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CN114106192B - 双特异性抗体及其应用 - Google Patents

双特异性抗体及其应用 Download PDF

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CN114106192B
CN114106192B CN202111585813.8A CN202111585813A CN114106192B CN 114106192 B CN114106192 B CN 114106192B CN 202111585813 A CN202111585813 A CN 202111585813A CN 114106192 B CN114106192 B CN 114106192B
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CN114106192A (zh
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张文军
李峰
刘敏
潘志豪
覃陆英
曹广灿
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Excelmab Inc
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Abstract

本发明提供了双特异性抗体及其应用，所述双特异性抗体含有抗体或抗体片段，所述抗体或抗体片段为具有第一重链和第一轻链以及第二重链和第二轻链的双特异性抗体或抗体片段；所述第一重链和第一轻链形成第一Fab区；所述第二重链和第二轻链形成第二Fab区；所述第一Fab区和第二Fab区中引入了多种突变。本申请通过对所述抗体或抗体片段的重链CH1和轻链CL界面氨基酸进行氨基酸大小和电荷突变，增强了针对不同抗原的重/轻链正确配对，保持特定抗原的结合能力，减少了错配并提高了结合特异性，提高了目的产物的产率。

Description

双特异性抗体及其应用
技术领域
本发明属于蛋白工程技术领域，涉及蛋白质分子内或分子间的改造方法。具体而言，本发明涉及对包含两种不同轻链的双特异性抗体的重链CH1和轻链CL的界面氨基酸进行氨基酸大小和电荷突变，防止错配以提高其结合特异性的方法，尤其涉及双特异性抗体及其应用。
背景技术
双特异性抗体能同时结合两种不同的抗原表位，具有特殊的生物学功能，并能产生优于单个抗体的加成或协同作用，是抗体工程领域以及肿瘤免疫治疗领域的一个研发热点。过往的一些研究针对新的双特异性形式(如DVD-Ig、CrossMab和BiTE等)进行过许多抗体工程化的尝试(Spiess等，Molecular Immunology，67(2)，pp.95-106(2015))，然而，这些形式可能在稳定性、溶解性、半衰周期和免疫原性等方面存在多种限制。
在双特异性抗体开发中，IgG型的双特异性抗体是较为通用的形式，结构上由靶向抗原A的一半抗体和靶向抗原B的一半抗体构成，具有与天然IgG相似的大小和形状。在制备IgG型双特异性抗体时，需要同时表达两条不同的重链以及两条不同的轻链，通过两对重链-轻链的精确配对形成识别两种不同抗原的两个Fab。然而，在此过程中，同源重链结合以及异源轻链结合产生的错配会产生十余种不同的产物，其中只有重链-轻链正确配对后的一种形式为所需要的正确产物。虽然可以通过下游纯化的方式获取目标双特异性抗体，但往往效率极低并且限制了大规模生产。因此，制备该种抗体需要解决的主要问题是不同链的随机装配，如同源重链错配、轻链错配以及其他非目的产物的污染等。
“knob-into-hole”是重链配对问题的第一个解决方案(Ridgway等，ProteinEngineering，9(7)，pp.617-21(1996)；Merchant等，Nature Biotechnology，16(7)，pp.677-681(1998))。此外，静电导向(Gunasekaran等，Journal of BiologicalChemistry，285(25)，pp.19637-19646(2010))或负状态设计(Kreudenstein等，mAbs，5(5)，pp.646-654(2013)；Leaver-Fay等，Structure，24(4)，pp.641-651(2016))等也是解决重链错配的方法。虽然这些策略有效降低了同源重链的错配，但并没有解决同源重链-轻链配对的问题。
轻-重链之间的界面包括可变结构域(VH-VL)和恒定结构域(CH1-CL)。已有数种方案应用于正交界面的设计以便于同源配对：Roche交换了CH1和CL的结构域，并且创造了CrossMab平台(Schaefer等，Proceedings of the National Academy of Sciences ofthe United States of America，108(27)，pp.11187-11192(2011))，MedImmune替代性地引入了二硫键(Mazor等，mAbs，7(2)，pp.377-389(2015))，Amgen在CH1-CL区进一步做了静电作用修饰(Liu等，Journal of Biological Chemistry，290(12)，pp.7535-7562(2015))，Lilly(Lewis等，Nature Biotechnology，32(2)，pp.191-198(2014))和Genentech(Dillon等，mAbs，9(2)，pp.213-230(2017))在可变结构域和恒定结构域均引入了突变。
通过设计VH:VL界面突变来解决异源轻链错配可能对抗体的亲和力及功能产生不利影响。转植TCRαβ恒定区界面的方法也可能影响亲和力或产生免疫源性。而对恒定区的CH1和CL结构域实施替换改造的方法还不足以有效地生产目标双特异性抗体。
因此，如何提供降低重链和轻链之间的错配的方法，提高目的产物的产率，已成为亟待解决的问题。
发明内容
针对现有技术的不足和实际需求，本发明提供双特异性抗体及其应用，通过对重链CH1和轻链CL界面氨基酸进行氨基酸大小和电荷突变，减少了轻重链之间的错配，提高了其对特定抗原的结合能力，提高了正确产物的产率，应用价值极高。
为达此目的，本发明采用如下技术方案：
第一方面，本发明提供了抗体或抗体片段，所述抗体或抗体片段为具有第一重链和第一轻链以及第二重链和第二轻链的双特异性抗体或抗体片段；
所述第一重链和第一轻链形成第一Fab区；
所述第二重链和第二轻链形成第二Fab区；
所述抗体或抗体片段的第一Fab区引入以下突变中的任意一种或至少两种的组合：
a)重链恒定区CH1中的
S186A/S186T/S186E/S186Q/S186M/S186D/S186K/S186V/S186W/S186Y；
和轻链恒定区CL中的
T178M/T178Y/T178L/T178I/T178A/T178V/T178S/T178R/T178E；
b)重链恒定区CH1中的
S188Y/S188M/S188T/S188F/S188V/S188L/S188I/S188R；
和轻链恒定区CL中的
S176M/S176L/S176Y/S176A/S176I/S176E/S176T/S176W/S176H/S176V；
c)重链恒定区CH1中的
V190T/V190I/V190W/V190M/V190Y/V190F/V190L/V190Q/V190S；
和轻链恒定区CL中的
S174F/S174N/S174M/S174A/S174T/S174W/S174R/S174C/S174K/S174H/S174Y/S174L；
所述抗体或抗体片段的第二Fab区引入以下突变中的任意一种或至少两种的组合：
a)重链恒定区CH1中的
S186A/S186T/S186E/S186Q/S186M/S186D/S186K/S186V/S186W/S186Y；
和轻链恒定区CL中的
T178M/T178Y/T178L/T178I/T178A/T178V/T178S/T178R/T178E；
b)重链恒定区CH1中的
S188Y/S188M/S188T/S188F/S188V/S188L/S188I/S188R；
和轻链恒定区CL中的
S176M/S176L/S176Y/S176A/S176I/S176E/S176T/S176W/S176H/S176V；
c)重链恒定区CH1中的
V190T/V190I/V190W/V190M/V190Y/V190F/V190L/V190Q/V190S；
和轻链恒定区CL中的
S174F/S174N/S174M/S174A/S174T/S174W/S174R/S174C/S174K/S174H/S174Y/S174L。
本发明中，如无特殊说明，根据Kabat编号对所述抗体重链和轻链的氨基酸进行编号。
本发明中，抗体的Fab区通过CH1/CL结合界面的氨基酸通过疏水作用、电荷作用以及氢键来维持侧链间的相互作用，当发生上述突变时，可以降低不同重/轻链之间的错配，提高其对特定抗原的结合能力，提高产率的同时增强其特异性，相应抗体的性能更优。
优选地，所述抗体或抗体片段的第一Fab区引入以下突变中的任意一种：
8D6：
a)重链恒定区CH1中的S186T和轻链恒定区CL中的T178A；
b)重链恒定区CH1中的S188Y和轻链恒定区CL中的S176E；
c)重链恒定区CH1中的V190F和轻链恒定区CL中的S174A；
或11B7：
a)重链恒定区CH1中的S186V和轻链恒定区CL中的T178L；
b)重链恒定区CH1中的S188L和轻链恒定区CL中的S176W；
c)重链恒定区CH1中的V190M和轻链恒定区CL中的S174R；
或11D6：
a)重链恒定区CH1中的S186W和轻链恒定区CL中的T178M；
b)重链恒定区CH1中的S188F和轻链恒定区CL中的S176V；
c)重链恒定区CH1中的V190T和轻链恒定区CL中的S174K；
或11H2：
a)重链恒定区CH1中的S186Y；
b)重链恒定区CH1中的S188M和轻链恒定区CL中的S176V；
c)重链恒定区CH1中的V190F。
优选地，所述抗体或抗体片段的第二Fab区引入以下突变中的任意一种：
11A10：
a)重链恒定区CH1中的S186M和轻链恒定区CL中的T178V；
b)重链恒定区CH1中的S188I和轻链恒定区CL中的S176H；
c)重链恒定区CH1中的V190W和轻链恒定区CL中的S174R；
或7A10：
a)重链恒定区CH1中的S186T和轻链恒定区CL中的T178V；
b)重链恒定区CH1中的S188R；
c)重链恒定区CH1中的V190F；
或11C12：
a)重链恒定区CH1中的S186K和轻链恒定区CL中的T178E；
b)重链恒定区CH1中的S188L和轻链恒定区CL中的S176M；
c)和轻链恒定区CL中的S174Y；
或11A10：
a)重链恒定区CH1中的S186M和轻链恒定区CL中的T178V；
b)重链恒定区CH1中的S188I和轻链恒定区CL中的S176H；
c)重链恒定区CH1中的V190W和轻链恒定区CL中的S174R。
优选地，所述第一Fab区的轻链恒定区CL还引入突变A178D，第二Fab区的重链恒定区CH1还引入突变I188D。
优选地，所述第一Fab区的轻链恒定区CL还引入突变L178K，第二Fab区的重链恒定区CH1还引入突变T192K。
优选地，所述抗体或抗体片段的第一Fab区和第二Fab区引入的突变相同或不同。
第二方面，本发明提供了双特异性抗体，所述双特异性抗体含有第一方面所述的抗体或抗体片段。
优选地，所述双特异性抗体轻链恒定区CL各自独立地选自以下序列中的任意一种：SEQ ID NO:3、SEQ ID NO:7、SEQ ID NO:11、SEQ ID NO:15、SEQ ID NO:19、SEQ ID NO:23、SEQ ID NO:27、SEQ ID NO:31、SEQ ID NO:35、SEQ ID NO:39、SEQ ID NO:43、SEQ IDNO:47、SEQ ID NO:51、SEQ ID NO:55、SEQ ID NO:59、SEQ ID NO:63、SEQ ID NO:67、SEQ IDNO:71、SEQ ID NO:75、SEQ ID NO:79、SEQ ID NO:83、SEQ ID NO:87、SEQ ID NO:91、SEQ IDNO:95、SEQ ID NO:99、SEQ ID NO:103、SEQ ID NO:107、SEQ ID NO:111或SEQ ID NO:115。
SEQ ID NO:3：
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLMSMLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC；
SEQ ID NO:7：
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYFLLSMLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC；
SEQ ID NO:11：
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYFLSSYLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC；
SEQ ID NO:15：
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYFLYSLLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC；
SEQ ID NO:19：
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYNLASTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC；
SEQ ID NO:23：
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYMLISILTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC；
SEQ ID NO:27：
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYNLLSALTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC；
SEQ ID NO:31：
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYNLYSLLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC；
SEQ ID NO:35：
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYALESALTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC；
SEQ ID NO:39：
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYTLASVLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC；
SEQ ID NO:43：
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYWLTSLLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC；
SEQ ID NO:47：
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLASILTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC；
SEQ ID NO:51：
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYTLYSSLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC；
SEQ ID NO:55：
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYRLWSLLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC；
SEQ ID NO:59：
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLTNYLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC；
SEQ ID NO:63：
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLYSVLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC；
SEQ ID NO:67：
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYNLSSLLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC；
SEQ ID NO:71：
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYCLTSVLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC；
SEQ ID NO:75：
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYFLSSLLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC；
SEQ ID NO:79：
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYRLHSVLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC；
SEQ ID NO:83：
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYKLVSMLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC；
SEQ ID NO:87：
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYHLVSRLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC；
SEQ ID NO:91：
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYYLMSELTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC；
SEQ ID NO:95：
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYNLESMLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC；
SEQ ID NO:99：
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYFLLSSLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC；
SEQ ID NO:103：
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYYLSSILTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC；
SEQ ID NO:107：
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLVSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC；
SEQ ID NO:111：
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYLLWSMLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC；
SEQ ID NO:115：
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSVLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。
优选地，所述双特异性抗体的重链恒定区CH1各自独立地选自以下序列中的任意一种：SEQ ID NO:1、SEQ ID NO:5、SEQ ID NO:9、SEQ ID NO:13、SEQ ID NO:17、SEQ ID NO:21、SEQ ID NO:25、SEQ ID NO:29、SEQ ID NO:33、SEQ ID NO:37、SEQ ID NO:41、SEQ IDNO:45、SEQ ID NO:49、SEQ ID NO:53、SEQ ID NO:57、SEQ ID NO:61、SEQ ID NO:65、SEQ IDNO:69、SEQ ID NO:73、SEQ ID NO:77、SEQ ID NO:81、SEQ ID NO:85、SEQ ID NO:89、SEQ IDNO:93、SEQ ID NO:97、SEQ ID NO:101、SEQ ID NO:105、SEQ ID NO:109或SEQ ID NO:113。
SEQ ID NO:1：
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYALYSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV；
SEQ ID NO:5：
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYTLMSTVTVPSSSLGTQTYICNVNHKPSNTKVDKKV；
SEQ ID NO:9：
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYELTSIVTVPSSSLGTQTYICNVNHKPSNTKVDKKV；
SEQ ID NO:13：
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYQLFSTVTVPSSSLGTQTYICNVNHKPSNTKVDKKV；
SEQ ID NO:17：
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYMLTSWVTVPSSSLGTQTYICNVNHKPSNTKVDKKV；
SEQ ID NO:21：
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLYSMVTVPSSSLGTQTYICNVNHKPSNTKVDKKV；
SEQ ID NO:25：
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYDLYSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV；
SEQ ID NO:29：
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYKLVSYVTVPSSSLGTQTYICNVNHKPSNTKVDKKV；
SEQ ID NO:33：
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYTLYSFVTVPSSSLGTQTYICNVNHKPSNTKVDKKV；
SEQ ID NO:37：
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYMLLSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV；
SEQ ID NO:41：
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYTLFSLVTVPSSSLGTQTYICNVNHKPSNTKVDKKV；
SEQ ID NO:45：
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYVLISQVTVPSSSLGTQTYICNVNHKPSNTKVDKKV；
SEQ ID NO:49：
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLVSYVTVPSSSLGTQTYICNVNHKPSNTKVDKKV；
SEQ ID NO:53：
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYVLLSMVTVPSSSLGTQTYICNVNHKPSNTKVDKKV；
SEQ ID NO:57：
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYMLVSTVTVPSSSLGTQTYICNVNHKPSNTKVDKKV；
SEQ ID NO:61：
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYALFSSVTVPSSSLGTQTYICNVNHKPSNTKVDKKV；
SEQ ID NO:65：
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYELLSIVTVPSSSLGTQTYICNVNHKPSNTKVDKKV；
SEQ ID NO:69：
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYTLVSFVTVPSSSLGTQTYICNVNHKPSNTKVDKKV；
SEQ ID NO:73：
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLLSIVTVPSSSLGTQTYICNVNHKPSNTKVDKKV；
SEQ ID NO:77：
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYMLISWVTVPSSSLGTQTYICNVNHKPSNTKVDKKV；
SEQ ID NO:81：
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYWLFSTVTVPSSSLGTQTYICNVNHKPSNTKVDKKV；
SEQ ID NO:85：
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYTLFSSVTVPSSSLGTQTYICNVNHKPSNTKVDKKV；
SEQ ID NO:89：
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYKLLSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV；
SEQ ID NO:93：
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYALISIVTVPSSSLGTQTYICNVNHKPSNTKVDKKV；
SEQ ID NO:97：
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLVSIVTVPSSSLGTQTYICNVNHKPSNTKVDKKV；
SEQ ID NO:101：
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYTLTSLVTVPSSSLGTQTYICNVNHKPSNTKVDKKV；
SEQ ID NO:105：
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYYLMSFVTVPSSSLGTQTYICNVNHKPSNTKVDKKV；
SEQ ID NO:109：
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYTLMSSVTVPSSSLGTQTYICNVNHKPSNTKVDKKV；
SEQ ID NO:113：
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYTLRSFVTVPSSSLGTQTYICNVNHKPSNTKVDKKV。
优选地，所述双特异性抗体的第一Fab区和第二Fab区各自独立地选自以下序列中的任意一种：
SEQ ID NO:35的轻链恒定区CL和SEQ ID NO:33的重链恒定区CH1；
或SEQ ID NO:79的轻链恒定区CL和SEQ ID NO:77的重链恒定区CH1；
或SEQ ID NO:55的轻链恒定区CL和SEQ ID NO:53的重链恒定区CH1；
或SEQ ID NO:115的轻链恒定区CL和SEQ ID NO:113的重链恒定区CH1；
或SEQ ID NO:83的轻链恒定区CL和SEQ ID NO:81的重链恒定区CH1；
或SEQ ID NO:91的轻链恒定区CL和SEQ ID NO:89的重链恒定区CH1；
或SEQ ID NO:107的轻链恒定区CL和SEQ ID NO:105的重链恒定区CH1；
或SEQ ID NO:79的轻链恒定区CL和SEQ ID NO:77的重链恒定区CH1。
优选地，所述双特异性抗体的第一Fab区和第二Fab区结合不同的抗原，或同一抗原的两个不同的表位。
优选地，所述抗原选自CD2，CD3，CD3E，CD4，CD11，CD11a，CD14，CD16，CD18，CD19，CD20，CD22，CD23，CD25，CD28，CD29，CD30，CD32a，CD32b，CD33(p67蛋白)，CD38，CD40，CD40L，CD52，CD54，CD56，CD64，CD80，CD147，GD3，IL-1α，IL-1β，IL-1R，IL-2，IL-2R，IL-4，IL-5，IL-5R，IL-6，IL-6R，IL-8，IL-9，IL-12，IL-13，IL-15，IL-17，IL-17R，IL-18，IL-23，干扰素α，干扰素β，干扰素γ，TNF-α，TNFβ，TNF-R1，TNF-RII，FasL，CD27L，CD30L，4-1BBL，TRAIL，RANKL，TWEAK，APRIL，BAFF，LIGHT，VEG1，OX40L，TRAIL受体-1，腺苷受体，淋巴毒素β受体，TACI，BAFF-R，EPO；LFA-3，ICAM-1，ICAM-3，EpCAM，整联蛋白β1，整联蛋白β2，整联蛋白α4/β7，整联蛋白α2，整联蛋白α3，整联蛋白α4，整联蛋白α5，整联蛋白α6，整联蛋白αv，整联蛋白αVβ3，FGFR-3，角质形成细胞生长因子，VLA-1，VLA-4，L-选择素，抗Id，E-选择素，HLA，HLA-DR，CTLA-4，T细胞受体，B7-1，B7-2，VNR整联蛋白，TGFβ1，TGFβ2，嗜酸性粒细胞趋化因子1(eotaxin1)，Blys(B淋巴细胞刺激因子),补体C5，IgE，因子VII，CD64，CBL，NCA 90，EGFR(ErbB-1)，Her1，Her2/neu(ErbB-2)，Her3(ErbB-3)，Her4(ErbB-4)，组织因子，内皮素受体，VLA-4，半抗原NP-cap，NIP-cap，E-选择素，地高辛，胎盘碱性磷酸酶(PLAP)，睾丸PLAP样碱性磷酸酶，转铁蛋白受体，癌胚抗原(CEA)，CEACAM5，HMFG1，PEM，粘蛋白MUC1，MUC18，肝素酶I，人心脏肌球蛋白，肿瘤相关糖蛋白-72(TAG-72)，肿瘤相关抗原CA125，前列腺特异性膜抗原(PSM-A)，高分子量黑色素瘤相关抗原(HMW-MAA)，癌(carcinoma)相关抗原，Gco蛋白Iib/IIIa(GPIIb/IIIa)，表达Lewis Y相关碳水化合物的肿瘤相关抗原，人巨细胞病毒(HCMV)gH包膜糖蛋白，HIV gp120，HCMV，呼吸合胞病毒RSV F，RSVF Fgp，细胞角蛋白肿瘤相关抗原，Hep B gp120，CMV，gpIIbIIIa，HIV IIIB gp120V3环，呼吸道合胞病毒(RSV)Fgp，单纯疱疹病毒(HSV)gD糖蛋白，HSV gB糖蛋白，HCMV g B包膜糖蛋白，产气荚膜梭菌毒素(Clostridium perfringens)，CD133，CD138，OX40，GITR，PD-1，PDL1，PD-L2，CTLA-4，KIR，LAG-3，TCRα，TCRβ，TCRγ，TCRδ，VEGF，EGF，VEGFR，EGFR，EpCAM，间皮素，Glypicans，Erbl，Erb2，B7-H3，ICOS，BMP1，BMP2，BMP3B，BMP4，CSF1，GM-CSF，FGF1，FGF2，FGF3，FGF4，PDGFR，TIGIT，CS1，TWEAK，CCL1，CCL2，CCL3，CCL13，CXCL1，CXCL2，CXCL3，IP-10，岩藻糖基-GM1，IGF1，IGF2，IGF1R，IGF2R，RANK配体，DLL-4，GM-CSFR，ADAMS，肌生成抑制素，PCSK9，CXCR4，MIF或PEG2中的任意一个或两个的组合。
优选地，所述双特异性抗体的第一Fab区结合CD3，CD3E，CD16，CD28，CD47，CD27，CD40，OX40，GITR，ICOS，4-1BB，PD-1，PD-L1，GITR，TIGIT，TIM3，LAG3，B7H3，CTLA4，VISTA，BTLA，HVEM，NKG2A或NECL中的任意一种。
优选地，所述双特异性抗体的第二Fab区结合CD2，CD4，CD11，CD11a，CD14，CD18，CD19，CD20，CD22，CD23，CD25，CD29，CD30，CD32a，CD32b，CD33，CD38，，CD52，CD54，CD56，CD64，CD80，CD147，GD3，IL-1α，IL-1β，IL-1R，IL-2，IL-2R，IL-4，IL-5，IL-5R，IL-6，IL-6R，IL-8，IL-9，IL-12，IL-13，IL-15，IL-17，IL-17R，IL-18，IL-23，干扰素α，干扰素β，干扰素γ，TNF-α，TNFβ，TNF-R1，TNF-RII，TACI，BAFF-R，EPO；LFA-3，GPC-3，ICAM-1，ICAM-3，补体C5，IgE，因子VII，CD64，CBL，NCA 90，Her1，Her2，Her3，Her4，癌胚抗原(CEA)，肿瘤相关糖蛋白-72(TAG-72)，肿瘤相关抗原CA125，前列腺特异性膜抗原(PSM-A)，高分子量黑色素瘤相关抗原(HMW-MAA)，癌(carcinoma)相关抗原或表达Lewis Y相关碳水化合物的肿瘤相关抗原中的任意一种。
优选地，所述双特异性抗体的第一Fab区结合的抗原为CD3，所述第二Fab区结合的抗原为GPC3。
优选地，所述双特异性抗体具有结合CD3的第一重链和第一轻链，和结合GPC3的第二重链和第二轻链。
优选地，所述双特异性抗体的选自以下氨基酸序列中的任意一种：
所述第一重链具有SEQ ID NO:129所示的氨基酸序列，且所述第一轻链具有SEQID NO:127所示的氨基酸序列；所述第二重链具有SEQ ID NO:133所示的氨基酸序列，且所述第二轻链具有SEQ ID NO:131所示的氨基酸序列；
或所述第一重链具有SEQ ID NO:145所示的氨基酸序列，且所述第一轻链具有SEQID NO:143所示的氨基酸序列；所述第二重链具有SEQ ID NO:149所示的氨基酸序列，且所述第二轻链具有SEQ ID NO:147所示的氨基酸序列；
或所述第一重链具有SEQ ID NO:137所示的氨基酸序列，且所述第一轻链具有SEQID NO:135所示的氨基酸序列；所述第二重链具有SEQ ID NO:141所示的氨基酸序列，且所述第二轻链具有SEQ ID NO:139所示的氨基酸序列；
或所述第一重链具有SEQ ID NO:153所示的氨基酸序列，且所述第一轻链具有SEQID NO:151所示的氨基酸序列；所述第二重链具有SEQ ID NO:157所示的氨基酸序列，且所述第二轻链具有SEQ ID NO:155所示的氨基酸序列。
SEQ ID NO:129：
EVQLVESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAMYYCVRHGNFGHSYVSWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYTLYSFVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTKKKLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK；
SEQ ID NO:127：
DIQMTQEPSLTTSPGGTVTLTCRSSTGAVTTSNYANWVQEKPGQAPRGLIGGTNKRAPGTPARFSGSLIGGKAALTITGVQPEDEAIYFCALWYSNLWVFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYALESALTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC；
SEQ ID NO:133：
QVQLVQSGAEVKKPGASVKLSCKASGYTFTDYEMHWVKQTPGKGLKWIGALDPKTGDTAYAQKFQGRATLTADTSTDTAYMELSSLRSEDSAVYYCTRFYSYTYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYMLISWVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTDDDVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK；
SEQ ID NO:131：
DIVLTQSPLSLTVTPGEPASISCRSSESLVHSNGNTYLSWLQQRPGQPPRLLIYKISNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCVQVSSFPYTFGQGTKVEIKTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYRLHSVLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC；
SEQ ID NO:145：
EVQLVESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAMYYCVRHGNFGHSYVSWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYTLYSFVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTKKKLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK；
SEQ ID NO:143：
DIQMTQEPSLTTSPGGTVTLTCRSSTGAVTTSNYANWVQEKPGQAPRGLIGGTNKRAPGTPARFSGSLIGGKAALTITGVQPEDEAIYFCALWYSNLWVFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYALESDLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC；
SEQ ID NO:149：
QVQLVQSGAEVKKPGASVKLSCKASGYTFTDYEMHWVKQTPGKGLKWIGALDPKTGDTAYAQKFQGRATLTADTSTDTAYMELSSLRSEDSAVYYCTRFYSYTYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYMLDSWVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTDDDVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK；
SEQ ID NO:147：
DIVLTQSPLSLTVTPGEPASISCRSSESLVHSNGNTYLSWLQQRPGQPPRLLIYKISNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCVQVSSFPYTFGQGTKVEIKTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYRLHSVLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC；
SEQ ID NO:137：
EVQLVESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAMYYCVRHGNFGHSYVSWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYVLLSMVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTKKKLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK；
SEQ ID NO:135：
DIQMTQEPSLTTSPGGTVTLTCRSSTGAVTTSNYANWVQEKPGQAPRGLIGGTNKRAPGTPARFSGSLIGGKAALTITGVQPEDEAIYFCALWYSNLWVFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYRLWSLLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC；
SEQ ID NO:141：
QVQLVQSGAEVKKPGASVKLSCKASGYTFTDYEMHWVKQTPGKGLKWIGALDPKTGDTAYAQKFQGRATLTADTSTDTAYMELSSLRSEDSAVYYCTRFYSYTYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYTLRSFVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTDDDVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK；
SEQ ID NO:139：
DIVLTQSPLSLTVTPGEPASISCRSSESLVHSNGNTYLSWLQQRPGQPPRLLIYKISNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCVQVSSFPYTFGQGTKVEIKTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC；
SEQ ID NO:153：
EVQLVESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAMYYCVRHGNFGHSYVSWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYVLLSMVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTKKKLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK；
SEQ ID NO:151：
DIQMTQEPSLTTSPGGTVTLTCRSSTGAVTTSNYANWVQEKPGQAPRGLIGGTNKRAPGTPARFSGSLIGGKAALTITGVQPEDEAIYFCALWYSNLWVFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYRLWSKLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC；
SEQ ID NO:157：
QVQLVQSGAEVKKPGASVKLSCKASGYTFTDYEMHWVKQTPGKGLKWIGALDPKTGDTAYAQKFQGRATLTADTSTDTAYMELSSLRSEDSAVYYCTRFYSYTYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYTLRSFVKVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTDDDVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK；
SEQ ID NO:155：
DIVLTQSPLSLTVTPGEPASISCRSSESLVHSNGNTYLSWLQQRPGQPPRLLIYKISNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCVQVSSFPYTFGQGTKVEIKTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。
优选地，所述双特异性抗体来源于IgG、IgA、IgM、IgE或IgD。
优选地，所述双特异性抗体来源于IgG1、IgG2、IgG3或IgG4。
第三方面，本发明提供了核酸分子，所述核酸分子编码第一方面所述的抗体或抗体片段和/或第二方面所述的双特异性抗体。
第四方面，本发明提供了表达载体，所述表达载体含有第三方面所述的核酸分子。
第五方面，本发明提供了重组细胞，所述重组细胞表达第一方面所述的抗体或抗体片段和/或第二方面所述的双特异性抗体。
优选地，所述重组细胞的基因组中整合有第三方面所述的核酸分子。
优选地，所述重组细胞中含有第四方面所述的表达载体。
第六方面，本发明提供了第一方面所述的抗体或抗体片段的制备方法，包括以下步骤：
将编码所述抗体或抗体片段的核酸分子连入质粒，转入感受态细胞中，随后挑选单克隆细胞进行筛选；
提取筛选的阳性克隆中的表达载体，转入受体细胞中，筛选得到重组细胞；
培养所述重组细胞，收集培养上清，分离纯化，得到所述抗体或抗体片段。
第七方面，本发明提供了抗体偶联物，其特征在于，所述抗体偶联物包括第一方面所述的抗体或抗体片段和/或第二方面所述的双特异性抗体，以及偶联的标记物。
优选地，所述标记物包括细胞毒素、放射性同位素、荧光标记物、发光物、显色物质或酶中的任意一种或至少两种的组合。
第八方面，本发明提供了药物组合物，其特征在于，所述药物组合物含有第一方面所述的抗体或抗体片段、第二方面所述的双特异性抗体或第七方面所述的抗体偶联物中的任意一种或至少两种的组合。
优选地，所述药物组合物还包括药学上可接受的载体、表面活性剂、稀释剂或赋形剂中的任意一种或至少两种的组合。
第九方面，本发明提供了第一方面所述的抗体或抗体片段、第二方面所述的双特异性抗体、第七方面所述的抗体偶联物或第八方面所述的药物组合物中的任意一种或至少两种的组合在制备疾病治疗药物中的应用。
优选地，所述疾病包括癌症。
相比于现有技术，本发明具有如下有益效果：
(1)发明通过对抗体的CH1/CL结合界面的氨基酸位点进行突变，提高了异二聚体双特异性抗体对特定抗原的重/轻链的正确配对的概率，进一步提高了其对特定抗体的结合能力，特异性及亲和能力更好，且提高了目标抗体的产率；
(2)本发明构建的抗GPC3×CD3四链双特异性抗体MutA、MutB、MutA+D-D以及MutB+K-K的轻链与相应的重链配对良好，能够有效地产生目标双抗分子，对靶抗原具有更好的结合活性，对T细胞的激活能力更强，对肿瘤细胞杀伤效果更好，应用价值极高。
附图说明
图1为CH1-CL结构域中E-strand的结合界面结构示意图；
图2为抗体的结构示意图，图中，I-两种Crossmab双特异性抗体的结构示意图，II-传统的三链双特异性抗体的结构示意图，III-本发明改造得到的双特异性抗体的结构示意图；
图3A为WT0的还原性和非还原性SDS-PAGE电泳结果；图3B为CM-C的还原性和非还原性SDS-PAGE电泳结果；图3C为CM-G的还原性和非还原性SDS-PAGE电泳结果；图3D为WT的还原性和非还原性SDS-PAGE电泳结果；图3E为MutA和MutB的还原性和非还原性SDS-PAGE电泳结果；图3F为MutA+D-D和MutB+K-K的还原性和非还原性SDS-PAGE电泳结果；
图4A为MutA、MutB与阴性对照WT0、CM-C以及CM-G对GPC3的ELISA检测结果；
图4B为MutA、MutB与阴性对照WT0、CM-C以及CM-G对CD3的ELISA检测结果；
图4C为MutA+D-D、MutB+K-K与阴性对照WT0、CM-C以及CM-G对GPC3的ELISA检测结果；
图4D为MutA+D-D、MutB+K-K与阴性对照WT0、CM-C以及CM-G对CD3的ELISA检测结果；
图5A为MutA、MutB与三链阳性抗体WT以及hIgG对GPC3的ELISA检测结果；
图5B为MutA、MutB与三链阳性抗体WT以及hIgG对CD3的ELISA检测结果；
图5C为MutA+D-D、MutB+K-K与三链阳性抗体WT以及hIgG对GPC3的ELISA检测结果；
图5D为MutA+D-D、MutB+K-K与三链阳性抗体WT以及hIgG对CD3的ELISA检测结果；
图6为MutA、MutB、MutA+D-D、MutB+K-K以及三链阳性抗体WT对GPC3结合活性的流式检测图片；
图7A为MutA、MutB与阴性对照WT0、CM-C以及CM-G激活T细胞活性的结果图片；
图7B为MutA+D-D、MutB+K-K与阴性对照WT0、CM-C以及CM-G激活T细胞活性的结果图片；
图8A为MutA、MutB与三链阳性抗体WT激活T细胞活性的结果图片；
图8B为MutA+D-D、MutB+K-K与三链阳性抗体WT激活T细胞活性的结果图片；
图9A为MutA+D-D与阴性对照WT0、CM-C和CM-G对肿瘤细胞的杀伤活性的结果图片；
图9B为MutA、MutB以及三链阳性抗体WT对肿瘤细胞的杀伤活性的结果图片；
图9C为MutA+D-D、MutB+K-K以及三链阳性抗体WT对肿瘤细胞的杀伤活性的结果图片。
具体实施方式
为进一步阐述本发明所采取的技术手段及其效果，以下结合实施例和附图对本发明作进一步地说明。可以理解的是，此处所描述的具体实施方式仅仅用于解释本发明，而非对本发明的限定。
实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件，或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道商购获得的常规产品。
除非本文另有定义，与本申请结合使用的科学和技术术语及其缩略语应具有本公开所属领域的普通技术人员通常理解的含义。以下列举了本文中使用的部分术语和缩略语。
抗体：antibody，Ab；
免疫球蛋白：immunoglobulin，Ig；
重链：heavy chain，HC；
轻链：light chain，LC；
重链可变区：heavy chain variable domain，VH；
重链恒定区：heavy chain constant domain，CH；
轻链可变区：light chain variable domain，VL；
轻链恒定区：light chain constant domain，CL；
抗原结合区：antigen binding fragment，Fab；
铰链区：hinge region；
Fc片段：fragment crystallizable region，Fc region；
单克隆抗体：monoclonal antibodies，mAbs；
抗体依赖性细胞毒作用：antibody-dependent cell-mediated cytotoxicity，ADCC；
补体依赖性细胞毒性作用：complement dependent cytotoxicity，CDC；
自然杀伤细胞：natural killing cell，NK细胞；
双特异性抗体：bispecific antibody，BsAb；
T细胞受体：T cell receptor，TCR；
主要组织相容性复合体：major histocompatibility complex，MHC；
互补决定区：complementarity determining region，CDR，是指抗体的抗原互补结合区；
免疫受体酪氨酸活化基序：immunoreceptor tyrosine-based activationmotif，ITAM；
单链可变区抗体片段(又称单链抗体)：single-chain variable fragment，scFv；
过继免疫治疗：adoptive cellular immunotherapy，ACI；
淋巴因子激活的杀伤细胞：lymphokine-activated killer cell，LAK细胞；
肿瘤浸润淋巴细胞：tumor infiltrating lymphocyte，TIL细胞；
细胞因子诱导的杀伤细胞：cytokine-induced killer cell，CIK细胞。
除非另外定义，否则在本发明中所使用的相关术语与该技术领域通常理解的含义相同。本发明所述分子克隆、细胞培养、蛋白纯化、免疫学实验、微生物学、动物模型等试验的操作步骤为该领域内被广泛应用的常规步骤。除非上下文另有说明，否则本发明单数术语包含复数且复数含义包括单数含义。除非另外指明，否则本发明所述核苷酸序列按照5’端至3’端的方向从左至右排列并书写。除非另外指明，否则本发明所述氨基酸序列按照从氨基端(N末端)到羧基端(C末端)的方向从左至右排列并书写。本发明所提及的氨基酸三字母缩写及核苷酸单字母缩写为该技术领域普遍接受的形式，氨基酸单字母缩写为IUPAC-IUB生物化学命名委员会(IUPAC-IUB Biochemical Nomenclature Commission)推荐的形式。
术语“氨基酸”是指20种天然存在的氨基酸之一或可能存在于特定的限定位置的任何非天然类似物。本发明所述“氨基酸突变”是指多肽序列中的氨基酸取代、插入、缺失和修饰，以及氨基酸取代、插入、缺失和修饰的任意组合。本文中优选的氨基酸修饰是取代。本发明中“氨基酸取代”或“取代”是指将亲本多肽序列中特定位置的氨基酸替代为另一种氨基酸。例如，取代C220S是指变体多肽，其中多肽的位置220处的氨基半胱氨酸已被氨基酸丝氨酸替代。氨基酸突变可以通过分子克隆或化学的方法实现，分子克隆的方法包括PCR、定点突变和全基因合成等。
术语“蛋白”、“肽链”和“多肽链”是指两个以上的氨基酸通过肽键连接的分子，包含天然蛋白、人工蛋白、蛋白片段、突变蛋白和融合蛋白等。
术语“结构域”是指生物大分子中具有独立功能的特异结构区域，结构域具有独立的三级结构，其功能不依赖于生物大分子中的其余部分，本发明中的结构域特指蛋白中这样的区域，如重链可变区VH结构域、轻链可变区VL结构域，结构域之间相互结合可以构成一个大的结构域。
术语“抗体”指包含至少一个抗原识别位点并能特异性结合抗原的免疫球蛋白分子。在此，术语“抗原”是在机体内能诱发免疫应答且与抗体特异性结合的物质，如蛋白质、多肽、肽、碳水化合物、多聚核苷酸、脂质、半抗原或上述物质的组合。抗体与抗原的结合依靠二者间形成的相互作用来介导，包括氢键、范德华力、离子键以及疏水键。抗原表面与抗体结合的区域为“抗原决定簇”或“表位”，一般来说，每个抗原有多个决定簇。本发明所提及的术语“抗体”包含单克隆抗体(包括全长单克隆抗体)、多克隆抗体、抗体片段、包含至少两个不同表位结合结构域的多特异性抗体(例如，双特异性抗体)、人抗体、人源化抗体、翻译后修饰抗体、骆驼抗体、嵌合抗体、包含抗体抗原决定簇的融合蛋白、以及包含抗原识别位点的任何其他修饰的免疫球蛋白分子，只要这些抗体展现出所期望的生物活性。具体来说，抗体包括免疫球蛋白分子和免疫球蛋白分子的免疫活性片段，即至少含有一个抗原结合位点的分子。
术语“Fab”、“Fab区域”、“Fab片段”或“Fab分子”是抗原结合片段，包含免疫球蛋白重链的VH结构域、CH1结构域以及轻链的VL结构域、CL结构域，重链第一个恒定区结构域CH1与轻链的恒定区结构域CL结合，重链的可变区结构域VH和轻链的可变区结构域VL结合。
术语“Fc”、“Fc区域”、“Fc片段”或“Fc分子”是抗体的效应区，能够引起如CDC、ADCC、ADCP以及细胞因子释放等。天然的抗体Fc通常是由两个相同的蛋白片段结合构成，该蛋白片段包含两个或三个免疫球蛋白恒定区结构域。本发明中所述Fc包含天然Fc以及突变的Fc。虽然Fc区的边界可以变化，但人IgG重链Fc区通常定义为包含从C226或P230开始到其羧基末端的残基。在实验条件下，免疫球蛋白单体经木瓜蛋白酶酶切生成的片段分别为Fab和Fc。
术语“铰链”或“铰链区”是指包含抗体的第一和第二恒定结构域(CH1和CH2)之间的氨基酸的柔性多肽。
除非另有说明，否则本发明所述抗体氨基酸编号使用Kabat等人在1991年所阐述的编码方案，即“Kabat索引”或“Kabat编号”(Kabat，E.A.et al.Sequences of Proteinsof Immunological Interest，5th ed.，NIH Publication No.91-3242，Bethesda，MD.:1991)。
术语“抗原结合位点”指抗原结合分子与抗原产生直接相互作用的一个或多个氨基酸残基，抗体的抗原结合位点由抗原互补决定区(CDR)构成，天然免疫球蛋白分子通常包含两个抗原结合位点，Fab分子通常包含一个抗原结合位点。
术语“T细胞激活”指T淋巴细胞尤其是杀伤性T淋巴细胞的一种或多种免疫反应，包括：增殖、分化、细胞因子的释放、杀伤性效应分子的分泌以及细胞杀伤等。
术语“EC50”即半最大效应浓度(concentration for 50％of maximal effect)，是指引起50％最大效应所对应的抗体浓度。
本文所用“特异性结合”是指，两种分子间的非随机的结合反应，如抗体及其所针对的抗原之间的反应。在某些实施方案中，特异性结合某抗原的抗体(或对某抗原具有特异性的抗体)是指，抗体以小于大约10-5M，例如小于大约10-6M、10-7M、10-8M、10-9M、10-10M或更小的亲和力(KD)结合该抗原。在本发明的一些实施方案中，术语“靶向”指特异性结合。
本文所用“KD”是指，特定抗体-抗原相互作用的解离平衡常数，用于描述抗体与抗原间的结合亲和力。平衡解离常数越小，抗体-抗原结合越紧密，抗体与抗原之间的亲和力越高。通常，抗体以小于大约10-5M，例如小于大约10-6M、10-7M、10-8M、10-9M、10-10M或更小的平衡解离常(KD)结合抗原。
术语“单链可变区抗体片段”或“scFv”指免疫球蛋白重链可变区VH和轻链可变区VL的融合蛋白，包括N端为VH以及N端为VL的不同组合形式，利用构建重组蛋白的常规分子克隆方法即可制备(Sambrook JF，E.F.et al.Molecular cloning:a laboratorymanual.4th ed.Cold Spring Harbor Laboratory Press，Cold Spring Harbor,NewYork:2012)。
术语“人源化抗体”是指人源免疫球蛋白(受体抗体)的部分或者全部CDR区被一非人源抗体(供体抗体)的CDR区替换后得到的抗体或者抗体片段，其中的供体抗体可以是具有预期特异性、亲和性或者反应性的非人源(如小鼠、大鼠或兔)抗体。此外，受体抗体的架构区(FR)的一些氨基酸残基也可被相应的非人源抗体的氨基酸残基替换，或者被其他抗体的氨基酸残基替换，以进一步改善或者优化抗体的一种或多种特性。
本发明涉及药物组合物，其包含本发明的抗体或抗体片段、双特异性抗体或抗体偶联物，和任选地药学上可接受的载体、表面活性剂和/或稀释剂。在一些实施方案中，除本发明的抗体、双特异性抗体或抗体偶联物外，所述药物组合物还包含一种或多种另外的治疗剂。在一些实施方案中，所述另外的治疗剂包括但不限于化疗剂、生长抑制剂、细胞毒性剂、用于放射疗法的试剂、抗血管生成剂、凋亡剂、抗微管蛋白剂和其它治疗癌症的试剂。
术语“宿主细胞”指导入外源核酸的细胞及其后代，可通过编码多肽的核苷酸转化或转染，从而表达外源多肽。本发明所述的宿主细胞包括但不限于CHO细胞(Chinesehamster ovary cells，中国仓鼠卵巢细胞)、HEK293细胞(Human embryonic kidney cells293，人胚肾细胞293)、BHK细胞(Baby Hamster Kidney，幼仓鼠肾细胞)、骨髓瘤细胞、酵母、昆虫细胞或原核细胞如大肠杆菌(Escherichia coli)等。应当指出，本发明所述“宿主细胞”不仅指导入了外源核酸的细胞，同时还包括了该细胞的后代，由于在细胞分裂过程中后代细胞会发生突变，但仍属于本发明所述的术语范围。
本发明还进一步包含编码这些多肽链的核酸序列。在表达抗体的过程中，将核酸序列插入合适的载体中，载体包括但不限于：质粒、噬菌表达载体、柯斯质粒、人工染色体、噬菌体以及动物病毒等。表达载体中包含用于调控表达的元件，包括但不限于启动子、转录起始序列、增强子以及信号肽序等。启动子包括但不限于T7启动子、T3启动子、SP6启动子、β-actin启动子、EF-1α启动子、CMV启动子以及SV40启动子。将表达载体转入宿主细胞中可使用本领域内已知的合适方法，包括但不限于：磷酸钙沉淀法、聚乙烯亚胺转染法、脂质体转染法、电穿孔法以及PEI(聚乙烯亚胺)转染法。
实施例1 CH1/CL结合界面可用于改造的氨基酸位点评估
抗体的Fab恒定区CH1/CL结合界面的氨基酸通过疏水性相互作用、电荷相互作用、氢键等来维持侧链间的相互作用，上述相互作用产生于氨基酸在侧链范围约内。本实施例通过分析CH1/CL结合界面蛋白晶体结构，获得了潜在用于双特异性抗体的各CH1和CL结构域中可用于改造的氨基酸位点。通过PyMOL软件分析蛋白晶体结构(PDB ID：3EO9)，CH1-CL结构域中E-strand的结合界面请见图1。其中，相互作用较强的氨基酸位点已汇总于表1(修饰位点的汇总)中。
本发明中，如无特殊说明，氨基酸编号根据Kabat编号来记载。在本实施例中，H链使用IgG1、L链使用IgK(Kappa)或Lambda(λ)。
表1
实施例2 CH1/CL结合界面氨基酸位点突变的筛选
为了筛选不同的可产生特异性结合的CH1/CL界面，利用下述方法筛选CH1/CL界面配对效果较好的突变组合：
参照Hust等(Methods in Molecular Biology-Phage Display Volume 1701||Construction of Synthetic Antibody Phage-Display Libraries[J].2018.)的方法，针对表1中的CH1-kCL结合界面可用于改造的氨基酸位点，设计氨基酸的随机突变引物，构建噬菌体CH1/CL界面氨基酸位点随机突变的Fab展示文库。
通过释放、包装、沉淀噬菌体文库，用相应的生物素化抗原进行3轮筛选后，对筛选得到的克隆进一步用酶联免疫吸附试验检测，以其在波长450nm下的吸光值(OD450)反映突变克隆与抗原的结合能力。展示文库中相互作用较强的突变组合，能够获得较好的抗原结合能力，通过野生型的CH1-kCL克隆做对照，最终获得一系列抗原结合能力增强的CH1-kCL界面氨基酸点突变的单克隆，筛选得到的抗原结合能力增强的CH1-kCL结合界面氨基酸位点突变的克隆已汇总在表2中，表2中还展示了野生型和相应点突变克隆号的OD450和ΔOD450，其中，OD450为噬菌体克隆与抗原的吸光值，ΔOD450是点突变克隆与野生型的OD450之差。
表2中克隆的CH1/CL结合界面氨基酸位点突变组合汇总在表3(氨基酸位点突变的汇总)中，这些突变组合可分别在异二聚体双特异性抗体中，增强针对特定抗原的重/轻链正确配对。
表2
表3
实施例3四链双特异性抗体的设计和表达载体构建
(1)四链双特异性抗体的设计
本实施例中设计和构建的示例性双特异性抗体为“Y”字型IgG抗体(结构示意图参见图2)，其包括2条完整的异源重链和2条完整的异源轻链，共同形成抗体的Fab和Fc结构域。
本实施例中选取的靶点组合是GPC3和CD3，但不限于此靶点组合。本实施例中的双特异性抗体的第一Fab区结合人CD3，第二Fab区结合人GPC3。其中特异性结合人CD3的部分具有第一重链和第一轻链，特异性结合人GPC3的部分具有第二重链和第二轻链，其序列来源于本公司的GPC3序列。抗体的Fc部分按照专利CN102471378B中的方法进行的改造，其中对结合人CD3部分的重链做如下突变：P395K、P396K、V397K，该突变标记为OA；对结合人GPC3部分的重链做如下突变：T394D、P395D、P396D，该突变标记为OB，使其形成重链异源二聚体。该双特异性抗体在本文中称为阴性对照WT0，其2条异源轻链与重链随机配对，结构示意图如图2中的III。
抗CD3轻链氨基酸序列(SEQ ID NO:117)：
DIQMTQEPSLTTSPGGTVTLTCRSSTGAVTTSNYANWVQEKPGQAPRGLIGGTNKRAPGTPARFSGSLIGGKAALTITGVQPEDEAIYFCALWYSNLWVFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC；
抗CD3重链氨基酸序列(SEQ ID NO:119)：
EVQLVESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAMYYCVRHGNFGTSYVSWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTKKKLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK；
抗GPC3轻链氨基酸序列(SEQ ID NO:121)：
DIVLTQSPLSLTVTPGEPASISCRSSESLVHSNGNTYLSWLQQRPGQPPRLLIYKISNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCVQVSSFPYTFGQGTKVEIKTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC；
抗GPC3重链氨基酸序列(SEQ ID NO:123)：
QVQLVQSGAEVKKPGASVKLSCKASGYTFTDYEMHWVKQTPGKGLKWIGALDPKTGDTAYAQKFQGRATLTADTSTDTAYMELSSLRSEDSAVYYCTRFYSYTYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTDDDVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。
除以上轻链天然配对组合WT0外，本文分别设计了三种重轻链防错配GPC3×CD3双特异性抗体分子，CM-C(GPC3 Fab×Crossmab-CD3)、CM-G(Crossmab-GPC3×CD3 Fab)及WT(GPC3 scFv×CD3 Fab)。其中，CM-C双特异性抗体的2条异源重链和2条异源轻链序列分别是：SEQ ID NO:163、SEQ ID NO:123和SEQ ID NO:161、SEQ ID NO:121，CM-G双特异性抗体的2条异源重链和2条异源轻链序列分别是：SEQ ID NO:119、SEQ ID NO:167和SEQ ID NO:117、SEQ ID NO:165，结构示意图如图2中的I。WT双特异性抗体中2条异源重链和1条轻链序列分别是：SEQ ID NO:119、SEQ ID NO:159和SEQ ID NO:117，结构示意图如图2中的II。本文共设计了以上4种对照GPC3×CD3双特异性抗体，所有对照组的分子的Fc均含有OA/OB，仅仅是两边抗原结合臂不同，详情已汇总于表4中。
表4 GPC3×CD3双特异性抗体的4种对照抗体重链及轻链序列
SEQ ID NO:163：
EVQLVESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAMYYCVRHGNFGTSYVSWFAYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTKKKLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK；
SEQ ID NO:161：
DIQMTQEPSLTTSPGGTVTLTCRSSTGAVTTSNYANWVQEKPGQAPRGLIGGTNKRAPGTPARFSGSLIGGKAALTITGVQPEDEAIYFCALWYSNLWVFGGGTKLEIKRSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV；
SEQ ID NO:167：
QVQLVQSGAEVKKPGASVKLSCKASGYTFTDYEMHWVKQTPGKGLKWIGALDPKTGDTAYAQKFQGRATLTADTSTDTAYMELSSLRSEDSAVYYCTRFYSYTYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTDDDVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK；
SEQ ID NO:165：
DIVLTQSPLSLTVTPGEPASISCRSSESLVHSNGNTYLSWLQQRPGQPPRLLIYKISNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCVQVSSFPYTFGQGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV；
SEQ ID NO:159：
QVQLVQSGAEVKKPGASVKLSCKASGYTFTDYEMHWVKQTPGKGLKWIGALDPKTGDTAYAQKFQGRATLTADTSTDTAYMELSSLRSEDSAVYYCTRFYSYTYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVLTQSPLSLTVTPGEPASISCRSSESLVHSNGNTYLSWLQQRPGQPPRLLIYKISNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCVQVSSFPYTFGQGTKVEIK。
(2)表达质粒的分子克隆
人工合成编码CD3轻链(SEQ ID NO:117)的核苷酸序列和编码CD3重链(SEQ IDNO:119)的核苷酸序列。利用通用的分子克隆的方法(Sambrook JF,E.F.et al.Molecularcloning:alaboratory manual.4th ed.Cold Spring Harbor Laboratory Press,ColdSpring Harbor,New York:2012)，将编码CD3轻链的核苷酸序列克隆到改造过的质粒pCDNA3.1(+)(Invitrogen，货号V790-20)上，所得的表达质粒命名为pCDNA3.1-CD3-LC。将编码CD3重链的核苷酸序列克隆到质粒pFUSE-hIgG1-Fc2(InvivoGene)上，所得的表达质粒命名为pFUSE-CD3-HC-OA。
人工合成编码抗GPC3轻链(SEQ ID NO:121)的核苷酸序列和编码抗GPC3重链(SEQID NO:123)的核苷酸序列。利用通用的分子克隆的方法，将编码抗GPC3轻链的核苷酸序列克隆到上述改造过的质粒pCDNA3.1(+)上，使其能够在细胞(例如HEK293细胞)中表达分泌抗体，所得的表达质粒命名为pCDNA3.1-GPC3-LC；将编码抗GPC3重链的核苷酸序列克隆到质粒pFUSE-hIgG1-Fc2(InvivoGene)上，所得的表达质粒命名pFUSE-GPC3-HC-OB。
(3)对四链双特异性抗体的改造
通过对CH1和CL界面氨基酸进行定突变筛选，获得了表3中的CH1-CL结合界面氨基酸位点突变组合，从表3的突变组合中引入2种示例性GPC3×CD3(11A10×8D6、7A10×11B7)四链双特异性抗体，分别命名为MutA、MutB(参见表5)。其中，MutA在双特异性抗体WT0的基础上，在抗CD3重链序列中引入T106H、S186T、S188Y和V190F突变，并在抗CD3轻链序列中引入S174A、S176E和T178A突变，在抗GPC3重链序列中引入S186M、S188I和V190W突变，并在抗GPC3轻链序列中引入S174R、S176H和T178V突变；MutB在抗CD3重链序列中引入T106H、S186V、S188L和V190M突变，并在抗CD3轻链序列中引入S174R、S176W和T178L突变，在抗GPC3重链序列中引入S186T、S188R和V190F突变。
表5 GPC3×CD3双特异性抗体突变组合I
为进一步增加轻链对重链的选择性，防止轻链错配，通过氨基酸大小和定点引入电荷突变，对抗体序列进行氨基酸点突变，得到表6中所示的GPC3×CD3四链双特异性抗体突变组合MutA+D-D和MutB+K-K。其中，MutA+D-D为在MutA的基础上，在抗CD3轻链序列中引入A178D突变，并在抗GPC3重链中引入I188D突变；MutB+K-K为在MutB的基础上，在抗CD3轻链中引入L178K突变，并在抗GPC3重链中引入T192K突变。
表6 GPC3×CD3双特异性抗体突变组合II
实施例4四链双特异性抗体的表达与纯化
(1)GPC3×CD3四链双特异性抗体的表达
采用无内毒素质粒大提试剂盒(Endo-Free-Plasmid Maxi Kit(100)，购自OMEGA公司，商品目录号为D6926-04)进行质粒大量提取，操作步骤按照试剂盒所提供的说明书进行。使用实施例1中所构建的表达载体即表5和表6所列的突变组合MutA、MutB、MutA+D-D和MutB+K-K分别对细胞进行共转染。将转染后的细胞悬浮液放置在37℃、5％CO2、120rpm的培养摇床中避光培养4～5天。
(2)GPC3×CD3四链双特异性抗体的纯化
离心收集表达上清并用0.22μm滤膜过滤细胞上清。使用已经平衡好的蛋白A亲和层析填料(MabSelectSuReTM，购自GE Healthcare公司，货号为17-5438-02)捕获上清液中的双特异性抗体蛋白，使用洗脱缓冲液洗脱5个柱体积，收集洗脱液，并用中和缓冲液调节pH至中性。将洗脱后的样品通过SDS-PAGE进行分析。
各个对照的双特异性抗体WT0、CM-C、CM-G和WT，经ProteinA纯化后目的蛋白的纯度示于图3A～图3D中。其中，WT0是阴性对照抗体，CM-C是抗CD3分子的Crossmab-CD3双特异性抗体，CM-G是抗GPC3分子的Crossmab-GPC3双特异性抗体，WT是三链阳性对照抗体。结果显示，由于所有的组合中Fc都含有OA/OB突变，所有双特异性抗体的两条重链都能以等摩尔比配成异源二聚体，即所有分子的两条异源重链在还原SDS-PAGE中的表达比例为1:1。但两条异源轻链的配比却相差甚远，在阴性对照WT0、Crossmab-CD3以及Crossmab-GPC3中(图3A、图3B和图3C)，两条轻链的配比相差较远，说明这些轻链与它们相应的重链不能形成1:1的配对，不能有效地产生目标双抗，然而在MutA、MutB、MutA+D-D以及MutB+K-K分子中(图3E和图3F)，两条轻链是等量的，说明它们轻链与相应的重链配对良好，能够有效地产生目标双抗分子。
实施例5四链双特异性抗体的抗原结合检测
为了验证本发明中根据氨基酸大小和定点引入电荷突变改造形成的四链双特异性抗体抗原结合能力，使用ELISA分别检测纯化的四链双特异性抗体对抗原CD3和抗原GPC3的结合检测。具体步骤如下。
(1)实验使用的抗原为人GPC3抗原(购自Acrobiosystems公司)和人CD3抗原(购自Sino Biological Inc.公司)。
(2)包被：用1×PBS将人GPC3抗原、人CD3抗原稀释，将稀释好的抗原加入酶标板中，每孔加200μL，用封板膜封住反应孔，室温包被1.5小时或者4℃包被过夜。使用0.05％PBST洗板6次。
(3)封闭：用脱脂奶粉和PBS Buffer配制3％M-PBS，向酶标板每孔加300μL 3％MPBS，用封板膜封住反应孔，室温孵育1小时。使用0.05％PBST洗板6次。
(4)抗体结合：以100μL/孔加入梯度浓度稀释的抗体，每个抗体浓度以2个复孔进行实验。添加抗体后，用封板膜封住反应孔，室温孵育1.5小时。使用0.05％PBST洗板6次。
(5)二抗：用3％M-PBS溶液按照1:2000稀释二抗即HRP标记的山羊抗人IgG(购自Peprotech公司，商品目录号为SA0001-17)，将稀释后的二抗添加到孔中。用封板膜封住反应孔，室温孵育1小时。使用0.05％PBST洗板6次。
(6)显色：以100μL/孔加入TMB显色液，室温避光孵育3～5min。
(7)终止：以100μL/孔加入终止液(1M HCl)，混匀后立即测量450nm处的OD值。
(8)数据分析：使用GraphPad Prism 5软件进行数据处理。生成以log(样品浓度)为横坐标、OD值为纵坐标绘制剂量反应曲线，计算EC50，以EC50代表样品与抗原的结合能力。
表7是GPC3×CD3四链双特异性抗体MutA、MutB与阴性对照WT0、CM-C以及CM-G对GPC3抗原和CD3抗原结合能力的对比结果，表8是GPC3×CD3四链双特异性抗体MutA+D-D、MutB+K-K与阴性对照WT0、CM-C和CM-G对GPC3抗原和CD3抗原结合能力的对比结果，表9是GPC3×CD3四链双特异性抗体MutA、MutB与三链阳性对照抗体WT对GPC3抗原和CD3抗原结合能力的对比结果，表10是GPC3×CD3四链双特异性抗体MutA+D-D、MutB+K-K与三链阳性对照抗体WT对GPC3抗原和CD3抗原结合能力的对比结果。
