
<210> 62
<211> 13
<212> PRT
<213> 人工序列(artificial sequence)
<400> 62
Leu Asn Ser Tyr Ser Gln Lys Asn Tyr Leu Ala Trp Tyr
1 5 10
<210> 63
<211> 7
<212> PRT
<213> 人工序列(artificial sequence)
<400> 63
Phe Ala Ser Thr Arg Glu Ser
1 5
<210> 64
<211> 10
<212> PRT
<213> 人工序列(artificial sequence)
<400> 64
Leu Leu Ile Tyr Phe Ala Ser Thr Arg Glu
1 5 10
<210> 65
<211> 9
<212> PRT
<213> 人工序列(artificial sequence)
<400> 65
Gln Gln His Tyr Asn Thr Pro Phe Thr
1 5
<210> 66
<211> 8
<212> PRT
<213> 人工序列(artificial sequence)
<400> 66
Gln Gln His Tyr Asn Thr Pro Phe
1 5
<210> 67
<211> 17
<212> PRT
<213> 人工序列(artificial sequence)
<400> 67
Lys Ser Ser Gln Ser Leu Leu Asn Thr Tyr Ser Gln Lys Asn Tyr Leu
1 5 10 15
Ala
<210> 68
<211> 5
<212> PRT
<213> 人工序列(artificial sequence)
<400> 68
Trp Gly Asp Ala Lys
1 5
<210> 69
<211> 5
<212> PRT
<213> 人工序列(artificial sequence)
<400> 69
Trp Gly Gly Gly Lys
1 5
<210> 70
<211> 7
<212> PRT
<213> 人工序列(artificial sequence)
<400> 70
Gly Tyr Thr Phe Thr Ser Tyr
1 5
<210> 71
<211> 10
<212> PRT
<213> 人工序列(artificial sequence)
<400> 71
Gly Tyr Thr Phe Thr Ser Tyr Tyr Ile His
1 5 10
<210> 72
<211> 5
<212> PRT
<213> 人工序列(artificial sequence)
<400> 72
Ser Tyr Tyr Ile His
1 5
<210> 73
<211> 6
<212> PRT
<213> 人工序列(artificial sequence)
<400> 73
Thr Ser Tyr Tyr Ile His
1 5
<210> 74
<211> 6
<212> PRT
<213> 人工序列(artificial sequence)
<400> 74
Tyr Pro Gly Asn Ala Asn
1 5
<210> 75
<211> 10
<212> PRT
<213> 人工序列(artificial sequence)
<400> 75
Trp Ile Tyr Pro Gly Asn Ala Asn Asn Lys
1 5 10
<210> 76
<211> 17
<212> PRT
<213> 人工序列(artificial sequence)
<400> 76
Trp Ile Tyr Pro Gly Asn Ala Asn Asn Lys Tyr Asn Glu Asn Phe Lys
1 5 10 15
Gly
<210> 77
<211> 13
<212> PRT
<213> 人工序列(artificial sequence)
<400> 77
Trp Ile Gly Trp Ile Tyr Pro Gly Asn Ala Asn Asn Lys
1 5 10
<210> 78
<211> 7
<212> PRT
<213> 人工序列(artificial sequence)
<400> 78
Ser Val Tyr Tyr Phe Asp Tyr
1 5
<210> 79
<211> 8
<212> PRT
<213> 人工序列(artificial sequence)
<400> 79
Ala Arg Ser Val Tyr Tyr Phe Asp
1 5
<210> 80
<211> 7
<212> PRT
<213> 人工序列(artificial sequence)
<400> 80