表7
表8
表9
表10
图4A～图4D是MutA、MutB、MutA+D-D以及MutB+K-K与阴性对照WT0、CM-C及CM-G对GPC3和CD3的亲和力的比较，图4A和图4B显示MutA、MutB对两个靶抗原的亲和力优于阴性对照WT0、CM-C以及CM-G，同样图4C和图4D显示MutA+D-D、MutB+K-K对两个靶抗原的亲和力也优于阴性对照WT0、CM-C以及CM-G。
图5A～图5D是MutA、MutB、MutA+D-D以及MutB+K-K与不会发生轻链错配的三链阳性对照抗体WT对GPC3和CD3的亲和力的比较，结果显示，MutA、MutB、MutA+D-D以MutB+K-K对两个靶抗原的结合活性都很接近三链阳性对照抗体WT，说明这些分子基本没有发生错配。
上述结果表明，通过与阴性对照WT0、CM-C、CM-G以及三链阳性对照抗体WT作比较，本发明的抗GPC3×抗CD3四链双特异性抗体MutA、MutB、MutA+D-D以及MutB+K-K比阴性对照WT0、CM-C以及CM-G对GPC3抗原和CD3抗原有具更强的结合活性，且与不会发生轻链错配的三链阳性对照抗体WT一致。由此可见，经本发明的方法改造的四链双特异性抗体保持了对抗原结合的能力。
实施例6四链双特异性抗体的流式结合活性验证
为了进一步研究GPC3×CD3四链双特异性抗体对GPC3的结合活性，本发明使用流式细胞术检测其与表达GPC3的细胞的结合活性。
首先，将GPC3克隆到pcDNA3.1载体中，感染、扩增。利用大提试剂盒(OMEGA)进行质粒提取，具体操作按照试剂盒说明书进行。用转染试剂PEI将上述质粒转染到293F细胞中，构建瞬时表达GPC3细胞，命名为293F/GPC3细胞，以293F细胞作为阴性对照细胞。
收集293F、293F/GPC3细胞，以4℃预冷的2％FBS/PBS(稀释液)洗涤一次，再以适量的稀释液重悬细胞，使细胞密度为5.0×106/mL。将细胞以100μL/孔接种于96孔板上，即接种量为5.0×105/well。以稀释液配制抗GPC3×抗CD3四链双特异性抗体，浓度为5μg/mL。取100μL抗体溶液分别与等体积的293F或293F/GPC3混合，即终浓度为2.5μg/mL，把96孔板置于2～8℃孵育1h。将96孔板在500×g、4℃下离心5min，去除上清，再以200μL稀释液洗涤两次。用PE抗人IgG FC(Invitrogen)稀释液重悬细胞，2～8℃避光孵育1h。将96孔板在500×g、4℃下离心5min，去除上清，再用200μL稀释液洗涤两次。用200μL稀释液重悬细胞后立刻上机检测。
结果如图6所示，GPC3×CD3四链双特异性抗体MutA、MutB、MutA+D-D以及MutB+K-K与GPC3均具有较高的结合活性，且结合活性与不会发生轻链错配的三链阳性对照抗体WT一致。
实施例7四链双特异性抗体的T细胞活化检测
采用Jurkat/NFAT-Luc报告基因细胞系和GPC3阳性的人肝癌细胞HepG2细胞组成的检测系统，来评价GPC3×CD3四链双特异性抗体对T细胞的激活作用。在Jurkat/NFAT-Luc细胞系中，荧光素酶基因受NFAT(Nuclear factor of activated T-cells)转录因子调控，作为效应细胞。GPC3阳性的HepG2细胞作为靶细胞。当Jurkat/NFAT-Luc细胞与HepG2细胞共培养时，荧光素酶不表达。而当加入GPC3×CD3四链双特异性抗体时，HepG2细胞上的GPC3通过GPC3×CD3四链双特异性抗体激活Jurkat/NFAT-Luc细胞内的CD3信号通路，促使荧光素酶表达，从而可以通过检测细胞中的荧光素酶表达量来判断Jurkat T细胞激活程度的强弱。
实验中，在96孔细胞培养板中依次加入适量HepG2细胞、Jurkat/NFAT-Luc细胞。再将抗GPC3×CD3四链双特异性抗体稀释到浓度为30、7.5、1.875、0.4688、0.1172、0.0293、0.0073、0.0018、0.0005、0.0001μg/mL。将不同浓度的抗GPC3×CD3四链双特异性抗体加入到上述细胞培养板中，37℃静置培养适当时间后，加入ONE-Glo Luciferase检测试剂，使用多功能酶标仪测定化学发光值。以抗体浓度的对数值为横坐标，平均化学发光值为纵坐标，进行四参数拟合，绘制剂量效应曲线，获得各曲线的EC50值，以EC50代表GPC3×CD3四链双特异性抗体激活T细胞的活性。
表11是GPC3×CD3四链双特异性抗体MutA、MutB与阴性对照WT0、CM-C和CM-G激活T细胞活性的对比结果，表12是GPC3×CD3四链双特异性抗体MutA+D-D、MutB+K-K与阴性对照WT0、CM-C和CM-G激活T细胞活性的对比结果，表13是GPC3×CD3四链双特异性抗体MutA、MutB与三链阳性对照抗体WT激活T细胞活性的对比结果，表14是GPC3×CD3四链双特异性抗体MutA+D-D、MutB+K-K与三链阳性对照抗体WT激活T细胞活性的对比结果。
表11
表12
表13
表14
图7A～图7B是MutA、MutB、MutA+D-D以及MutB+K-K与阴性对照WT0、CM-C以及CM-G激活T细胞活性的比较结果，结果显示，MutA、MutB、MutA+D-D以及MutB+K-K对T细胞的激活活性高于阴性对照WT0、CM-C以及CM-G。
图8A～图8B是MutA、MutB、MutA+D-D以及MutB+K-K与不会发生轻链错配的三链阳性对照抗体WT激活T细胞活性的比较结果，结果显示，MutA、MutB、MutA+D-D以及MutB+K-K对T细胞的激活活性与不会发生轻链错配的三链阳性对照抗体WT一致。
上述结果表明，当与GPC3阳性肿瘤细胞HepG2、Jurkat/NFAT-Luc细胞共同孵育时，本文发明的GPC3×CD3四链双特异性抗体MutA、MutB、MutA+D-D以及MutB+K-K比阴性对照WT0、CM-C以及CM-G对Jurkat T细胞有更好的激活作用，且激活效果与不会发生轻链错配的三链阳性对照抗体WT一致。
实施例8四链双特异性抗体的T细胞肿瘤杀伤检测(CTL)
GPC3×CD3四链双特异性抗体的CD3结合臂特异性地结合T细胞表面的CD3复合物，另一端GPC3结合臂特异性地结合肿瘤细胞表面GPC3分子，使T细胞与肿瘤细胞之间形成免疫桥联，继而激活T细胞，释放穿孔素(Perforin)和颗粒酶B(Granzyme B)等细胞杀伤蛋白，从而杀伤肿瘤细胞。当肿瘤细胞膜受损后，细胞膜通透性增大，胞浆中的乳酸脱氢酶(LDH)被释放到培养上清中。取一定量的上清，加入乳酸脱氢酶的反应底物乳酸后，乳酸脱氢酶催化脱氢反应，产生红色产物甲臜(Formazan)，可在490nm波长下产生吸收峰。细胞上清的乳酸脱氢酶含量越多，其颜色越深，吸光值越大；故可通过测定吸光值来定量肿瘤细胞释放乳酸脱氢酶的量，并计算抗GPC3×抗CD3四链双特异性抗体介导PBMC杀伤肿瘤细胞的杀伤活性。
杀伤率的计算公式为：细胞毒性(％)＝(OD样品-SR)/(MR-SR)×100％
其中，SR＝OD自发释放孔(靶细胞+效应细胞)，MR＝OD最大释放孔(靶细胞)。
(1)PBMC的准备
外周血单核细胞(Peripheral Blood Mononuclear Cell，PBMC)指外周血中具有单个核的细胞，包括淋巴细胞、单个核细胞、树突细胞和少量其他细胞(造血干细胞等)。用Ficoll离心法分离PBMC，吸取中间的白膜并通过反复离心洗涤，可获得相对较纯的PBMC。以无酚红RPMI 1640(含5％FBS)把PBMC的密度调整为2.00×106个/mL，待用。
(2)靶细胞的准备
本发明所用的GPC3阳性靶细胞为人肝癌细胞HepG2。为了获得生长状态良好的靶细胞，需在实验前1～2天进行传代培养。取生长良好的HepG2，吸出培养基，以适量1×PBS(pH 7.4)洗涤2次培养瓶；加入2mL 0.05％胰蛋白酶-EDTA在37℃下消化后，以相应培养基终止消化，1000rpm(190×g)水平离心5min收集细胞。用1×PBS(pH 7.4)洗涤2次后用无酚红RPMI 1640完全培养基调整细胞浓度为2.00×105个/mL。
(3)样品的准备
用1×PBS缓冲液(pH 7.4)将样品稀释为30μg/mL，再进行4倍梯度稀释，共10个梯度，其浓度依次为30、7.5、1.875、0.4688、0.1172、0.0293、0.0073、0.0018、0.0005和0.0001μg/mL。
(4)孵育和检测
取96孔细胞培养板，依次加入50μL/well靶细胞、100μL/well PBMC和50μL/well梯度稀释的样品，混匀，使得PBMC与靶细胞的比例为20:1，样品的起始浓度为10μg/mL。对照孔用无酚红RPMI 1640培养基补足200μL。把培养板置于37℃、5％CO2培养箱内孵育。用乳酸脱氢酶细胞毒性试剂盒检测细胞的杀伤毒性。以抗体浓度的对数值为横坐标，杀伤活性为纵坐标，进行四参数拟合，绘制剂量效应曲线，获得各曲线的EC50值，以EC50代表GPC3×CD3四链双特异性抗体对靶细胞的杀伤活性。
表15是GPC3×CD3四链双特异性抗体MutA+D-D与阴性对照WT0、CM-C、CM-G对GPC3高表达的HepG2细胞杀伤效果的对比结果，表16是GPC3×CD3四链双特异性抗体MutA、MutB以及三链阳性对照抗体WT对GPC3高表达的HepG2细胞杀伤效果的对比结果，表17是MutA+D-D、MutB+K-K与三链阳性对照抗体WT对GPC3高表达的HepG2细胞杀伤效果的对比结果。
表15
表16
表17
图9A为MutA+D-D与阴性对照WT0、CM-C和CM-G对肿瘤细胞的杀伤活性的结果对比；图9B为MutA、MutB以及三链阳性抗体WT对肿瘤细胞的杀伤活性的结果对比；图9C为MutA+D-D、MutB+K-K以及三链阳性抗体WT对肿瘤细胞的杀伤活性的结果对比。图9A显示MutA+D-D的杀肿瘤活性高于阴性对照WT0、CM-C以及CM-G。图9B和图9C显示MutA、MutB、MutA+D-D以及MutB+K-K的肿瘤杀伤活性与不会发生轻链错配的三链阳性对照抗体WT一致，进一步说明本发明中的四链改造有效地去除了两个不同轻链错配的问题。
上述结果表明，通过与阴性对照WT0、CM-C、CM-G以及三链阳性对照抗体WT作比较，本文发明的抗GPC3×CD3四链双特异性抗体MutA、MutB、MutA+D-D以及MutB+K-K比阴性对照WT0、CM-C以及CM-G对GPC3高表达的HepG2细胞有更好的杀伤效果，且杀伤效果与不会发生轻链错配的三链阳性对照抗体WT一致。
综上所述，本发明通过对抗体的重链CH1和轻链CL的界面氨基酸进行突变，显著降低了重链与轻链发生错配的概率，提高了其对特定抗体的结合能力，特异性更好，提高了目标抗体的产率；抗GPC3×CD3四链双特异性抗体MutA、MutB、MutA+D-D以及MutB+K-K对靶抗原具有更好的结合活性，更强的T细胞活性激活能力以及肿瘤细胞杀伤能力，具有广阔的应用前景。
申请人声明，本发明通过上述实施例来说明本发明的详细方法，但本发明并不局限于上述详细方法，即不意味着本发明必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了，对本发明的任何改进，对本发明产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本发明的保护范围和公开范围之内。
序列表
<110> 广州爱思迈生物医药科技有限公司
<120> 双特异性抗体及其应用
<130> 2021
<160> 168
<170> PatentIn version 3.3
<210> 1
<211> 98
<212> PRT
<213> 人工序列 7D5 CH1
<400> 1
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 Ala
50 55 60
Leu Tyr 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
Lys Val
<210> 2
<211> 294
<212> DNA
<213> 人工序列 7D5 CH1
<400> 2
gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60
ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180
ggactctacg cgctctatag cgttgtgacc gtgccctcca gcagcttggg cacccagacc 240
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agtt 294
<210> 3
<211> 106
<212> PRT
<213> 人工序列 7D5 CL
<400> 3
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
1 5 10 15
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
20 25 30
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
35 40 45
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
50 55 60
Tyr Ser Leu Met Ser Met Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
65 70 75 80
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
85 90 95
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 105
<210> 4
<211> 318
<212> DNA
<213> 人工序列 7D5 CL
<400> 4
actgtcgcag ctccatccgt gttcatcttt ccaccctccg acgagcaact gaaaagtggc 60
acagcctccg tcgtctgtct gctgaacaat ttctaccctc gggaggccaa ggtccagtgg 120
aaggtggaca acgctctcca gagtggaaat agccaagagt ccgttactga acaggactct 180
aaggattcaa cctacagtct catgagcatg ctgactctga gcaaggcaga ttatgagaag 240
cacaaagtct acgcatgtga agtgactcat cagggactca gctcacccgt taccaagtct 300
ttcaacaggg gagagtgt 318
<210> 5
<211> 98
<212> PRT
<213> 人工序列 7D10 CH1
<400> 5
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 Thr
50 55 60
Leu Met Ser Thr 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
Lys Val
<210> 6
<211> 294
<212> DNA
<213> 人工序列 7D10 CH1
<400> 6
gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60
ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180
ggactctaca cgctcatgag cacggtgacc gtgccctcca gcagcttggg cacccagacc 240
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agtt 294
<210> 7
<211> 106
<212> PRT
<213> 人工序列 7D10 CL
<400> 7
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
1 5 10 15
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
20 25 30
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
35 40 45
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
50 55 60
Tyr Phe Leu Leu Ser Met Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
65 70 75 80
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
85 90 95
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 105
<210> 8
<211> 318
<212> DNA
<213> 人工序列 7D10 CL
<400> 8
actgtcgcag ctccatccgt gttcatcttt ccaccctccg acgagcaact gaaaagtggc 60
acagcctccg tcgtctgtct gctgaacaat ttctaccctc gggaggccaa ggtccagtgg 120
aaggtggaca acgctctcca gagtggaaat agccaagagt ccgttactga gcaggactct 180
aaggattcaa cctactttct cctgagcatg ctgactctga gcaaggcaga ttatgagaag 240
cacaaagtct acgcatgtga agtgactcat cagggactca gctcacccgt taccaagtct 300
ttcaacaggg gagagtgt 318
<210> 9
<211> 98
<212> PRT
<213> 人工序列 7D11 CH1
<400> 9
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 Glu
50 55 60
Leu Thr Ser Ile 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
Lys Val
<210> 10
<211> 294
<212> DNA
<213> 人工序列 7D11 CH1
<400> 10
gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60
ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180
ggactctacg agctcacgag cattgtgacc gtgccctcca gcagcttggg cacccagacc 240
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agtt 294
<210> 11
<211> 106
<212> PRT
<213> 人工序列 7D11 CL
<400> 11
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
1 5 10 15
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
20 25 30
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
35 40 45
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
50 55 60
Tyr Phe Leu Ser Ser Tyr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
65 70 75 80
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
85 90 95
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 105
<210> 12
<211> 318
<212> DNA
<213> 人工序列 7D11 CL
<400> 12
actgtcgcag ctccatccgt gttcatcttt ccaccctccg acgagcaact gaaaagtggc 60
acagcctccg tcgtctgtct gctgaacaat ttctaccctc gggaggccaa ggtccagtgg 120
aaggtggaca acgctctcca gagtggaaat agccaagagt ccgttactga gcaggactct 180
aaggattcaa cctactttct cagtagctat ctgactctga gcaaggcaga ttatgagaag 240
cacaaagtct acgcatgtga agtgactcat cagggactca gctcacccgt taccaagtct 300
ttcaacaggg gagagtgt 318
<210> 13
<211> 98
<212> PRT
<213> 人工序列 7F4 CH1
<400> 13
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 Gln
50 55 60
Leu Phe Ser Thr 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
Lys Val
<210> 14
<211> 294
<212> DNA
<213> 人工序列 7F4 CH1
<400> 14
gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60
ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180
ggactctacc agctctttag cacggtgacc gtgccctcca gcagcttggg cacccagacc 240
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agtt 294
<210> 15
<211> 106
<212> PRT
<213> 人工序列 7F4 CL
<400> 15
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
1 5 10 15
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
20 25 30
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
35 40 45
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
50 55 60
Tyr Phe Leu Tyr Ser Leu Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
65 70 75 80
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
85 90 95
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 105
<210> 16
<211> 318
<212> DNA
<213> 人工序列 7F4 CL
<400> 16
actgtcgcag ctccatccgt gttcatcttt ccaccctccg acgagcaact gaaaagtggc 60
acagcctccg tcgtctgtct gctgaacaat ttctaccctc gggaggccaa ggtccagtgg 120
aaggtggaca acgctctcca gagtggaaat agccaagagt ccgttactga gcaggactct 180
aaggattcaa cctactttct ctatagcctg ctgactctga gcaaggcaga ttatgagaag 240
cacaaagtct acgcatgtga agtgactcat cagggactca gctcacccgt taccaagtct 300
ttcaacaggg gagagtgt 318
<210> 17
<211> 98
<212> PRT
<213> 人工序列 7F10 CH1
<400> 17
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 Met
50 55 60
Leu Thr Ser Trp 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
Lys Val
<210> 18
<211> 294
<212> DNA
<213> 人工序列 7F10 CH1
<400> 18
gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60
ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180
ggactctaca tgctcacgag ctgggtgacc gtgccctcca gcagcttggg cacccagacc 240
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agtt 294
<210> 19
<211> 106
<212> PRT
<213> 人工序列 7F10 CL
<400> 19
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
1 5 10 15
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
20 25 30
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
35 40 45
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
50 55 60
Tyr Asn Leu Ala Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
65 70 75 80
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
85 90 95
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 105
<210> 20
<211> 318
<212> DNA
<213> 人工序列 7F10 CL
<400> 20
actgtcgcag ctccatccgt gttcatcttt ccaccctccg acgagcaact gaaaagtggc 60
acagcctccg tcgtctgtct gctgaacaat ttctaccctc gggaggccaa ggtccagtgg 120
aaggtggaca acgctctcca gagtggaaat agccaagagt ccgttactga gcaggactct 180
aaggattcaa cctacaatct cgctagcacg ctgactctga gcaaggcaga ttatgagaag 240
cacaaagtct acgcatgtga agtgactcat cagggactca gctcacccgt taccaagtct 300
ttcaacaggg gagagtgt 318
<210> 21
<211> 98
<212> PRT
<213> 人工序列 7G1 CH1
<400> 21
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 Tyr Ser Met 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
Lys Val
<210> 22
<211> 294
<212> DNA
<213> 人工序列 7G1 CH1
<400> 22
gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60
ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180
ggactctact cgctctatag catggtgacc gtgccctcca gcagcttggg cacccagacc 240
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agtt 294
<210> 23
<211> 106
<212> PRT
<213> 人工序列 7G1 CL
<400> 23
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
1 5 10 15
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
20 25 30
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
35 40 45
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
50 55 60
Tyr Met Leu Ile Ser Ile Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
65 70 75 80
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
85 90 95
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 105
<210> 24
<211> 318
<212> DNA
<213> 人工序列 7G1 CL
<400> 24
actgtcgcag ctccatccgt gttcatcttt ccaccctccg acgagcaact gaaaagtggc 60
acagcctccg tcgtctgtct gctgaacaat ttctaccctc gggaggccaa ggtccagtgg 120
aaggtggaca acgctctcca gagtggaaat agccaagagt ccgttactga gcaggactct 180
aaggattcaa cctacatgct cattagcatt ctgactctga gcaaggcaga ttatgagaag 240
cacaaagtct acgcatgtga agtgactcat cagggactca gctcacccgt taccaagtct 300
ttcaacaggg gagagtgt 318
<210> 25
<211> 98
<212> PRT
<213> 人工序列 7H11 CH1
<400> 25
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 Asp
50 55 60
Leu Tyr 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
Lys Val
<210> 26
<211> 294
<212> DNA
<213> 人工序列 7H11 CH1
<400> 26
gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60
ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180
ggactctacg atctctatag cgttgtgacc gtgccctcca gcagcttggg cacccagacc 240
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agtt 294
<210> 27
<211> 106
<212> PRT
<213> 人工序列 7H11 CL
<400> 27
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
1 5 10 15
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
20 25 30
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
35 40 45
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
50 55 60
Tyr Asn Leu Leu Ser Ala Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
65 70 75 80
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
85 90 95
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 105
<210> 28
<211> 318
<212> DNA
<213> 人工序列 7H11 CL
<400> 28
actgtcgcag ctccatccgt gttcatcttt ccaccctccg acgagcaact gaaaagtggc 60
acagcctccg tcgtctgtct gctgaacaat ttctaccctc gggaggccaa ggtccagtgg 120
aaggtggaca acgctctcca gagtggaaat agccaagagt ccgttactga gcaggactct 180
aaggattcaa cctacaatct cctgagcgcg ctgactctga gcaaggcaga ttatgagaag 240
cacaaagtct acgcatgtga agtgactcat cagggactca gctcacccgt taccaagtct 300
ttcaacaggg gagagtgt 318
<210> 29
<211> 98
<212> PRT
<213> 人工序列 8C10 CH1
<400> 29
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 Lys
50 55 60
Leu Val Ser Tyr 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
Lys Val
<210> 30
<211> 294
<212> DNA
<213> 人工序列 8C10 CH1
<400> 30
gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60
ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180
ggactctaca agctcgtgag ctatgtgacc gtgccctcca gcagcttggg cacccagacc 240
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agtt 294
<210> 31
<211> 106
<212> PRT
<213> 人工序列 8C10 CL
<400> 31
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
1 5 10 15
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
20 25 30
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
35 40 45
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
50 55 60
Tyr Asn Leu Tyr Ser Leu Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
65 70 75 80
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
85 90 95
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 105
<210> 32
<211> 318
<212> DNA
<213> 人工序列 8C10 CL
<400> 32
actgtcgcag ctccatccgt gttcatcttt ccaccctccg acgagcaact gaaaagtggc 60
acagcctccg tcgtctgtct gctgaacaat ttctaccctc gggaggccaa ggtccagtgg 120
aaggtggaca acgctctcca gagtggaaat agccaagagt ccgttactga gcaggactct 180
aaggattcaa cctacaatct ctatagcctg ctgactctga gcaaggcaga ttatgagaag 240
cacaaagtct acgcatgtga agtgactcat cagggactca gctcacccgt taccaagtct 300
ttcaacaggg gagagtgt 318
<210> 33
<211> 98
<212> PRT
<213> 人工序列 8D6 CH1
<400> 33
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 Thr
50 55 60
Leu Tyr Ser Phe 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
Lys Val
<210> 34
<211> 294
<212> DNA
<213> 人工序列 8D6 CH1
<400> 34
gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60
ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180
ggactctaca cgctctatag ctttgtgacc gtgccctcca gcagcttggg cacccagacc 240
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agtt 294
<210> 35
<211> 106
<212> PRT
<213> 人工序列 8D6 CL
<400> 35
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
1 5 10 15
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
20 25 30
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
35 40 45
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
50 55 60
Tyr Ala Leu Glu Ser Ala Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
65 70 75 80
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
85 90 95
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 105
<210> 36
<211> 318
<212> DNA
<213> 人工序列 8D6 CL
<400> 36
actgtcgcag ctccatccgt gttcatcttt ccaccctccg acgagcaact gaaaagtggc 60
acagcctccg tcgtctgtct gctgaacaat ttctaccctc gggaggccaa ggtccagtgg 120
aaggtggaca acgctctcca gagtggaaat agccaagagt ccgttactga gcaggactct 180
aaggattcaa cctacgctct cgagagcgct ctgactctga gcaaggcaga ttatgagaag 240
cacaaagtct acgcatgtga agtgactcat cagggactca gctcacccgt taccaagtct 300
ttcaacaggg gagagtgt 318
<210> 37
<211> 98
<212> PRT
<213> 人工序列 8E10 CH1
<400> 37
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 Met
50 55 60
Leu Leu 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
Lys Val
<210> 38
<211> 294
<212> DNA
<213> 人工序列 8E10 CH1
<400> 38
gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60
ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180
ggactctaca tgctcttgag cgttgtgacc gtgccctcca gcagcttggg cacccagacc 240
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agtt 294
<210> 39
<211> 106
<212> PRT
<213> 人工序列 8E10 CL
<400> 39
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
1 5 10 15
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
20 25 30
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
35 40 45
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
50 55 60
Tyr Thr Leu Ala Ser Val Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
65 70 75 80
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
85 90 95
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 105
<210> 40
<211> 318
<212> DNA
<213> 人工序列 8E10 CL
<400> 40
actgtcgcag ctccatccgt gttcatcttt ccaccctccg acgagcaact gaaaagtggc 60
acagcctccg tcgtctgtct gctgaacaat ttctaccctc gggaggccaa ggtccagtgg 120
aaggtggaca acgctctcca gagtggaaat agccaagagt ccgttactga gcaggactct 180
aaggattcaa cctacacgct cgctagcgtt ctgactctga gcaaggcaga ttatgagaag 240
cacaaagtct acgcatgtga agtgactcat cagggactca gctcacccgt taccaagtct 300
ttcaacaggg gagagtgt 318
<210> 41
<211> 98
<212> PRT
<213> 人工序列 8F11 CH1
<400> 41
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 Thr
50 55 60
Leu Phe Ser Leu 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
Lys Val
<210> 42
<211> 294
<212> DNA
<213> 人工序列 8F11 CH1
<400> 42
gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60
ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180
ggactctaca ctctctttag ccttgtgacc gtgccctcca gcagcttggg cacccagacc 240
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agtt 294
<210> 43
<211> 106
<212> PRT
<213> 人工序列 8F11 CL
<400> 43
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
1 5 10 15
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
20 25 30
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
35 40 45
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
50 55 60
Tyr Trp Leu Thr Ser Leu Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
65 70 75 80
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
85 90 95
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 105
<210> 44
<211> 318
<212> DNA
<213> 人工序列 8F11 CL
<400> 44
actgtcgcag ctccatccgt gttcatcttt ccaccctccg acgagcaact gaaaagtggc 60
acagcctccg tcgtctgtct gctgaacaat ttctaccctc gggaggccaa ggtccagtgg 120
aaggtggaca acgctctcca gagtggaaat agccaagagt ccgttactga gcaggactct 180
aaggattcaa cctactggct cacgagcttg ctgactctga gcaaggcaga ttatgagaag 240
cacaaagtct acgcatgtga agtgactcat cagggactca gctcacccgt taccaagtct 300
ttcaacaggg gagagtgt 318
<210> 45
<211> 98
<212> PRT
<213> 人工序列 8G5 CH1
<400> 45
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 Val
50 55 60
Leu Ile Ser Gln 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
Lys Val
<210> 46
<211> 294
<212> DNA
<213> 人工序列 8G5 CH1
<400> 46
gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60
ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180
ggactctacg ttctcattag ccaggtgacc gtgccctcca gcagcttggg cacccagacc 240
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agtt 294
<210> 47
<211> 106
<212> PRT
<213> 人工序列 8G5 CL
<400> 47
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
1 5 10 15
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
20 25 30
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
35 40 45
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
50 55 60
Tyr Ser Leu Ala Ser Ile Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
65 70 75 80
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
85 90 95
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 105
<210> 48
<211> 318
<212> DNA
<213> 人工序列 8G5 CL
<400> 48
actgtcgcag ctccatccgt gttcatcttt ccaccctccg acgagcaact gaaaagtggc 60
acagcctccg tcgtctgtct gctgaacaat ttctaccctc gggaggccaa ggtccagtgg 120
aaggtggaca acgctctcca gagtggaaat agccaagagt ccgttactga gcaggactct 180
aaggattcaa cctactctct cgcgagcatt ctgactctga gcaaggcaga ttatgagaag 240
cacaaagtct acgcatgtga agtgactcat cagggactca gctcacccgt taccaagtct 300
ttcaacaggg gagagtgt 318
<210> 49
<211> 98
<212> PRT
<213> 人工序列 8H5 CH1
<400> 49
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 Val Ser Tyr 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
Lys Val
<210> 50
<211> 294
<212> DNA
<213> 人工序列 8H5 CH1
<400> 50
gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60
ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180
ggactctact cgctcgtgag ctatgtgacc gtgccctcca gcagcttggg cacccagacc 240
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agtt 294
<210> 51
<211> 106
<212> PRT
<213> 人工序列 8H5 CL
<400> 51
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
1 5 10 15
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
20 25 30
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
35 40 45
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
50 55 60
Tyr Thr Leu Tyr Ser Ser Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
65 70 75 80
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
85 90 95
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 105
<210> 52
<211> 318
<212> DNA
<213> 人工序列 8H5 CL
<400> 52
actgtcgcag ctccatccgt gttcatcttt ccaccctccg acgagcaact gaaaagtggc 60
acagcctccg tcgtctgtct gctgaacaat ttctaccctc gggaggccaa ggtccagtgg 120
aaggtggaca acgctctcca gagtggaaat agccaagagt ccgttactga gcaggactct 180
aaggattcaa cctacactct ctatagctcg ctgactctga gcaaggcaga ttatgagaag 240
cacaaagtct acgcatgtga agtgactcat cagggactca gctcacccgt taccaagtct 300
ttcaacaggg gagagtgt 318
<210> 53
<211> 98
<212> PRT
<213> 人工序列 11B7 CH1
<400> 53
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 Val
50 55 60
Leu Leu Ser Met 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
Lys Val
<210> 54
<211> 294
<212> DNA
<213> 人工序列 11B7 CH1
<400> 54
gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60
ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180
ggactctacg tgctcttgag catggtgacc gtgccctcca gcagcttggg cacccagacc 240
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agtt 294
<210> 55
<211> 106
<212> PRT
<213> 人工序列 11B7 CL
<400> 55
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
1 5 10 15
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
20 25 30
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
35 40 45
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
50 55 60
Tyr Arg Leu Trp Ser Leu Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
65 70 75 80
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
85 90 95
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 105
<210> 56
<211> 318
<212> DNA
<213> 人工序列 11B7 CL
<400> 56
actgtcgcag ctccatccgt gttcatcttt ccaccctccg acgagcaact gaaaagtggc 60
acagcctccg tcgtctgtct gctgaacaat ttctaccctc gggaggccaa ggtccagtgg 120
aaggtggaca acgctctcca gagtggaaat agccaagagt ccgttactga gcaggactct 180
aaggattcaa cctaccgtct ctggagcctt ctgactctga gcaaggcaga ttatgagaag 240
cacaaagtct acgcatgtga agtgactcat cagggactca gctcacccgt taccaagtct 300
ttcaacaggg gagagtgt 318
<210> 57
<211> 98
<212> PRT
<213> 人工序列 11C8 CH1
<400> 57
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 Met
50 55 60
Leu Val Ser Thr 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
Lys Val
<210> 58
<211> 294
<212> DNA
<213> 人工序列 11C8 CH1
<400> 58
gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60
ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180
ggactctaca tgctcgttag cacggtgacc gtgccctcca gcagcttggg cacccagacc 240
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agtt 294
<210> 59
<211> 106
<212> PRT
<213> 人工序列 11C8 CL
<400> 59
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
1 5 10 15
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
20 25 30
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
35 40 45
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
50 55 60
Tyr Ser Leu Thr Asn Tyr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
65 70 75 80
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
85 90 95
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 105
<210> 60
<211> 318
<212> DNA
<213> 人工序列 11C8 CL
<400> 60
actgtcgcag ctccatccgt gttcatcttt ccaccctccg acgagcaact gaaaagtggc 60
acagcctccg tcgtctgtct gctgaacaat ttctaccctc gggaggccaa ggtccagtgg 120
aaggtggaca acgctctcca gagtggaaat agccaagagt ccgttactga gcaggactct 180
aaggattcaa cctacagtct cactaactat ctgactctga gcaaggcaga ttatgagaag 240
cacaaagtct acgcatgtga agtgactcat cagggactca gctcacccgt taccaagtct 300
ttcaacaggg gagagtgt 318
<210> 61
<211> 98
<212> PRT
<213> 人工序列 11F5 CH1
<400> 61
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 Ala
50 55 60
Leu Phe Ser Ser 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
Lys Val
<210> 62
<211> 294
<212> DNA
<213> 人工序列 11F5 CH1
<400> 62
gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60
ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180
ggactctacg ctctctttag ctctgtgacc gtgccctcca gcagcttggg cacccagacc 240
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agtt 294
<210> 63
<211> 106
<212> PRT
<213> 人工序列 11F5 CL
<400> 63
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
1 5 10 15
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
20 25 30
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
35 40 45
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
50 55 60
Tyr Ser Leu Tyr Ser Val Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
65 70 75 80
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
85 90 95
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 105
<210> 64
<211> 318
<212> DNA
<213> 人工序列 11F5 CL
<400> 64
actgtcgcag ctccatccgt gttcatcttt ccaccctccg acgagcaact gaaaagtggc 60
acagcctccg tcgtctgtct gctgaacaat ttctaccctc gggaggccaa ggtccagtgg 120
aaggtggaca acgctctcca gagtggaaat agccaagagt ccgttactga gcaggactct 180
aaggattcaa cctacagtct ctatagcgtg ctgactctga gcaaggcaga ttatgagaag 240
cacaaagtct acgcatgtga agtgactcat cagggactca gctcacccgt taccaagtct 300
ttcaacaggg gagagtgt 318
<210> 65
<211> 98
<212> PRT
<213> 人工序列 11H7 CH1
<400> 65
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 Glu
50 55 60
Leu Leu Ser Ile 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
Lys Val
<210> 66
<211> 294
<212> DNA
<213> 人工序列 11H7 CH1
<400> 66
gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60
ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180
ggactctacg agctccttag cattgtgacc gtgccctcca gcagcttggg cacccagacc 240
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agtt 294
<210> 67
<211> 106
<212> PRT
<213> 人工序列 11H7 CL
<400> 67
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
1 5 10 15
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
20 25 30
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
35 40 45
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
50 55 60
Tyr Asn Leu Ser Ser Leu Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
65 70 75 80
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
85 90 95
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 105
<210> 68
<211> 318
<212> DNA
<213> 人工序列 11H7 CL
<400> 68
actgtcgcag ctccatccgt gttcatcttt ccaccctccg acgagcaact gaaaagtggc 60
acagcctccg tcgtctgtct gctgaacaat ttctaccctc gggaggccaa ggtccagtgg 120
aaggtggaca acgctctcca gagtggaaat agccaagagt ccgttactga gcaggactct 180
aaggattcaa cctacaatct ctctagcctg ctgactctga gcaaggcaga ttatgagaag 240
cacaaagtct acgcatgtga agtgactcat cagggactca gctcacccgt taccaagtct 300
ttcaacaggg gagagtgt 318
<210> 69
<211> 98
<212> PRT
<213> 人工序列 12B2 CH1
<400> 69
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 Thr
50 55 60
Leu Val Ser Phe 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
Lys Val
<210> 70
<211> 294
<212> DNA
<213> 人工序列 12B2 CH1
<400> 70
gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60
ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180
ggactctaca cgctcgttag ctttgtgacc gtgccctcca gcagcttggg cacccagacc 240
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agtt 294
<210> 71
<211> 106
<212> PRT
<213> 人工序列 12B2 CL
<400> 71
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
1 5 10 15
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
20 25 30
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
35 40 45
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
50 55 60
Tyr Cys Leu Thr Ser Val Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
65 70 75 80
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
85 90 95
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 105
<210> 72
<211> 318
<212> DNA
<213> 人工序列 12B2 CL
<400> 72
actgtcgcag ctccatccgt gttcatcttt ccaccctccg acgagcaact gaaaagtggc 60
acagcctccg tcgtctgtct gctgaacaat ttctaccctc gggaggccaa ggtccagtgg 120
aaggtggaca acgctctcca gagtggaaat agccaagagt ccgttactga gcaggactct 180
aaggattcaa cctactgtct cactagcgtt ctgactctga gcaaggcaga ttatgagaag 240
cacaaagtct acgcatgtga agtgactcat cagggactca gctcacccgt taccaagtct 300
ttcaacaggg gagagtgt 318
<210> 73
<211> 98
<212> PRT
<213> 人工序列 12D6 CH1
<400> 73
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 Leu Ser Ile 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
Lys Val
<210> 74
<211> 294
<212> DNA
<213> 人工序列 12D6 CH1
<400> 74
gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60
ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180
ggactctact ctctccttag cattgtgacc gtgccctcca gcagcttggg cacccagacc 240
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agtt 294
<210> 75
<211> 106
<212> PRT
<213> 人工序列 12D6 CL
<400> 75
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
1 5 10 15
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
20 25 30
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
35 40 45
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
50 55 60
Tyr Phe Leu Ser Ser Leu Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
65 70 75 80
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
85 90 95
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 105
<210> 76
<211> 318
<212> DNA
<213> 人工序列 12D6 CL
<400> 76
actgtcgcag ctccatccgt gttcatcttt ccaccctccg acgagcaact gaaaagtggc 60
acagcctccg tcgtctgtct gctgaacaat ttctaccctc gggaggccaa ggtccagtgg 120
aaggtggaca acgctctcca gagtggaaat agccaagagt ccgttactga gcaggactct 180
aaggattcaa cctactttct ctctagcttg ctgactctga gcaaggcaga ttatgagaag 240
cacaaagtct acgcatgtga agtgactcat cagggactca gctcacccgt taccaagtct 300
ttcaacaggg gagagtgt 318
<210> 77
<211> 98
<212> PRT
<213> 人工序列 11A10 CH1
<400> 77
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 Met
50 55 60
Leu Ile Ser Trp 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
Lys Val
<210> 78
<211> 294
<212> DNA
<213> 人工序列 11A10 CH1
<400> 78
gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60
ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180
ggactctaca tgctcattag ctgggtgacc gtgccctcca gcagcttggg cacccagacc 240
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agtt 294
<210> 79
<211> 106
<212> PRT
<213> 人工序列 11A10 CL
<400> 79
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
1 5 10 15
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
20 25 30
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
35 40 45
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
50 55 60
Tyr Arg Leu His Ser Val Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
65 70 75 80
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
85 90 95
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 105
<210> 80
<211> 318
<212> DNA
<213> 人工序列 11A10 CL
<400> 80
actgtcgcag ctccatccgt gttcatcttt ccaccctccg acgagcaact gaaaagtggc 60
acagcctccg tcgtctgtct gctgaacaat ttctaccctc gggaggccaa ggtccagtgg 120
aaggtggaca acgctctcca gagtggaaat agccaagagt ccgttactga gcaggactct 180
aaggattcaa cctacaggct ccatagcgtt ctgactctga gcaaggcaga ttatgagaag 240
cacaaagtct acgcatgtga agtgactcat cagggactca gctcacccgt taccaagtct 300
ttcaacaggg gagagtgt 318
<210> 81
<211> 98
<212> PRT
<213> 人工序列 11D6 CH1
<400> 81
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 Trp
50 55 60
Leu Phe Ser Thr 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
Lys Val
<210> 82
<211> 294
<212> DNA
<213> 人工序列 11D6 CH1
<400> 82
gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60
ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180
ggactctact ggctctttag cacggtgacc gtgccctcca gcagcttggg cacccagacc 240
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agtt 294
<210> 83
<211> 106
<212> PRT
<213> 人工序列 11D6 CL
<400> 83
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
1 5 10 15
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
20 25 30
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
35 40 45
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
50 55 60
Tyr Lys Leu Val Ser Met Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
65 70 75 80
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
85 90 95
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 105
<210> 84
<211> 318
<212> DNA
<213> 人工序列 11D6 CL
<400> 84
actgtcgcag ctccatccgt gttcatcttt ccaccctccg acgagcaact gaaaagtggc 60
acagcctccg tcgtctgtct gctgaacaat ttctaccctc gggaggccaa ggtccagtgg 120
aaggtggaca acgctctcca gagtggaaat agccaagagt ccgttactga gcaggactct 180
aaggattcaa cctacaagct cgttagcatg ctgactctga gcaaggcaga ttatgagaag 240
cacaaagtct acgcatgtga agtgactcat cagggactca gctcacccgt taccaagtct 300
ttcaacaggg gagagtgt 318
<210> 85
<211> 98
<212> PRT
<213> 人工序列 11C3 CH1
<400> 85
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 Thr
50 55 60
Leu Phe Ser Ser 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
Lys Val
<210> 86
<211> 294
<212> DNA
<213> 人工序列 11C3 CH1
<400> 86
gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60
ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180
ggactctaca cgctctttag ctcggtgacc gtgccctcca gcagcttggg cacccagacc 240
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agtt 294
<210> 87
<211> 106
<212> PRT
<213> 人工序列 11C3 CL
<400> 87
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
1 5 10 15
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
20 25 30
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
35 40 45
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
50 55 60
Tyr His Leu Val Ser Arg Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
65 70 75 80
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
85 90 95
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 105
<210> 88
<211> 318
<212> DNA
<213> 人工序列 11C3 CL
<400> 88
actgtcgcag ctccatccgt gttcatcttt ccaccctccg acgagcaact gaaaagtggc 60
acagcctccg tcgtctgtct gctgaacaat ttctaccctc gggaggccaa ggtccagtgg 120
aaggtggaca acgctctcca gagtggaaat agccaagagt ccgttactga gcaggactct 180
aaggattcaa cctaccatct cgtgagccgg ctgactctga gcaaggcaga ttatgagaag 240
cacaaagtct acgcatgtga agtgactcat cagggactca gctcacccgt taccaagtct 300
ttcaacaggg gagagtgt 318
<210> 89
<211> 98
<212> PRT
<213> 人工序列 11C12 CH1
<400> 89
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 Lys
50 55 60
Leu Leu 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
Lys Val
<210> 90
<211> 294
<212> DNA
<213> 人工序列 11C12 CH1
<400> 90
gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60
ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180
ggactctaca agctccttag cgttgtgacc gtgccctcca gcagcttggg cacccagacc 240
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agtt 294
<210> 91
<211> 106
<212> PRT
<213> 人工序列 11C12 CL
<400> 91
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
1 5 10 15
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
20 25 30
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
35 40 45
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
50 55 60
Tyr Tyr Leu Met Ser Glu Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
65 70 75 80
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
85 90 95
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 105
<210> 92
<211> 318
<212> DNA
<213> 人工序列 11C12 CL
<400> 92
actgtcgcag ctccatccgt gttcatcttt ccaccctccg acgagcaact gaaaagtggc 60
acagcctccg tcgtctgtct gctgaacaat ttctaccctc gggaggccaa ggtccagtgg 120
aaggtggaca acgctctcca gagtggaaat agccaagagt ccgttactga gcaggactct 180
aaggattcaa cctactatct catgagcgag ctgactctga gcaaggcaga ttatgagaag 240
cacaaagtct acgcatgtga agtgactcat cagggactca gctcacccgt taccaagtct 300
ttcaacaggg gagagtgt 318
<210> 93
<211> 98
<212> PRT
<213> 人工序列 11F6 CH1
<400> 93
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 Ala
50 55 60
Leu Ile Ser Ile 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
Lys Val
<210> 94
<211> 294
<212> DNA
<213> 人工序列 11F6 CH1
<400> 94
gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60
ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180
ggactctacg cgctcattag cattgtgacc gtgccctcca gcagcttggg cacccagacc 240
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agtt 294
<210> 95
<211> 106
<212> PRT
<213> 人工序列 11F6 CL
<400> 95
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
1 5 10 15
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
20 25 30
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
35 40 45
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
50 55 60
Tyr Asn Leu Glu Ser Met Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
65 70 75 80
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
85 90 95
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 105
<210> 96
<211> 318
<212> DNA
<213> 人工序列 11F6 CL
<400> 96
actgtcgcag ctccatccgt gttcatcttt ccaccctccg acgagcaact gaaaagtggc 60
acagcctccg tcgtctgtct gctgaacaat ttctaccctc gggaggccaa ggtccagtgg 120
aaggtggaca acgctctcca gagtggaaat agccaagagt ccgttactga gcaggactct 180
aaggattcaa cctacaatct cgagagcatg ctgactctga gcaaggcaga ttatgagaag 240
cacaaagtct acgcatgtga agtgactcat cagggactca gctcacccgt taccaagtct 300
ttcaacaggg gagagtgt 318
<210> 97
<211> 98
<212> PRT
<213> 人工序列 11F8 CH1
<400> 97
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 Val Ser Ile 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
Lys Val
<210> 98
<211> 294
<212> DNA
<213> 人工序列 11F8 CH1
<400> 98
gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60
ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180
ggactctaca gtctcgtgag cattgtgacc gtgccctcca gcagcttggg cacccagacc 240
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agtt 294
<210> 99
<211> 106
<212> PRT
<213> 人工序列 11F8 CL
<400> 99
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
1 5 10 15
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
20 25 30
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
35 40 45
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
50 55 60
Tyr Phe Leu Leu Ser Ser Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
65 70 75 80
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
85 90 95
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 105
<210> 100
<211> 318
<212> DNA
<213> 人工序列 11F8 CL
<400> 100
actgtcgcag ctccatccgt gttcatcttt ccaccctccg acgagcaact gaaaagtggc 60
acagcctccg tcgtctgtct gctgaacaat ttctaccctc gggaggccaa ggtccagtgg 120
aaggtggaca acgctctcca gagtggaaat agccaagagt ccgttactga gcaggactct 180
aaggattcaa cctactttct cttgagctcg ctgactctga gcaaggcaga ttatgagaag 240
cacaaagtct acgcatgtga agtgactcat cagggactca gctcacccgt taccaagtct 300
ttcaacaggg gagagtgt 318
<210> 101
<211> 98
<212> PRT
<213> 人工序列 11H5 CH1
<400> 101
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 Thr
50 55 60
Leu Thr Ser Leu 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
Lys Val
<210> 102
<211> 294
<212> DNA
<213> 人工序列 11H5 CH1
<400> 102
gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60
ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180
ggactctaca cgctcactag cttggtgacc gtgccctcca gcagcttggg cacccagacc 240
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agtt 294
<210> 103
<211> 106
<212> PRT
<213> 人工序列 11H5 CL
<400> 103
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
1 5 10 15
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
20 25 30
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
35 40 45
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
50 55 60
Tyr Tyr Leu Ser Ser Ile Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
65 70 75 80
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
85 90 95
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 105
<210> 104
<211> 318
<212> DNA
<213> 人工序列 11H5 CL
<400> 104
actgtcgcag ctccatccgt gttcatcttt ccaccctccg acgagcaact gaaaagtggc 60
acagcctccg tcgtctgtct gctgaacaat ttctaccctc gggaggccaa ggtccagtgg 120
aaggtggaca acgctctcca gagtggaaat agccaagagt ccgttactga gcaggactct 180
aaggattcaa cctactatct cagtagcatt ctgactctga gcaaggcaga ttatgagaag 240
cacaaagtct acgcatgtga agtgactcat cagggactca gctcacccgt taccaagtct 300
ttcaacaggg gagagtgt 318
<210> 105
<211> 98
<212> PRT
<213> 人工序列 11H2 CH1
<400> 105
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 Tyr
50 55 60
Leu Met Ser Phe 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
Lys Val
<210> 106
<211> 294
<212> DNA
<213> 人工序列 11H2 CH1
<400> 106
gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60
ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180
ggactctact atctcatgag ctttgtgacc gtgccctcca gcagcttggg cacccagacc 240
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agtt 294
<210> 107
<211> 106
<212> PRT
<213> 人工序列 11H2 CL
<400> 107
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
1 5 10 15
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
20 25 30
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
35 40 45
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
50 55 60
Tyr Ser Leu Val Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
65 70 75 80
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
85 90 95
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 105
<210> 108
<211> 318
<212> DNA
<213> 人工序列 11H2 CL
<400> 108
actgtcgcag ctccatccgt gttcatcttt ccaccctccg acgagcaact gaaaagtggc 60
acagcctccg tcgtctgtct gctgaacaat ttctaccctc gggaggccaa ggtccagtgg 120
aaggtggaca acgctctcca gagtggaaat agccaagagt ccgttactga gcaggactct 180
aaggattcaa cctactcgct cgtgagcacg ctgactctga gcaaggcaga ttatgagaag 240
cacaaagtct acgcatgtga agtgactcat cagggactca gctcacccgt taccaagtct 300
ttcaacaggg gagagtgt 318
<210> 109
<211> 98
<212> PRT
<213> 人工序列 12C3 CH1
<400> 109
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 Thr
50 55 60
Leu Met Ser Ser 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
Lys Val
<210> 110
<211> 294
<212> DNA
<213> 人工序列 12C3 CH1
<400> 110
gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60
ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180
ggactctaca ctctcatgag cagtgtgacc gtgccctcca gcagcttggg cacccagacc 240
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agtt 294
<210> 111
<211> 106
<212> PRT
<213> 人工序列 12C3 CL
<400> 111
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
1 5 10 15
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
20 25 30
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
35 40 45
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
50 55 60
Tyr Leu Leu Trp Ser Met Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
65 70 75 80
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
85 90 95
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 105
<210> 112
<211> 318
<212> DNA
<213> 人工序列 12C3 CL
<400> 112
actgtcgcag ctccatccgt gttcatcttt ccaccctccg acgagcaact gaaaagtggc 60
acagcctccg tcgtctgtct gctgaacaat ttctaccctc gggaggccaa ggtccagtgg 120
aaggtggaca acgctctcca gagtggaaat agccaagagt ccgttactga gcaggactct 180
aaggattcaa cctacctgct ctggagcatg ctgactctga gcaaggcaga ttatgagaag 240
cacaaagtct acgcatgtga agtgactcat cagggactca gctcacccgt taccaagtct 300
ttcaacaggg gagagtgt 318
<210> 113
<211> 98
<212> PRT
<213> 人工序列 7A10 CH1
<400> 113
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 Thr
50 55 60
Leu Arg Ser Phe 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
Lys Val
<210> 114
<211> 294
<212> DNA
<213> 人工序列 7A10 CH1
<400> 114
gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60
ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180
ggactctaca cgctccgtag ctttgtgacc gtgccctcca gcagcttggg cacccagacc 240
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agtt 294
<210> 115
<211> 106
<212> PRT
<213> 人工序列 7A10 CL
<400> 115
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
1 5 10 15
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
20 25 30
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
35 40 45
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
50 55 60
Tyr Ser Leu Ser Ser Val Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
65 70 75 80
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
85 90 95
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 105
<210> 116
<211> 318
<212> DNA
<213> 人工序列 7A10 CL
<400> 116
actgtcgcag ctccatccgt gttcatcttt ccaccctccg acgagcaact gaaaagtggc 60
acagcctccg tcgtctgtct gctgaacaat ttctaccctc gggaggccaa ggtccagtgg 120
aaggtggaca acgctctcca gagtggaaat agccaagagt ccgttactga gcaggactct 180
aaggattcaa cctacagcct ctccagcgtg ctgactctga gcaaggcaga ttatgagaag 240
cacaaagtct acgcatgtga agtgactcat cagggactca gctcacccgt taccaagtct 300
ttcaacaggg gagagtgt 318
<210> 117
<211> 216
<212> PRT
<213> 人工序列 抗CD3轻链
<400> 117
Asp Ile Gln Met Thr Gln Glu Pro Ser Leu Thr Thr Ser Pro Gly Gly
1 5 10 15
Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser
20 25 30
Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Gly Gln Ala Pro Arg Gly
35 40 45
Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe
50 55 60
Ser Gly Ser Leu Ile Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Val
65 70 75 80
Gln Pro Glu Asp Glu Ala Ile Tyr Phe Cys Ala Leu Trp Tyr Ser Asn
85 90 95
Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val
100 105 110
Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys
115 120 125
Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg
130 135 140
Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn
145 150 155 160
Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser
165 170 175
Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys
180 185 190
Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr
195 200 205
Lys Ser Phe Asn Arg Gly Glu Cys
210 215
<210> 118
<211> 648
<212> DNA
<213> 人工序列 抗CD3轻链
<400> 118
gatattcaga tgacacaaga accaagtctc actacaagcc ctggaggcac cgttacactg 60
acctgtcgct cttccactgg cgccgtgacc acttccaact atgcaaactg ggtgcaggag 120
aaacctggac aggctcctag aggtctgatt ggaggcacta acaagagagc tccagggact 180
cctgccaggt tcagcggatc tctgatcggt gggaaggcag ctctgacaat cactggagtg 240
caacccgagg atgaggctat ctacttctgt gctctctggt actcaaatct gtgggtgttc 300
ggaggtggaa caaagctgga gatcaagcgg actgtcgcag ctccatccgt gttcatcttt 360
ccaccctccg acgagcaact gaaaagtggc acagcctccg tcgtctgtct gctgaacaat 420
ttctaccctc gggaggccaa ggtccagtgg aaggtggaca acgctctcca gagtggaaat 480
agccaagagt ccgttactga gcaggactct aaggattcaa cctacagcct ctccagcacc 540
ctgactctga gcaaggcaga ttatgagaag cacaaagtct acgcatgtga agtgactcat 600
cagggactca gctcacccgt taccaagtct ttcaacaggg gagagtgt 648
<210> 119
<211> 455
<212> PRT
<213> 人工序列 抗CD3重链
<400> 119
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr
20 25 30
Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp
50 55 60
Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr
65 70 75 80
Ala Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr
85 90 95
Tyr Cys Val Arg His Gly Asn Phe Gly Thr Ser Tyr Val Ser Trp Phe
100 105 110
Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr
115 120 125
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
130 135 140
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
145 150 155 160
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
165 170 175
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
180 185 190
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
195 200 205
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
210 215 220
Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
225 230 235 240
Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
245 250 255
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
260 265 270
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
275 280 285
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
290 295 300
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
305 310 315 320
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
325 330 335
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
340 345 350
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
355 360 365
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
370 375 380
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
385 390 395 400
Thr Thr Lys Lys Lys Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
405 410 415
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
420 425 430
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
435 440 445
Leu Ser Leu Ser Pro Gly Lys
450 455
<210> 120
<211> 1365
<212> DNA
<213> 人工序列 抗CD3重链
<400> 120
gaagtgcaac tggttgaaag cggtggagga ctggttcagc ctggaggctc cctcaaactc 60
agttgcgccg cttccggttt tacattcaac acttatgcca tgaattgggt gcgccaggct 120
cccggaaaag gcctggaatg ggtggcacgg atcaggagca aatacaacaa ttacgccaca 180
tattacgctg actccgttaa agacagattc acaatttcca gagatgattc caagaatact 240
gcttatctcc agatgaacaa cctgaagaca gaggatacag ctatgtacta ttgcgtgcgg 300
catggcaatt tcggaaattc ctatgtgagt tggtttgcct actggggaca gggaactctc 360
gttactgtca gctccgctag caccaagggc ccatcggtct tccccctggc accctcctcc 420
aagagcacct ctgggggcac agcggccctg ggctgcctgg tcaaggacta cttccccgaa 480