Tyr Ala Ser Asn Arg Asn Thr
1 5
<210> 81
<211> 10
<212> PRT
<213> 人工序列(artificial sequence)
<400> 81
Leu Leu Ile Tyr Tyr Ala Ser Asn Arg Asn
1 5 10
<210> 82
<211> 7
<212> PRT
<213> 人工序列(artificial sequence)
<400> 82
Gly Tyr Ser Phe Thr Asp Tyr
1 5
<210> 83
<211> 10
<212> PRT
<213> 人工序列(artificial sequence)
<400> 83
Gly Tyr Ser Phe Thr Asp Tyr Tyr Met His
1 5 10
<210> 84
<211> 5
<212> PRT
<213> 人工序列(artificial sequence)
<400> 84
Asp Tyr Tyr Met His
1 5
<210> 85
<211> 6
<212> PRT
<213> 人工序列(artificial sequence)
<400> 85
Thr Asp Tyr Tyr Met His
1 5
<210> 86
<211> 6
<212> PRT
<213> 人工序列(artificial sequence)
<400> 86
Asn Pro Asn Asn Gly Asn
1 5
<210> 87
<211> 10
<212> PRT
<213> 人工序列(artificial sequence)
<400> 87
Arg Val Asn Pro Asn Asn Gly Asn Thr Leu
1 5 10
<210> 88
<211> 17
<212> PRT
<213> 人工序列(artificial sequence)
<400> 88
Arg Val Asn Pro Asn Asn Gly Asn Thr Leu Tyr Asn Gln Lys Phe Arg
1 5 10 15
Gly
<210> 89
<211> 13
<212> PRT
<213> 人工序列(artificial sequence)
<400> 89
Trp Ile Gly Arg Val Asn Pro Asn Asn Gly Asn Thr Leu
1 5 10
<210> 90
<211> 8
<212> PRT
<213> 人工序列(artificial sequence)
<400> 90
Glu Asp Arg Tyr Ala Phe Ala Tyr
1 5
<210> 91
<211> 9
<212> PRT
<213> 人工序列(artificial sequence)
<400> 91
Ala Arg Glu Asp Arg Tyr Ala Phe Ala
1 5
<210> 92
<211> 15
<212> PRT
<213> 人工序列(artificial sequence)
<400> 92
Arg Ala Ser Gln Ser Val Ser Thr Ser Ser Tyr Thr Tyr Met His
1 5 10 15
<210> 93
<211> 11
<212> PRT
<213> 人工序列(artificial sequence)
<400> 93
Ser Thr Ser Ser Tyr Thr Tyr Met His Trp Tyr
1 5 10
<210> 94
<211> 7
<212> PRT
<213> 人工序列(artificial sequence)
<400> 94
Tyr Ala Ser Asn Leu Glu Ser
1 5
<210> 95
<211> 10
<212> PRT
<213> 人工序列(artificial sequence)
<400> 95
Leu Leu Ile Lys Tyr Ala Ser Asn Leu Glu
1 5 10
<210> 96
<211> 9
<212> PRT
<213> 人工序列(artificial sequence)
<400> 96
Gln His Thr Trp Glu Ile Pro Tyr Thr
1 5
<210> 97
<211> 8
<212> PRT
<213> 人工序列(artificial sequence)
<400> 97
Gln His Thr Trp Glu Ile Pro Tyr
1 5

Claims (14)

1.一种抗体分子或其片段，所述抗体分子或其片段包含重链可变区(VH)和轻链可变区(VL)，所述重链可变区和轻链可变区分别包含选自以下的重链CDR和轻链CDR的组合：