ccggtgacgg tgtcgtggaa ctcaggcgcc ctgaccagcg gcgtgcacac cttcccggct 540
gtcctacagt cctcaggact ctactccctc agcagcgtgg tgaccgtgcc ctccagcagc 600
ttgggcaccc agacctacat ctgcaacgtg aatcacaagc ccagcaacac caaggtggac 660
aagaaagttg agcccaaatc ttgtgacaaa actcacacat gcccaccgtg cccagcacct 720
gaagccgcag ggggaccgtc agtcttcctc ttccccccaa aacccaagga caccctcatg 780
atctcccgga cccctgaggt cacatgcgtg gtggtggacg tgagccacga agaccctgag 840
gtcaagttca actggtacgt ggacggcgtg gaggtgcata atgccaagac aaagccgcgg 900
gaggagcagt acaacagcac gtaccgtgtg gtcagcgtcc tcaccgtcct gcaccaggac 960
tggctgaatg gcaaggagta caagtgcaag gtctccaaca aagccctccc agcccccatc 1020
gagaaaacca tctccaaagc caaagggcag ccccgagaac cacaggtgta caccctgccc 1080
ccatcccggg aggagatgac caagaaccag gtcagcctga cctgcctggt caaaggcttc 1140
tatcccagcg acatcgccgt ggagtgggag agcaatgggc agccggagaa caactacaag 1200
accacgaaga agaagctgga ctccgacggc tccttcttcc tctacagcaa gctcaccgtg 1260
gacaagagca ggtggcagca ggggaacgtc ttctcatgct ccgtgatgca tgaggctctg 1320
cacaaccact acacgcagaa gagcctctcc ctgtctccgg gtaaa 1365
<210> 121
<211> 218
<212> PRT
<213> 人工序列 抗GPC3轻链
<400> 121
Asp Ile Val Leu Thr Gln Ser Pro Leu Ser Leu Thr Val Thr Pro Gly
1 5 10 15
Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Glu Ser Leu Val His Ser
20 25 30
Asn Gly Asn Thr Tyr Leu Ser Trp Leu Gln Gln Arg Pro Gly Gln Pro
35 40 45
Pro Arg Leu Leu Ile Tyr Lys Ile Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Val Gln Val
85 90 95
Ser Ser Phe Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105 110
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
115 120 125
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
130 135 140
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
145 150 155 160
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
165 170 175
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
180 185 190
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
195 200 205
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215
<210> 122
<211> 654
<212> DNA
<213> 人工序列 抗GPC3轻链
<400> 122
gatattgtgc tgacgcagtc tccactctca ctgaccgtca cccctggaga gccggcctcc 60
atctcctgca ggtctagtga aagcctcgta cacagtaatg gaaacaccta cttgagttgg 120
cttcagcaga ggccaggcca gcctccaaga ctcctaattt ataagatttc taaccggttc 180
tctggggtcc cagacagatt cagtggcagt ggggcaggga cagatttcac actgaaaatc 240
agcagggtgg aagctgagga tgtcggggtt tattactgcg ttcaggtgtc gtcgtttccg 300
tacacttttg gccaggggac caaggtggag atcaaaactg tcgcagctcc atccgtgttc 360
atctttccac cctccgacga gcaactgaaa agtggcacag cctccgtcgt ctgtctgctg 420
aacaatttct accctcggga ggccaaggtc cagtggaagg tggacaacgc tctccagagt 480
ggaaatagcc aagagtccgt tactgagcag gactctaagg attcaaccta cagcctctcc 540
agcaccctga ctctgagcaa ggcagattat gagaagcaca aagtctacgc atgtgaagtg 600
actcatcagg gactcagctc acccgttacc aagtctttca acaggggaga gtgt 654
<210> 123
<211> 445
<212> PRT
<213> 人工序列 抗GPC3重链
<400> 123
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Glu Met His Trp Val Lys Gln Thr Pro Gly Lys Gly Leu Lys Trp Ile
35 40 45
Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Ala Thr Leu Thr Ala Asp Thr Ser Thr Asp Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
115 120 125
Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val
130 135 140
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
145 150 155 160
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
165 170 175
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
180 185 190
Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys
195 200 205
Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys
210 215 220
Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu
225 230 235 240
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
245 250 255
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
260 265 270
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
275 280 285
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
290 295 300
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
305 310 315 320
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
325 330 335
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
340 345 350
Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
355 360 365
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
370 375 380
Pro Glu Asn Asn Tyr Lys Thr Asp Asp Asp Val Leu Asp Ser Asp Gly
385 390 395 400
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
405 410 415
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
420 425 430
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 124
<211> 1335
<212> DNA
<213> 人工序列 抗GPC3重链
<400> 124
caagtgcaac tcgttcaatc aggagctgag gtgaagaaac ctggagcatc tgtcaagctc 60
tcctgtaagg cctccggata cacctttacc gattacgaga tgcactgggt gaagcagact 120
cctggaaagg gtctgaagtg gatcggcgcc ctggacccta agaccggaga caccgcttat 180
gcccagaagt ttcagggaag agccactctg accgctgaca catcaaccga taccgcttac 240
atggagctca gctccctgcg cagtgaggat tctgctgtgt actattgtac acgcttttat 300
agctacacct actggggaca gggcactctc gtgaccgtct cctccgctag caccaagggc 360
ccatcggtct tccccctggc accctcctcc aagagcacct ctgggggcac agcggccctg 420
ggctgcctgg tcaaggacta cttccccgaa ccggtgacgg tgtcgtggaa ctcaggcgcc 480
ctgaccagcg gcgtgcacac cttcccggct gtcctacagt cctcaggact ctactccctc 540
agcagcgtgg tgaccgtgcc ctccagcagc ttgggcaccc agacctacat ctgcaacgtg 600
aatcacaagc ccagcaacac caaggtggac aagaaagttg agcccaaatc ttgtgacaaa 660
actcacacat gcccaccgtg cccagcacct gaagccgcag ggggaccgtc agtcttcctc 720
ttccccccaa aacccaagga caccctcatg atctcccgga cccctgaggt cacatgcgtg 780
gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt ggacggcgtg 840
gaggtgcata atgccaagac aaagccgcgg gaggagcagt acaacagcac gtaccgtgtg 900
gtcagcgtcc tcaccgtcct gcaccaggac tggctgaatg gcaaggagta caagtgcaag 960
gtctccaaca aagccctccc agcccccatc gagaaaacca tctccaaagc caaagggcag 1020
ccccgagaac cacaggtgta caccctgccc ccatcccggg aggagatgac caagaaccag 1080
gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtgggag 1140
agcaatgggc agccggagaa caactacaag accgacgatg acgtgctgga ctccgacggc 1200
tccttcttcc tctacagcaa gctcaccgtg gacaagagca ggtggcagca ggggaacgtc 1260
ttctcatgct ccgtgatgca tgaggctctg cacaaccact acacgcagaa gagcctctcc 1320
ctgtctccgg gtaaa 1335
<210> 125
<211> 20
<212> PRT
<213> 人工序列 human interleukin-2 信号肽
<400> 125
Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu
1 5 10 15
Val Thr Asn Ser
20
<210> 126
<211> 60
<212> DNA
<213> 人工序列 human interleukin-2 信号肽
<400> 126
atgtacagga tgcaactcct gtcttgcatt gcactaagtc ttgcacttgt cacgaattcg 60
<210> 127
<211> 216
<212> PRT
<213> 人工序列 MutA的CD3轻链
<400> 127
Asp Ile Gln Met Thr Gln Glu Pro Ser Leu Thr Thr Ser Pro Gly Gly
1 5 10 15
Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser
20 25 30
Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Gly Gln Ala Pro Arg Gly
35 40 45
Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe
50 55 60
Ser Gly Ser Leu Ile Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Val
65 70 75 80
Gln Pro Glu Asp Glu Ala Ile Tyr Phe Cys Ala Leu Trp Tyr Ser Asn
85 90 95
Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val
100 105 110
Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys
115 120 125
Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg
130 135 140
Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn
145 150 155 160
Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ala
165 170 175
Leu Glu Ser Ala Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys
180 185 190
Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr
195 200 205
Lys Ser Phe Asn Arg Gly Glu Cys
210 215
<210> 128
<211> 648
<212> DNA
<213> 人工序列 MutA的CD3轻链
<400> 128
gatattcaga tgacacaaga accaagtctc actacaagcc ctggaggcac cgttacactg 60
acctgtcgct cttccactgg cgccgtgacc acttccaact atgcaaactg ggtgcaggag 120
aaacctggac aggctcctag aggtctgatt ggaggcacta acaagagagc tccagggact 180
cctgccaggt tcagcggatc tctgatcggt gggaaggcag ctctgacaat cactggagtg 240
caacccgagg atgaggctat ctacttctgt gctctctggt actcaaatct gtgggtgttc 300
ggaggtggaa caaagctgga gatcaagcgg actgtcgcag ctccatccgt gttcatcttt 360
ccaccctccg acgagcaact gaaaagtggc acagcctccg tcgtctgtct gctgaacaat 420
ttctaccctc gggaggccaa ggtccagtgg aaggtggaca acgctctcca gagtggaaat 480
agccaagagt ccgttactga gcaggactct aaggattcaa cctacgcact cgagagcgct 540
ctgactctga gcaaggcaga ttatgagaag cacaaagtct acgcatgtga agtgactcat 600
cagggactca gctcacccgt taccaagtct ttcaacaggg gagagtgt 648
<210> 129
<211> 455
<212> PRT
<213> 人工序列 MutA的CD3重链
<400> 129
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr
20 25 30
Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp
50 55 60
Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr
65 70 75 80
Ala Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr
85 90 95
Tyr Cys Val Arg His Gly Asn Phe Gly His Ser Tyr Val Ser Trp Phe
100 105 110
Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr
115 120 125
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
130 135 140
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
145 150 155 160
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
165 170 175
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Thr Leu Tyr Ser
180 185 190
Phe Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
195 200 205
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
210 215 220
Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
225 230 235 240
Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
245 250 255
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
260 265 270
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
275 280 285
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
290 295 300
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
305 310 315 320
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
325 330 335
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
340 345 350
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
355 360 365
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
370 375 380
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
385 390 395 400
Thr Thr Lys Lys Lys Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
405 410 415
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
420 425 430
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
435 440 445
Leu Ser Leu Ser Pro Gly Lys
450 455
<210> 130
<211> 1365
<212> DNA
<213> 人工序列 MutA的CD3重链
<400> 130
gaagtgcaac tggttgaaag cggtggagga ctggttcagc ctggaggctc cctcaaactc 60
agttgcgccg cttccggttt tacattcaac acttatgcca tgaattgggt gcgccaggct 120
cccggaaaag gcctggaatg ggtggcacgg atcaggagca aatacaacaa ttacgccaca 180
tattacgctg actccgttaa agacagattc acaatttcca gagatgattc caagaatact 240
gcttatctcc agatgaacaa cctgaagaca gaggatacag ctatgtacta ttgcgtgcgg 300
catggcaatt tcggacattc ctatgtgagt tggtttgcct actggggaca gggaactctc 360
gttactgtca gctccgctag caccaagggc ccatcggtct tccccctggc accctcctcc 420
aagagcacct ctgggggcac agcggccctg ggctgcctgg tcaaggacta cttccccgaa 480
ccggtgacgg tgtcgtggaa ctcaggcgcc ctgaccagcg gcgtgcacac cttcccggct 540
gtcctacagt cctcaggact ctacacgctc tatagctttg tgaccgtgcc ctccagcagc 600
ttgggcaccc agacctacat ctgcaacgtg aatcacaagc ccagcaacac caaggtggac 660
aagaaagttg agcccaaatc ttgtgacaaa actcacacat gcccaccgtg cccagcacct 720
gaagccgcag ggggaccgtc agtcttcctc ttccccccaa aacccaagga caccctcatg 780
atctcccgga cccctgaggt cacatgcgtg gtggtggacg tgagccacga agaccctgag 840
gtcaagttca actggtacgt ggacggcgtg gaggtgcata atgccaagac aaagccgcgg 900
gaggagcagt acaacagcac gtaccgtgtg gtcagcgtcc tcaccgtcct gcaccaggac 960
tggctgaatg gcaaggagta caagtgcaag gtctccaaca aagccctccc agcccccatc 1020
gagaaaacca tctccaaagc caaagggcag ccccgagaac cacaggtgta caccctgccc 1080
ccatcccggg aggagatgac caagaaccag gtcagcctga cctgcctggt caaaggcttc 1140
tatcccagcg acatcgccgt ggagtgggag agcaatgggc agccggagaa caactacaag 1200
accacgaaga agaagctgga ctccgacggc tccttcttcc tctacagcaa gctcaccgtg 1260
gacaagagca ggtggcagca ggggaacgtc ttctcatgct ccgtgatgca tgaggctctg 1320
cacaaccact acacgcagaa gagcctctcc ctgtctccgg gtaaa 1365
<210> 131
<211> 218
<212> PRT
<213> 人工序列 MutA的GPC3轻链
<400> 131
Asp Ile Val Leu Thr Gln Ser Pro Leu Ser Leu Thr Val Thr Pro Gly
1 5 10 15
Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Glu Ser Leu Val His Ser
20 25 30
Asn Gly Asn Thr Tyr Leu Ser Trp Leu Gln Gln Arg Pro Gly Gln Pro
35 40 45
Pro Arg Leu Leu Ile Tyr Lys Ile Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Val Gln Val
85 90 95
Ser Ser Phe Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105 110
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
115 120 125
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
130 135 140
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
145 150 155 160
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
165 170 175
Tyr Arg Leu His Ser Val Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
180 185 190
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
195 200 205
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215
<210> 132
<211> 654
<212> DNA
<213> 人工序列 MutA的GPC3轻链
<400> 132
gatattgtgc tgacgcagtc tccactctca ctgaccgtca cccctggaga gccggcctcc 60
atctcctgca ggtctagtga aagcctcgta cacagtaatg gaaacaccta cttgagttgg 120
cttcagcaga ggccaggcca gcctccaaga ctcctaattt ataagatttc taaccggttc 180
tctggggtcc cagacagatt cagtggcagt ggggcaggga cagatttcac actgaaaatc 240
agcagggtgg aagctgagga tgtcggggtt tattactgcg ttcaggtgtc gtcgtttccg 300
tacacttttg gccaggggac caaggtggag atcaaaactg tcgcagctcc atccgtgttc 360
atctttccac cctccgacga gcaactgaaa agtggcacag cctccgtcgt ctgtctgctg 420
aacaatttct accctcggga ggccaaggtc cagtggaagg tggacaacgc tctccagagt 480
ggaaatagcc aagagtccgt tactgagcag gactctaagg attcaaccta caggctccat 540
agcgttctga ctctgagcaa ggcagattat gagaagcaca aagtctacgc atgtgaagtg 600
actcatcagg gactcagctc acccgttacc aagtctttca acaggggaga gtgt 654
<210> 133
<211> 445
<212> PRT
<213> 人工序列 MutA的GPC3重链
<400> 133
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Glu Met His Trp Val Lys Gln Thr Pro Gly Lys Gly Leu Lys Trp Ile
35 40 45
Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Ala Thr Leu Thr Ala Asp Thr Ser Thr Asp Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
115 120 125
Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val
130 135 140
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
145 150 155 160
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
165 170 175
Leu Tyr Met Leu Ile Ser Trp Val Thr Val Pro Ser Ser Ser Leu Gly
180 185 190
Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys
195 200 205
Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys
210 215 220
Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu
225 230 235 240
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
245 250 255
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
260 265 270
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
275 280 285
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
290 295 300
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
305 310 315 320
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
325 330 335
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
340 345 350
Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
355 360 365
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
370 375 380
Pro Glu Asn Asn Tyr Lys Thr Asp Asp Asp Val Leu Asp Ser Asp Gly
385 390 395 400
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
405 410 415
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
420 425 430
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 134
<211> 1335
<212> DNA
<213> 人工序列 MutA的GPC3重链
<400> 134
caagtgcaac tcgttcaatc aggagctgag gtgaagaaac ctggagcatc tgtcaagctc 60
tcctgtaagg cctccggata cacctttacc gattacgaga tgcactgggt gaagcagact 120
cctggaaagg gtctgaagtg gatcggcgcc ctggacccta agaccggaga caccgcttat 180
gcccagaagt ttcagggaag agccactctg accgctgaca catcaaccga taccgcttac 240
atggagctca gctccctgcg cagtgaggat tctgctgtgt actattgtac acgcttttat 300
agctacacct actggggaca gggcactctc gtgaccgtct cctccgctag caccaagggc 360
ccatcggtct tccccctggc accctcctcc aagagcacct ctgggggcac agcggccctg 420
ggctgcctgg tcaaggacta cttccccgaa ccggtgacgg tgtcgtggaa ctcaggcgcc 480
ctgaccagcg gcgtgcacac cttcccggct gtcctacagt cctcaggact ctacatgctc 540
attagctggg tgaccgtgcc ctccagcagc ttgggcaccc agacctacat ctgcaacgtg 600
aatcacaagc ccagcaacac caaggtggac aagaaagttg agcccaaatc ttgtgacaaa 660
actcacacat gcccaccgtg cccagcacct gaagccgcag ggggaccgtc agtcttcctc 720
ttccccccaa aacccaagga caccctcatg atctcccgga cccctgaggt cacatgcgtg 780
gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt ggacggcgtg 840
gaggtgcata atgccaagac aaagccgcgg gaggagcagt acaacagcac gtaccgtgtg 900
gtcagcgtcc tcaccgtcct gcaccaggac tggctgaatg gcaaggagta caagtgcaag 960
gtctccaaca aagccctccc agcccccatc gagaaaacca tctccaaagc caaagggcag 1020
ccccgagaac cacaggtgta caccctgccc ccatcccggg aggagatgac caagaaccag 1080
gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtgggag 1140
agcaatgggc agccggagaa caactacaag accgacgatg acgtgctgga ctccgacggc 1200
tccttcttcc tctacagcaa gctcaccgtg gacaagagca ggtggcagca ggggaacgtc 1260
ttctcatgct ccgtgatgca tgaggctctg cacaaccact acacgcagaa gagcctctcc 1320
ctgtctccgg gtaaa 1335
<210> 135
<211> 216
<212> PRT
<213> 人工序列 MutB的CD3轻链
<400> 135
Asp Ile Gln Met Thr Gln Glu Pro Ser Leu Thr Thr Ser Pro Gly Gly
1 5 10 15
Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser
20 25 30
Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Gly Gln Ala Pro Arg Gly