(1)依次示于SEQ ID NO:35、39、43的CDR-H1、CDR-H2、CDR-H3；和，依次示于SEQ ID NO:45、47、49的CDR-L1、CDR-L2、CDR-L3；
(2)依次示于SEQ ID NO:36、40、43的CDR-H1、CDR-H2、CDR-H3；和，依次示于SEQ ID NO:45、47、49的CDR-L1、CDR-L2、CDR-L3；
(3)依次示于SEQ ID NO:37、41、43的CDR-H1、CDR-H2、CDR-H3；和，依次示于SEQ ID NO:45、47、49的CDR-L1、CDR-L2、CDR-L3；
(4)依次示于SEQ ID NO:38、42、44的CDR-H1、CDR-H2、CDR-H3；和，依次示于SEQ ID NO:46、48、50的CDR-L1、CDR-L2、CDR-L3；
(5)依次示于SEQ ID NO:51、55、59的CDR-H1、CDR-H2、CDR-H3；和，依次示于SEQ ID NO:61、63、65的CDR-L1、CDR-L2、CDR-L3；
(6)依次示于SEQ ID NO:52、56、59的CDR-H1、CDR-H2、CDR-H3；和，依次示于SEQ ID NO:61、63、65的CDR-L1、CDR-L2、CDR-L3；
(7)依次示于SEQ ID NO:53、57、59的CDR-H1、CDR-H2、CDR-H3；和，依次示于SEQ ID NO:61、63、65的CDR-L1、CDR-L2、CDR-L3；
(8)依次示于SEQ ID NO:54、58、60的CDR-H1、CDR-H2、CDR-H3；和，依次示于SEQ ID NO:62、64、66的CDR-L1、CDR-L2、CDR-L3；
(9)依次示于SEQ ID NO:51、55、59的CDR-H1、CDR-H2、CDR-H3；和，依次示于SEQ ID NO:67、63、65的CDR-L1、CDR-L2、CDR-L3；
(10)依次示于SEQ ID NO:51、68、59的CDR-H1、CDR-H2、CDR-H3；和，依次示于SEQ IDNO:67、63、65的CDR-L1、CDR-L2、CDR-L3；
(11)依次示于SEQ ID NO:51、69、59的CDR-H1、CDR-H2、CDR-H3；和，依次示于SEQ IDNO:67、63、65的CDR-L1、CDR-L2、CDR-L3；
(12)依次示于SEQ ID NO:70、74、78的CDR-H1、CDR-H2、CDR-H3；和，依次示于SEQ IDNO:45、80、49的CDR-L1、CDR-L2、CDR-L3；
(13)依次示于SEQ ID NO:71、75、78的CDR-H1、CDR-H2、CDR-H3；和，依次示于SEQ IDNO:45、80、49的CDR-L1、CDR-L2、CDR-L3；
(14)依次示于SEQ ID NO:72、76、78的CDR-H1、CDR-H2、CDR-H3；和，依次示于SEQ IDNO:45、80、49的CDR-L1、CDR-L2、CDR-L3；
(15)依次示于SEQ ID NO:73、77、79的CDR-H1、CDR-H2、CDR-H3；和，依次示于SEQ IDNO:46、81、50的CDR-L1、CDR-L2、CDR-L3；
(16)依次示于SEQ ID NO:82、86、90的CDR-H1、CDR-H2、CDR-H3；和，依次示于SEQ IDNO:92、94、96的CDR-L1、CDR-L2、CDR-L3；
(17)依次示于SEQ ID NO:83、87、90的CDR-H1、CDR-H2、CDR-H3；和，依次示于SEQ IDNO:92、94、96的CDR-L1、CDR-L2、CDR-L3；
(18)依次示于SEQ ID NO:84、88、90的CDR-H1、CDR-H2、CDR-H3；和，依次示于SEQ IDNO:92、94、96的CDR-L1、CDR-L2、CDR-L3；
(19)依次示于SEQ ID NO:85、89、91的CDR-H1、CDR-H2、CDR-H3；和，依次示于SEQ IDNO:93、95、97的CDR-L1、CDR-L2、CDR-L3。
2.根据权利要求1所述的抗体分子或其片段，其特征在于，所述抗体分子或其片段中，所述重链可变区包含示于SEQ ID NO:7、9或10的氨基酸序列或与所述氨基酸序列具有至少75％同一性的氨基酸序列；和，所述轻链可变区包含示于SEQ ID NO:8、11或12的氨基酸序列或与所述氨基酸序列具有至少75％同一性的氨基酸序列；或者，
所述重链可变区包含示于SEQ ID NO:13、15、16、18、19、20或21的氨基酸序列或与所述氨基酸序列具有至少75％同一性的氨基酸序列；和，所述轻链可变区包含示于SEQ ID NO:14、22、23或25的氨基酸序列或与所述氨基酸序列具有至少75％同一性的氨基酸序列；或者