35 40 45
Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe
50 55 60
Ser Gly Ser Leu Ile Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Val
65 70 75 80
Gln Pro Glu Asp Glu Ala Ile Tyr Phe Cys Ala Leu Trp Tyr Ser Asn
85 90 95
Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val
100 105 110
Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys
115 120 125
Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg
130 135 140
Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn
145 150 155 160
Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Arg
165 170 175
Leu Trp Ser Leu Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys
180 185 190
Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr
195 200 205
Lys Ser Phe Asn Arg Gly Glu Cys
210 215
<210> 136
<211> 648
<212> DNA
<213> 人工序列 MutB的CD3轻链
<400> 136
gatattcaga tgacacaaga accaagtctc actacaagcc ctggaggcac cgttacactg 60
acctgtcgct cttccactgg cgccgtgacc acttccaact atgcaaactg ggtgcaggag 120
aaacctggac aggctcctag aggtctgatt ggaggcacta acaagagagc tccagggact 180
cctgccaggt tcagcggatc tctgatcggt gggaaggcag ctctgacaat cactggagtg 240
caacccgagg atgaggctat ctacttctgt gctctctggt actcaaatct gtgggtgttc 300
ggaggtggaa caaagctgga gatcaagcgg actgtcgcag ctccatccgt gttcatcttt 360
ccaccctccg acgagcaact gaaaagtggc acagcctccg tcgtctgtct gctgaacaat 420
ttctaccctc gggaggccaa ggtccagtgg aaggtggaca acgctctcca gagtggaaat 480
agccaagagt ccgttactga gcaggactct aaggattcaa cctaccgtct ctggagcctt 540
ctgactctga gcaaggcaga ttatgagaag cacaaagtct acgcatgtga agtgactcat 600
cagggactca gctcacccgt taccaagtct ttcaacaggg gagagtgt 648
<210> 137
<211> 455
<212> PRT
<213> 人工序列 MutB的CD3重链
<400> 137
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr
20 25 30
Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp
50 55 60
Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr
65 70 75 80
Ala Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr
85 90 95
Tyr Cys Val Arg His Gly Asn Phe Gly His Ser Tyr Val Ser Trp Phe
100 105 110
Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr
115 120 125
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
130 135 140
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
145 150 155 160
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
165 170 175
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Val Leu Leu Ser
180 185 190
Met Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
195 200 205
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
210 215 220
Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
225 230 235 240
Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
245 250 255
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
260 265 270
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
275 280 285
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
290 295 300
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
305 310 315 320
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
325 330 335
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
340 345 350
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
355 360 365
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
370 375 380
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
385 390 395 400
Thr Thr Lys Lys Lys Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
405 410 415
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
420 425 430
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
435 440 445
Leu Ser Leu Ser Pro Gly Lys
450 455
<210> 138
<211> 1365
<212> DNA
<213> 人工序列 MutB的CD3重链
<400> 138
gaagtgcaac tggttgaaag cggtggagga ctggttcagc ctggaggctc cctcaaactc 60
agttgcgccg cttccggttt tacattcaac acttatgcca tgaattgggt gcgccaggct 120
cccggaaaag gcctggaatg ggtggcacgg atcaggagca aatacaacaa ttacgccaca 180
tattacgctg actccgttaa agacagattc acaatttcca gagatgattc caagaatact 240
gcttatctcc agatgaacaa cctgaagaca gaggatacag ctatgtacta ttgcgtgcgg 300
catggcaatt tcggacattc ctatgtgagt tggtttgcct actggggaca gggaactctc 360
gttactgtca gctccgctag caccaagggc ccatcggtct tccccctggc accctcctcc 420
aagagcacct ctgggggcac agcggccctg ggctgcctgg tcaaggacta cttccccgaa 480
ccggtgacgg tgtcgtggaa ctcaggcgcc ctgaccagcg gcgtgcacac cttcccggct 540
gtcctacagt cctcaggact ctacgtgctc ttgagcatgg tgaccgtgcc ctccagcagc 600
ttgggcaccc agacctacat ctgcaacgtg aatcacaagc ccagcaacac caaggtggac 660
aagaaagttg agcccaaatc ttgtgacaaa actcacacat gcccaccgtg cccagcacct 720
gaagccgcag ggggaccgtc agtcttcctc ttccccccaa aacccaagga caccctcatg 780
atctcccgga cccctgaggt cacatgcgtg gtggtggacg tgagccacga agaccctgag 840
gtcaagttca actggtacgt ggacggcgtg gaggtgcata atgccaagac aaagccgcgg 900
gaggagcagt acaacagcac gtaccgtgtg gtcagcgtcc tcaccgtcct gcaccaggac 960
tggctgaatg gcaaggagta caagtgcaag gtctccaaca aagccctccc agcccccatc 1020
gagaaaacca tctccaaagc caaagggcag ccccgagaac cacaggtgta caccctgccc 1080
ccatcccggg aggagatgac caagaaccag gtcagcctga cctgcctggt caaaggcttc 1140
tatcccagcg acatcgccgt ggagtgggag agcaatgggc agccggagaa caactacaag 1200
accacgaaga agaagctgga ctccgacggc tccttcttcc tctacagcaa gctcaccgtg 1260
gacaagagca ggtggcagca ggggaacgtc ttctcatgct ccgtgatgca tgaggctctg 1320
cacaaccact acacgcagaa gagcctctcc ctgtctccgg gtaaa 1365
<210> 139
<211> 218
<212> PRT
<213> 人工序列 MutB的GPC3轻链
<400> 139
Asp Ile Val Leu Thr Gln Ser Pro Leu Ser Leu Thr Val Thr Pro Gly
1 5 10 15
Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Glu Ser Leu Val His Ser
20 25 30
Asn Gly Asn Thr Tyr Leu Ser Trp Leu Gln Gln Arg Pro Gly Gln Pro
35 40 45
Pro Arg Leu Leu Ile Tyr Lys Ile Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Val Gln Val
85 90 95
Ser Ser Phe Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105 110
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
115 120 125
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
130 135 140
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
145 150 155 160
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
165 170 175
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
180 185 190
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
195 200 205
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215
<210> 140
<211> 654
<212> DNA
<213> 人工序列 MutB的GPC3轻链
<400> 140
gatattgtgc tgacgcagtc tccactctca ctgaccgtca cccctggaga gccggcctcc 60
atctcctgca ggtctagtga aagcctcgta cacagtaatg gaaacaccta cttgagttgg 120
cttcagcaga ggccaggcca gcctccaaga ctcctaattt ataagatttc taaccggttc 180
tctggggtcc cagacagatt cagtggcagt ggggcaggga cagatttcac actgaaaatc 240
agcagggtgg aagctgagga tgtcggggtt tattactgcg ttcaggtgtc gtcgtttccg 300
tacacttttg gccaggggac caaggtggag atcaaaactg tcgcagctcc atccgtgttc 360
atctttccac cctccgacga gcaactgaaa agtggcacag cctccgtcgt ctgtctgctg 420
aacaatttct accctcggga ggccaaggtc cagtggaagg tggacaacgc tctccagagt 480
ggaaatagcc aagagtccgt tactgagcag gactctaagg attcaaccta cagcctctcc 540
agcaccctga ctctgagcaa ggcagattat gagaagcaca aagtctacgc atgtgaagtg 600
actcatcagg gactcagctc acccgttacc aagtctttca acaggggaga gtgt 654
<210> 141
<211> 445
<212> PRT
<213> 人工序列 MutB的GPC3重链
<400> 141
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Glu Met His Trp Val Lys Gln Thr Pro Gly Lys Gly Leu Lys Trp Ile
35 40 45
Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Ala Thr Leu Thr Ala Asp Thr Ser Thr Asp Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
115 120 125
Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val
130 135 140
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
145 150 155 160
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
165 170 175
Leu Tyr Thr Leu Arg Ser Phe Val Thr Val Pro Ser Ser Ser Leu Gly
180 185 190
Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys
195 200 205
Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys
210 215 220
Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu
225 230 235 240
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
245 250 255
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
260 265 270
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
275 280 285
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
290 295 300
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
305 310 315 320
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
325 330 335
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
340 345 350
Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
355 360 365
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
370 375 380
Pro Glu Asn Asn Tyr Lys Thr Asp Asp Asp Val Leu Asp Ser Asp Gly
385 390 395 400
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
405 410 415
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
420 425 430
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 142
<211> 1335
<212> DNA
<213> 人工序列 MutB的GPC3重链
<400> 142
caagtgcaac tcgttcaatc aggagctgag gtgaagaaac ctggagcatc tgtcaagctc 60
tcctgtaagg cctccggata cacctttacc gattacgaga tgcactgggt gaagcagact 120
cctggaaagg gtctgaagtg gatcggcgcc ctggacccta agaccggaga caccgcttat 180
gcccagaagt ttcagggaag agccactctg accgctgaca catcaaccga taccgcttac 240
atggagctca gctccctgcg cagtgaggat tctgctgtgt actattgtac acgcttttat 300
agctacacct actggggaca gggcactctc gtgaccgtct cctccgctag caccaagggc 360
ccatcggtct tccccctggc accctcctcc aagagcacct ctgggggcac agcggccctg 420
ggctgcctgg tcaaggacta cttccccgaa ccggtgacgg tgtcgtggaa ctcaggcgcc 480
ctgaccagcg gcgtgcacac cttcccggct gtcctacagt cctcaggact ctacacgctc 540
cgtagctttg tgaccgtgcc ctccagcagc ttgggcaccc agacctacat ctgcaacgtg 600
aatcacaagc ccagcaacac caaggtggac aagaaagttg agcccaaatc ttgtgacaaa 660
actcacacat gcccaccgtg cccagcacct gaagccgcag ggggaccgtc agtcttcctc 720
ttccccccaa aacccaagga caccctcatg atctcccgga cccctgaggt cacatgcgtg 780
gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt ggacggcgtg 840
gaggtgcata atgccaagac aaagccgcgg gaggagcagt acaacagcac gtaccgtgtg 900
gtcagcgtcc tcaccgtcct gcaccaggac tggctgaatg gcaaggagta caagtgcaag 960
gtctccaaca aagccctccc agcccccatc gagaaaacca tctccaaagc caaagggcag 1020
ccccgagaac cacaggtgta caccctgccc ccatcccggg aggagatgac caagaaccag 1080
gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtgggag 1140
agcaatgggc agccggagaa caactacaag accgacgatg acgtgctgga ctccgacggc 1200
tccttcttcc tctacagcaa gctcaccgtg gacaagagca ggtggcagca ggggaacgtc 1260
ttctcatgct ccgtgatgca tgaggctctg cacaaccact acacgcagaa gagcctctcc 1320
ctgtctccgg gtaaa 1335
<210> 143
<211> 216
<212> PRT
<213> 人工序列 MutA+D-D的CD3轻链
<400> 143
Asp Ile Gln Met Thr Gln Glu Pro Ser Leu Thr Thr Ser Pro Gly Gly
1 5 10 15
Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser
20 25 30
Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Gly Gln Ala Pro Arg Gly
35 40 45
Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe
50 55 60
Ser Gly Ser Leu Ile Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Val
65 70 75 80
Gln Pro Glu Asp Glu Ala Ile Tyr Phe Cys Ala Leu Trp Tyr Ser Asn
85 90 95
Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val
100 105 110
Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys
115 120 125
Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg
130 135 140
Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn
145 150 155 160
Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ala
165 170 175
Leu Glu Ser Asp Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys
180 185 190
Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr
195 200 205
Lys Ser Phe Asn Arg Gly Glu Cys
210 215
<210> 144
<211> 648
<212> DNA
<213> 人工序列 MutA+D-D的CD3轻链
<400> 144
gatattcaga tgacacaaga accaagtctc actacaagcc ctggaggcac cgttacactg 60
acctgtcgct cttccactgg cgccgtgacc acttccaact atgcaaactg ggtgcaggag 120
aaacctggac aggctcctag aggtctgatt ggaggcacta acaagagagc tccagggact 180
cctgccaggt tcagcggatc tctgatcggt gggaaggcag ctctgacaat cactggagtg 240
caacccgagg atgaggctat ctacttctgt gctctctggt actcaaatct gtgggtgttc 300
ggaggtggaa caaagctgga gatcaagcgg actgtcgcag ctccatccgt gttcatcttt 360
ccaccctccg acgagcaact gaaaagtggc acagcctccg tcgtctgtct gctgaacaat 420
ttctaccctc gggaggccaa ggtccagtgg aaggtggaca acgctctcca gagtggaaat 480
agccaagagt ccgttactga gcaggactct aaggattcaa cctacgctct cgagagcgat 540
ctgactctga gcaaggcaga ttatgagaag cacaaagtct acgcatgtga agtgactcat 600
cagggactca gctcacccgt taccaagtct ttcaacaggg gagagtgt 648
<210> 145
<211> 455
<212> PRT
<213> 人工序列 MutA+D-D的CD3重链
<400> 145
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr
20 25 30
Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp
50 55 60
Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr
65 70 75 80
Ala Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr
85 90 95
Tyr Cys Val Arg His Gly Asn Phe Gly His Ser Tyr Val Ser Trp Phe
100 105 110
Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr
115 120 125
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
130 135 140
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
145 150 155 160
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
165 170 175
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Thr Leu Tyr Ser
180 185 190
Phe Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
195 200 205
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
210 215 220
Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
225 230 235 240
Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
245 250 255
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
260 265 270
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
275 280 285
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
290 295 300
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
305 310 315 320
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
325 330 335
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
340 345 350
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
355 360 365
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
370 375 380
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
385 390 395 400
Thr Thr Lys Lys Lys Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
405 410 415
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
420 425 430
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
435 440 445
Leu Ser Leu Ser Pro Gly Lys
450 455
<210> 146
<211> 1365
<212> DNA
<213> 人工序列 MutA+D-D的CD3重链
<400> 146
gaagtgcaac tggttgaaag cggtggagga ctggttcagc ctggaggctc cctcaaactc 60
agttgcgccg cttccggttt tacattcaac acttatgcca tgaattgggt gcgccaggct 120
cccggaaaag gcctggaatg ggtggcacgg atcaggagca aatacaacaa ttacgccaca 180
tattacgctg actccgttaa agacagattc acaatttcca gagatgattc caagaatact 240
gcttatctcc agatgaacaa cctgaagaca gaggatacag ctatgtacta ttgcgtgcgg 300
catggcaatt tcggacattc ctatgtgagt tggtttgcct actggggaca gggaactctc 360
gttactgtca gctccgctag caccaagggc ccatcggtct tccccctggc accctcctcc 420
aagagcacct ctgggggcac agcggccctg ggctgcctgg tcaaggacta cttccccgaa 480
ccggtgacgg tgtcgtggaa ctcaggcgcc ctgaccagcg gcgtgcacac cttcccggct 540
gtcctacagt cctcaggact ctacacgctc tatagctttg tgaccgtgcc ctccagcagc 600
ttgggcaccc agacctacat ctgcaacgtg aatcacaagc ccagcaacac caaggtggac 660
aagaaagttg agcccaaatc ttgtgacaaa actcacacat gcccaccgtg cccagcacct 720
gaagccgcag ggggaccgtc agtcttcctc ttccccccaa aacccaagga caccctcatg 780
atctcccgga cccctgaggt cacatgcgtg gtggtggacg tgagccacga agaccctgag 840
gtcaagttca actggtacgt ggacggcgtg gaggtgcata atgccaagac aaagccgcgg 900
gaggagcagt acaacagcac gtaccgtgtg gtcagcgtcc tcaccgtcct gcaccaggac 960
tggctgaatg gcaaggagta caagtgcaag gtctccaaca aagccctccc agcccccatc 1020
gagaaaacca tctccaaagc caaagggcag ccccgagaac cacaggtgta caccctgccc 1080
ccatcccggg aggagatgac caagaaccag gtcagcctga cctgcctggt caaaggcttc 1140
tatcccagcg acatcgccgt ggagtgggag agcaatgggc agccggagaa caactacaag 1200
accacgaaga agaagctgga ctccgacggc tccttcttcc tctacagcaa gctcaccgtg 1260
gacaagagca ggtggcagca ggggaacgtc ttctcatgct ccgtgatgca tgaggctctg 1320
cacaaccact acacgcagaa gagcctctcc ctgtctccgg gtaaa 1365
<210> 147
<211> 218
<212> PRT
<213> 人工序列 MutA+D-D的GPC3轻链
<400> 147
Asp Ile Val Leu Thr Gln Ser Pro Leu Ser Leu Thr Val Thr Pro Gly
1 5 10 15
Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Glu Ser Leu Val His Ser
20 25 30
Asn Gly Asn Thr Tyr Leu Ser Trp Leu Gln Gln Arg Pro Gly Gln Pro
35 40 45
Pro Arg Leu Leu Ile Tyr Lys Ile Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Val Gln Val
85 90 95
Ser Ser Phe Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105 110
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
115 120 125
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
130 135 140
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
145 150 155 160
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
165 170 175
Tyr Arg Leu His Ser Val Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
180 185 190
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
195 200 205
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215
<210> 148
<211> 654
<212> DNA
<213> 人工序列 MutA+D-D的GPC3轻链
<400> 148
gatattgtgc tgacgcagtc tccactctca ctgaccgtca cccctggaga gccggcctcc 60
atctcctgca ggtctagtga aagcctcgta cacagtaatg gaaacaccta cttgagttgg 120
cttcagcaga ggccaggcca gcctccaaga ctcctaattt ataagatttc taaccggttc 180
tctggggtcc cagacagatt cagtggcagt ggggcaggga cagatttcac actgaaaatc 240
agcagggtgg aagctgagga tgtcggggtt tattactgcg ttcaggtgtc gtcgtttccg 300
tacacttttg gccaggggac caaggtggag atcaaaactg tcgcagctcc atccgtgttc 360
atctttccac cctccgacga gcaactgaaa agtggcacag cctccgtcgt ctgtctgctg 420