所述重链可变区包含示于SEQ ID NO:27、29或30的氨基酸序列或与所述氨基酸序列具有至少75％同一性的氨基酸序列；和，所述轻链可变区包含示于SEQ ID NO:28、31或32的氨基酸序列或与所述氨基酸序列具有至少75％同一性的氨基酸序列；或者，
所述重链可变区包含示于SEQ ID NO:33的氨基酸序列或与所述氨基酸序列具有至少75％同一性的氨基酸序列；和，所述轻链可变区包含示于SEQ ID NO:34的氨基酸序列或与所述氨基酸序列具有至少75％同一性的氨基酸序列。
3.根据权利要求1或2所述的抗体分子或其片段，其特征在于，所述抗体分子或其片段中的重链可变区和轻链可变区选自以下氨基酸序列的组合：
(1)如SEQ ID NO:7所示的氨基酸序列或与如SEQ ID NO:7所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO:8所示的氨基酸序列或与如SEQ ID NO:8所示的氨基酸序列具有至少75％同一性的氨基酸序列；
(2)如SEQ ID NO:9所示的氨基酸序列或与如SEQ ID NO:9所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO:11所示的氨基酸序列或与如SEQ ID NO:11所示的氨基酸序列具有至少75％同一性的氨基酸序列；
(3)如SEQ ID NO:9所示的氨基酸序列或与如SEQ ID NO:9所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO:12所示的氨基酸序列或与如SEQ ID NO:12所示的氨基酸序列具有至少75％同一性的氨基酸序列；
(4)如SEQ ID NO:10所示的氨基酸序列或与如SEQ ID NO:10所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO:11所示的氨基酸序列或与如SEQ ID NO:11所示的氨基酸序列具有至少75％同一性的氨基酸序列；
(5)如SEQ ID NO:10所示的氨基酸序列或与如SEQ ID NO:10所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO:12所示的氨基酸序列或与如SEQ ID NO:12所示的氨基酸序列具有至少75％同一性的氨基酸序列；
(6)如SEQ ID NO:13所示的氨基酸序列或与如SEQ ID NO:13所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO:14所示的氨基酸序列或与如SEQ ID NO:14所示的氨基酸序列具有至少75％同一性的氨基酸序列；
(7)如SEQ ID NO:16所示的氨基酸序列或与如SEQ ID NO:16所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO:22所示的氨基酸序列或与如SEQ ID NO:22所示的氨基酸序列具有至少75％同一性的氨基酸序列；
(8)如SEQ ID NO:16所示的氨基酸序列或与如SEQ ID NO:16所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO:23所示的氨基酸序列或与如SEQ ID NO:23所示的氨基酸序列具有至少75％同一性的氨基酸序列；
(9)如SEQ ID NO:16所示的氨基酸序列或与如SEQ ID NO:16所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO:25所示的氨基酸序列或与如SEQ ID NO:25所示的氨基酸序列具有至少75％同一性的氨基酸序列；
(10)如SEQ ID NO:19所示的氨基酸序列或与如SEQ ID NO:19所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO:25所示的氨基酸序列或与如SEQ ID NO:25所示的氨基酸序列具有至少75％同一性的氨基酸序列；
(11)如SEQ ID NO:21所示的氨基酸序列或与如SEQ ID NO:21所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO:25所示的氨基酸序列或与如SEQ ID NO:25所示的氨基酸序列具有至少75％同一性的氨基酸序列；
(12)如SEQ ID NO:27所示的氨基酸序列或与如SEQ ID NO:27所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO:28所示的氨基酸序列或与如SEQ ID NO:28所示的氨基酸序列具有至少75％同一性的氨基酸序列；
(13)如SEQ ID NO:29所示的氨基酸序列或与如SEQ ID NO:29所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO:32所示的氨基酸序列或与如SEQ ID NO:32所示的氨基酸序列具有至少75％同一性的氨基酸序列；
(14)如SEQ ID NO:30所示的氨基酸序列或与如SEQ ID NO:30所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO:32所示的氨基酸序列或与如SEQ ID NO:32所示的氨基酸序列具有至少75％同一性的氨基酸序列；或者
(15)如SEQ ID NO:33所示的氨基酸序列或与如SEQ ID NO:33所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO:34所示的氨基酸序列或与如SEQ ID NO:34所示的氨基酸序列具有至少75％同一性的氨基酸序列。
4.根据权利要求1至3中任一项所述的抗体分子或其片段，其特征在于，所述抗体分子或其片段结合脊髓灰质炎病毒受体样分子4(Nectin-4)，优选哺乳动物Nectin-4，更优选灵长类动物Nectin-4，进一步优选人或猴Nectin-4，特别是人Nectin-4。
5.根据权利要求1至4中任一项所述的抗体分子或其片段，其特征在于，所述抗体分子为鼠源抗体、嵌合抗体或者完全或部分人源化抗体；所述片段为所述抗体分子的scFv、dsFv、(dsFv)2、Fab、Fab'、F(ab')2或Fv片段；
优选地，所述抗体分子为单克隆抗体或单链抗体；
优选地，所述抗体分子或其片段还包含恒定区，优选包含鼠或人的重链恒定区(CH)和/或轻链恒定区(CL)；优选地，所述抗体分子或其片段包含重链和轻链；
更优选地，所述抗体分子或其片段包含IgG、IgA、IgM、IgD或IgE的重链恒定区和/或κ或λ型轻链恒定区。
6.根据权利要求1至5中任一项所述的抗体分子或其片段，其特征在于，所述抗体分子为单克隆抗体，优选为人源化的单克隆抗体；
优选地，所述单克隆抗体的重链恒定区为IgG1型，轻链恒定区为κ型。
7.一种核酸分子，其包含编码权利要求1至6中任一项所述的抗体分子或其片段中包含的轻链可变区、重链可变区、重链或轻链的核苷酸序列。
8.一种载体，其包含权利要求7所述的核酸分子。
9.一种宿主细胞，其包含权利要求7所述的核酸分子和/或权利要求8所述的载体，或者所述宿主细胞被权利要求7所述的核酸分子和/或权利要求8所述的载体转化或转染。
10.一种组合物，其包含权利要求1至6中任一项所述的抗体分子或其片段、权利要求7所述的核酸分子、权利要求8所述的载体或权利要求9所述的宿主细胞；
优选地，所述组合物为药物组合物，其可选地还包含药学上可接受的载体、辅料或赋形剂。
11.权利要求1至6中任一项所述的抗体分子或其片段、权利要求7所述的核酸分子、权利要求8所述的载体、权利要求9所述的宿主细胞或权利要求10所述的组合物在制备预防或治疗癌症的药物中的用途；
优选地，所述癌症为膀胱癌、乳腺癌、卵巢癌或肺癌。
12.一种试剂盒，所述试剂盒包括权利要求1至6中任一项所述的抗体分子或其片段、权利要求7所述的核酸分子、权利要求8所述的载体、权利要求9所述的宿主细胞或者权利要求10所述的组合物。
13.权利要求1至6中任一项所述的抗体分子或其片段在制备抗体药物偶联物中的用途。
14.一种抗体药物偶联物，其由权利要求1至6中任一项所述的抗体分子或其片段与细胞毒性部分偶联形成；
优选地，所述细胞毒性部分为微管蛋白抑制剂、拓扑异构酶抑制剂或DNA结合剂；
更优选地，所述微管蛋白抑制剂选自美登素衍生物、Monomethyl auristatin E(MMAE)、Monomethylauristatin F(MMAF)、Monomethyl Dolastatin 10、Tubulysin类衍生物、Cryptophycin类衍生物和Taltobulin；
更优选地，所述拓扑异构酶抑制剂选自阿霉素(Doxorubicin)代谢产物PNU-159682衍生物和伊立替康(irinotecan,CPT-11)代谢产物SN38衍生物；
更优选地，所述DNA结合剂选自PBD类衍生物和Duocarmycin类衍生物。
CN202010320420.3A 2020-04-21 2020-04-21 一种抗Nectin-4的抗体及其应用 Pending CN113527486A (zh)

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KR1020227040582A KR20230007406A (ko) 2020-04-21 2021-04-21 넥틴-4 (nectin-4) 에 대한 항체 및 이의 응용
PCT/CN2021/088661 WO2021213434A1 (zh) 2020-04-21 2021-04-21 一种抗Nectin-4的抗体及其应用
US17/996,403 US20230265183A1 (en) 2020-04-21 2021-04-21 Antibody against nectin-4 and application thereof
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CN114702588A (zh) * 2022-04-27 2022-07-05 博际生物医药科技(杭州)有限公司 抗Nectin-4抗体和双特异性抗体
WO2022228563A1 (zh) * 2021-04-30 2022-11-03 江苏迈威康新药研发有限公司 靶向Nectin-4的抗体药物偶联物及其制备方法和用途
WO2023025243A1 (zh) * 2021-08-27 2023-03-02 石药集团巨石生物制药有限公司 抗Nectin-4抗体、药物缀合物及其制备方法和用途
CN115877008A (zh) * 2022-12-05 2023-03-31 中国科学院自动化研究所 用于膀胱癌检测的光学分子成像探针及其制备方法和应用
CN116063498A (zh) * 2022-04-27 2023-05-05 博际生物医药科技(杭州)有限公司 单域抗Nectin-4抗体
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WO2023221971A1 (zh) * 2022-05-16 2023-11-23 江苏恒瑞医药股份有限公司 一种含抗Nectin-4抗体药物偶联物的药物组合物及其用途
WO2024012539A1 (zh) * 2022-07-14 2024-01-18 百奥泰生物制药股份有限公司 抗Nectin-4抗体及其应用
WO2024088390A1 (zh) * 2022-10-28 2024-05-02 江苏迈威康新药研发有限公司 一种包含抗Nectin-4抗体药物偶联物的制剂及其应用
CN116381251B (zh) * 2023-03-13 2024-06-25 柏定生物工程(北京)有限公司 一种肿瘤标志物诊断试剂盒及其诊断方法

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WO2022228563A1 (zh) * 2021-04-30 2022-11-03 江苏迈威康新药研发有限公司 靶向Nectin-4的抗体药物偶联物及其制备方法和用途
WO2023025243A1 (zh) * 2021-08-27 2023-03-02 石药集团巨石生物制药有限公司 抗Nectin-4抗体、药物缀合物及其制备方法和用途
CN114702589A (zh) * 2022-04-27 2022-07-05 博际生物医药科技(杭州)有限公司 治疗癌症的组合物和方法
CN114702588A (zh) * 2022-04-27 2022-07-05 博际生物医药科技(杭州)有限公司 抗Nectin-4抗体和双特异性抗体
CN114702588B (zh) * 2022-04-27 2022-11-22 博际生物医药科技(杭州)有限公司 抗Nectin-4抗体和双特异性抗体
CN116063498A (zh) * 2022-04-27 2023-05-05 博际生物医药科技(杭州)有限公司 单域抗Nectin-4抗体
WO2023221971A1 (zh) * 2022-05-16 2023-11-23 江苏恒瑞医药股份有限公司 一种含抗Nectin-4抗体药物偶联物的药物组合物及其用途
WO2024012539A1 (zh) * 2022-07-14 2024-01-18 百奥泰生物制药股份有限公司 抗Nectin-4抗体及其应用
WO2024088390A1 (zh) * 2022-10-28 2024-05-02 江苏迈威康新药研发有限公司 一种包含抗Nectin-4抗体药物偶联物的制剂及其应用
CN115877008A (zh) * 2022-12-05 2023-03-31 中国科学院自动化研究所 用于膀胱癌检测的光学分子成像探针及其制备方法和应用
CN116381251A (zh) * 2023-03-13 2023-07-04 广州鹏翔生物技术有限公司 一种肿瘤标志物诊断试剂盒及其诊断方法
CN116381251B (zh) * 2023-03-13 2024-06-25 柏定生物工程(北京)有限公司 一种肿瘤标志物诊断试剂盒及其诊断方法

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