aacaatttct accctcggga ggccaaggtc cagtggaagg tggacaacgc tctccagagt 480
ggaaatagcc aagagtccgt tactgagcag gactctaagg attcaaccta caggctccat 540
agcgttctga ctctgagcaa ggcagattat gagaagcaca aagtctacgc atgtgaagtg 600
actcatcagg gactcagctc acccgttacc aagtctttca acaggggaga gtgt 654
<210> 149
<211> 445
<212> PRT
<213> 人工序列 MutA+D-D的GPC3重链
<400> 149
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Glu Met His Trp Val Lys Gln Thr Pro Gly Lys Gly Leu Lys Trp Ile
35 40 45
Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Ala Thr Leu Thr Ala Asp Thr Ser Thr Asp Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
115 120 125
Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val
130 135 140
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
145 150 155 160
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
165 170 175
Leu Tyr Met Leu Asp Ser Trp Val Thr Val Pro Ser Ser Ser Leu Gly
180 185 190
Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys
195 200 205
Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys
210 215 220
Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu
225 230 235 240
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
245 250 255
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
260 265 270
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
275 280 285
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
290 295 300
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
305 310 315 320
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
325 330 335
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
340 345 350
Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
355 360 365
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
370 375 380
Pro Glu Asn Asn Tyr Lys Thr Asp Asp Asp Val Leu Asp Ser Asp Gly
385 390 395 400
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
405 410 415
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
420 425 430
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 150
<211> 1335
<212> DNA
<213> 人工序列 MutA+D-D的GPC3重链
<400> 150
caagtgcaac tcgttcaatc aggagctgag gtgaagaaac ctggagcatc tgtcaagctc 60
tcctgtaagg cctccggata cacctttacc gattacgaga tgcactgggt gaagcagact 120
cctggaaagg gtctgaagtg gatcggcgcc ctggacccta agaccggaga caccgcttat 180
gcccagaagt ttcagggaag agccactctg accgctgaca catcaaccga taccgcttac 240
atggagctca gctccctgcg cagtgaggat tctgctgtgt actattgtac acgcttttat 300
agctacacct actggggaca gggcactctc gtgaccgtct cctccgctag caccaagggc 360
ccatcggtct tccccctggc accctcctcc aagagcacct ctgggggcac agcggccctg 420
ggctgcctgg tcaaggacta cttccccgaa ccggtgacgg tgtcgtggaa ctcaggcgcc 480
ctgaccagcg gcgtgcacac cttcccggct gtcctacagt cctcaggact ctacatgctc 540
gacagctggg tgaccgtgcc ctccagcagc ttgggcaccc agacctacat ctgcaacgtg 600
aatcacaagc ccagcaacac caaggtggac aagaaagttg agcccaaatc ttgtgacaaa 660
actcacacat gcccaccgtg cccagcacct gaagccgcag ggggaccgtc agtcttcctc 720
ttccccccaa aacccaagga caccctcatg atctcccgga cccctgaggt cacatgcgtg 780
gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt ggacggcgtg 840
gaggtgcata atgccaagac aaagccgcgg gaggagcagt acaacagcac gtaccgtgtg 900
gtcagcgtcc tcaccgtcct gcaccaggac tggctgaatg gcaaggagta caagtgcaag 960
gtctccaaca aagccctccc agcccccatc gagaaaacca tctccaaagc caaagggcag 1020
ccccgagaac cacaggtgta caccctgccc ccatcccggg aggagatgac caagaaccag 1080
gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtgggag 1140
agcaatgggc agccggagaa caactacaag accgacgatg acgtgctgga ctccgacggc 1200
tccttcttcc tctacagcaa gctcaccgtg gacaagagca ggtggcagca ggggaacgtc 1260
ttctcatgct ccgtgatgca tgaggctctg cacaaccact acacgcagaa gagcctctcc 1320
ctgtctccgg gtaaa 1335
<210> 151
<211> 216
<212> PRT
<213> 人工序列 MutB+K-K的CD3轻链
<400> 151
Asp Ile Gln Met Thr Gln Glu Pro Ser Leu Thr Thr Ser Pro Gly Gly
1 5 10 15
Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser
20 25 30
Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Gly Gln Ala Pro Arg Gly
35 40 45
Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe
50 55 60
Ser Gly Ser Leu Ile Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Val
65 70 75 80
Gln Pro Glu Asp Glu Ala Ile Tyr Phe Cys Ala Leu Trp Tyr Ser Asn
85 90 95
Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val
100 105 110
Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys
115 120 125
Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg
130 135 140
Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn
145 150 155 160
Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Arg
165 170 175
Leu Trp Ser Lys Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys
180 185 190
Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr
195 200 205
Lys Ser Phe Asn Arg Gly Glu Cys
210 215
<210> 152
<211> 648
<212> DNA
<213> 人工序列 MutB+K-K的CD3轻链
<400> 152
gatattcaga tgacacaaga accaagtctc actacaagcc ctggaggcac cgttacactg 60
acctgtcgct cttccactgg cgccgtgacc acttccaact atgcaaactg ggtgcaggag 120
aaacctggac aggctcctag aggtctgatt ggaggcacta acaagagagc tccagggact 180
cctgccaggt tcagcggatc tctgatcggt gggaaggcag ctctgacaat cactggagtg 240
caacccgagg atgaggctat ctacttctgt gctctctggt actcaaatct gtgggtgttc 300
ggaggtggaa caaagctgga gatcaagcgg actgtcgcag ctccatccgt gttcatcttt 360
ccaccctccg acgagcaact gaaaagtggc acagcctccg tcgtctgtct gctgaacaat 420
ttctaccctc gggaggccaa ggtccagtgg aaggtggaca acgctctcca gagtggaaat 480
agccaagagt ccgttactga gcaggactct aaggattcaa cctaccgtct ctggagcaag 540
ctgactctga gcaaggcaga ttatgagaag cacaaagtct acgcatgtga agtgactcat 600
cagggactca gctcacccgt taccaagtct ttcaacaggg gagagtgt 648
<210> 153
<211> 455
<212> PRT
<213> 人工序列 MutB+K-K的CD3重链
<400> 153
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr
20 25 30
Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp
50 55 60
Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr
65 70 75 80
Ala Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr
85 90 95
Tyr Cys Val Arg His Gly Asn Phe Gly His Ser Tyr Val Ser Trp Phe
100 105 110
Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr
115 120 125
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
130 135 140
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
145 150 155 160
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
165 170 175
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Val Leu Leu Ser
180 185 190
Met Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
195 200 205
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
210 215 220
Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
225 230 235 240
Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
245 250 255
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
260 265 270
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
275 280 285
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
290 295 300
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
305 310 315 320
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
325 330 335
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
340 345 350
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
355 360 365
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
370 375 380
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
385 390 395 400
Thr Thr Lys Lys Lys Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
405 410 415
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
420 425 430
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
435 440 445
Leu Ser Leu Ser Pro Gly Lys
450 455
<210> 154
<211> 1365
<212> DNA
<213> 人工序列 MutB+K-K的CD3重链
<400> 154
gaagtgcaac tggttgaaag cggtggagga ctggttcagc ctggaggctc cctcaaactc 60
agttgcgccg cttccggttt tacattcaac acttatgcca tgaattgggt gcgccaggct 120
cccggaaaag gcctggaatg ggtggcacgg atcaggagca aatacaacaa ttacgccaca 180
tattacgctg actccgttaa agacagattc acaatttcca gagatgattc caagaatact 240
gcttatctcc agatgaacaa cctgaagaca gaggatacag ctatgtacta ttgcgtgcgg 300
catggcaatt tcggacattc ctatgtgagt tggtttgcct actggggaca gggaactctc 360
gttactgtca gctccgctag caccaagggc ccatcggtct tccccctggc accctcctcc 420
aagagcacct ctgggggcac agcggccctg ggctgcctgg tcaaggacta cttccccgaa 480
ccggtgacgg tgtcgtggaa ctcaggcgcc ctgaccagcg gcgtgcacac cttcccggct 540
gtcctacagt cctcaggact ctacgtgctc ttgagcatgg tgaccgtgcc ctccagcagc 600
ttgggcaccc agacctacat ctgcaacgtg aatcacaagc ccagcaacac caaggtggac 660
aagaaagttg agcccaaatc ttgtgacaaa actcacacat gcccaccgtg cccagcacct 720
gaagccgcag ggggaccgtc agtcttcctc ttccccccaa aacccaagga caccctcatg 780
atctcccgga cccctgaggt cacatgcgtg gtggtggacg tgagccacga agaccctgag 840
gtcaagttca actggtacgt ggacggcgtg gaggtgcata atgccaagac aaagccgcgg 900
gaggagcagt acaacagcac gtaccgtgtg gtcagcgtcc tcaccgtcct gcaccaggac 960
tggctgaatg gcaaggagta caagtgcaag gtctccaaca aagccctccc agcccccatc 1020
gagaaaacca tctccaaagc caaagggcag ccccgagaac cacaggtgta caccctgccc 1080
ccatcccggg aggagatgac caagaaccag gtcagcctga cctgcctggt caaaggcttc 1140
tatcccagcg acatcgccgt ggagtgggag agcaatgggc agccggagaa caactacaag 1200
accacgaaga agaagctgga ctccgacggc tccttcttcc tctacagcaa gctcaccgtg 1260
gacaagagca ggtggcagca ggggaacgtc ttctcatgct ccgtgatgca tgaggctctg 1320
cacaaccact acacgcagaa gagcctctcc ctgtctccgg gtaaa 1365
<210> 155
<211> 218
<212> PRT
<213> 人工序列 MutB+K-K的GPC3轻链
<400> 155
Asp Ile Val Leu Thr Gln Ser Pro Leu Ser Leu Thr Val Thr Pro Gly
1 5 10 15
Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Glu Ser Leu Val His Ser
20 25 30
Asn Gly Asn Thr Tyr Leu Ser Trp Leu Gln Gln Arg Pro Gly Gln Pro
35 40 45
Pro Arg Leu Leu Ile Tyr Lys Ile Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Val Gln Val
85 90 95
Ser Ser Phe Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105 110
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
115 120 125
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
130 135 140
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
145 150 155 160
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
165 170 175
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
180 185 190
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
195 200 205
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215
<210> 156
<211> 654
<212> DNA
<213> 人工序列 MutB+K-K的GPC3轻链
<400> 156
gatattgtgc tgacgcagtc tccactctca ctgaccgtca cccctggaga gccggcctcc 60
atctcctgca ggtctagtga aagcctcgta cacagtaatg gaaacaccta cttgagttgg 120
cttcagcaga ggccaggcca gcctccaaga ctcctaattt ataagatttc taaccggttc 180
tctggggtcc cagacagatt cagtggcagt ggggcaggga cagatttcac actgaaaatc 240
agcagggtgg aagctgagga tgtcggggtt tattactgcg ttcaggtgtc gtcgtttccg 300
tacacttttg gccaggggac caaggtggag atcaaaactg tcgcagctcc atccgtgttc 360
atctttccac cctccgacga gcaactgaaa agtggcacag cctccgtcgt ctgtctgctg 420
aacaatttct accctcggga ggccaaggtc cagtggaagg tggacaacgc tctccagagt 480
ggaaatagcc aagagtccgt tactgagcag gactctaagg attcaaccta cagcctctcc 540
agcaccctga ctctgagcaa ggcagattat gagaagcaca aagtctacgc atgtgaagtg 600
actcatcagg gactcagctc acccgttacc aagtctttca acaggggaga gtgt 654
<210> 157
<211> 445
<212> PRT
<213> 人工序列 MutB+K-K的GPC3重链
<400> 157
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Glu Met His Trp Val Lys Gln Thr Pro Gly Lys Gly Leu Lys Trp Ile
35 40 45
Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Ala Thr Leu Thr Ala Asp Thr Ser Thr Asp Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
115 120 125
Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val
130 135 140
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
145 150 155 160
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
165 170 175
Leu Tyr Thr Leu Arg Ser Phe Val Lys Val Pro Ser Ser Ser Leu Gly
180 185 190
Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys
195 200 205
Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys
210 215 220
Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu
225 230 235 240
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
245 250 255
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
260 265 270
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
275 280 285
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
290 295 300
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
305 310 315 320
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
325 330 335
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
340 345 350
Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
355 360 365
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
370 375 380
Pro Glu Asn Asn Tyr Lys Thr Asp Asp Asp Val Leu Asp Ser Asp Gly
385 390 395 400
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
405 410 415
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
420 425 430
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 158
<211> 1335
<212> DNA
<213> 人工序列 MutB+K-K的GPC3重链
<400> 158
caagtgcaac tcgttcaatc aggagctgag gtgaagaaac ctggagcatc tgtcaagctc 60
tcctgtaagg cctccggata cacctttacc gattacgaga tgcactgggt gaagcagact 120
cctggaaagg gtctgaagtg gatcggcgcc ctggacccta agaccggaga caccgcttat 180
gcccagaagt ttcagggaag agccactctg accgctgaca catcaaccga taccgcttac 240
atggagctca gctccctgcg cagtgaggat tctgctgtgt actattgtac acgcttttat 300
agctacacct actggggaca gggcactctc gtgaccgtct cctccgctag caccaagggc 360
ccatcggtct tccccctggc accctcctcc aagagcacct ctgggggcac agcggccctg 420
ggctgcctgg tcaaggacta cttccccgaa ccggtgacgg tgtcgtggaa ctcaggcgcc 480
ctgaccagcg gcgtgcacac cttcccggct gtcctacagt cctcaggact ctacacgctc 540
cgtagctttg tgaaggtgcc ctccagcagc ttgggcaccc agacctacat ctgcaacgtg 600
aatcacaagc ccagcaacac caaggtggac aagaaagttg agcccaaatc ttgtgacaaa 660
actcacacat gcccaccgtg cccagcacct gaagccgcag ggggaccgtc agtcttcctc 720
ttccccccaa aacccaagga caccctcatg atctcccgga cccctgaggt cacatgcgtg 780
gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt ggacggcgtg 840
gaggtgcata atgccaagac aaagccgcgg gaggagcagt acaacagcac gtaccgtgtg 900
gtcagcgtcc tcaccgtcct gcaccaggac tggctgaatg gcaaggagta caagtgcaag 960
gtctccaaca aagccctccc agcccccatc gagaaaacca tctccaaagc caaagggcag 1020
ccccgagaac cacaggtgta caccctgccc ccatcccggg aggagatgac caagaaccag 1080
gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtgggag 1140
agcaatgggc agccggagaa caactacaag accgacgatg acgtgctgga ctccgacggc 1200
tccttcttcc tctacagcaa gctcaccgtg gacaagagca ggtggcagca ggggaacgtc 1260
ttctcatgct ccgtgatgca tgaggctctg cacaaccact acacgcagaa gagcctctcc 1320
ctgtctccgg gtaaa 1335
<210> 159
<211> 242
<212> PRT
<213> 人工序列 抗GPC3的ScFv
<400> 159
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Glu Met His Trp Val Lys Gln Thr Pro Gly Lys Gly Leu Lys Trp Ile
35 40 45
Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Ala Thr Leu Thr Ala Asp Thr Ser Thr Asp Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu 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 Val Leu Thr Gln Ser Pro Leu Ser Leu Thr Val Thr
130 135 140
Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Glu Ser Leu Val
145 150 155 160
His Ser Asn Gly Asn Thr Tyr Leu Ser Trp Leu Gln Gln Arg Pro Gly
165 170 175
Gln Pro Pro Arg Leu Leu Ile Tyr Lys Ile Ser Asn Arg Phe Ser Gly
180 185 190
Val Pro Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu
195 200 205
Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Val
210 215 220
Gln Val Ser Ser Phe Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu
225 230 235 240
Ile Lys
<210> 160
<211> 726
<212> DNA
<213> 人工序列 抗GPC3的ScFv
<400> 160
caagtgcaac tcgttcaatc aggagctgag gtgaagaaac ctggagcatc tgtcaagctc 60
tcctgtaagg cctccggata cacctttacc gattacgaga tgcactgggt gaagcagact 120
cctggaaagg gtctgaagtg gatcggcgcc ctggacccta agaccggaga caccgcttat 180
gcccagaagt ttcagggaag agccactctg accgctgaca catcaaccga taccgcttac 240
atggagctca gctccctgcg cagtgaggat tctgctgtgt actattgtac acgcttttat 300
agctacacct actggggaca gggcactctc gtgaccgtct cctccggtgg aggtggatct 360
ggcggcggcg gctcaggcgg aggaggttcc gatattgtgc tgacgcagtc tccactctca 420
ctgaccgtca cccctggaga gccggcctcc atctcctgca ggtctagtga aagcctcgta 480
cacagtaatg gaaacaccta cttgagttgg cttcagcaga ggccaggcca gcctccaaga 540
ctcctaattt ataagatttc taaccggttc tctggggtcc cagacagatt cagtggcagt 600
ggggcaggga cagatttcac actgaaaatc agcagggtgg aagctgagga tgtcggggtt 660
tattactgcg ttcaggtgtc gtcgtttccg tacacttttg gccaggggac caaggtggag 720
atcaaa 726
<210> 161
<211> 210
<212> PRT
<213> 人工序列 Crossmab-CD3双特异性抗体CD3轻链
<400> 161
Asp Ile Gln Met Thr Gln Glu Pro Ser Leu Thr Thr Ser Pro Gly Gly
1 5 10 15
Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser
20 25 30
Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Gly Gln Ala Pro Arg Gly
35 40 45
Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe
50 55 60
Ser Gly Ser Leu Ile Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Val
65 70 75 80
Gln Pro Glu Asp Glu Ala Ile Tyr Phe Cys Ala Leu Trp Tyr Ser Asn
85 90 95
Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ser Ser
100 105 110
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
115 120 125
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
130 135 140
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
145 150 155 160
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
165 170 175
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
180 185 190
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
195 200 205
Lys Val
210
<210> 162
<211> 630
<212> DNA
<213> 人工序列 Crossmab-CD3双特异性抗体CD3轻链
<400> 162
gatattcaga tgacacaaga accaagtctc actacaagcc ctggaggcac cgttacactg 60
acctgtcgct cttccactgg cgccgtgacc acttccaact atgcaaactg ggtgcaggag 120
aaacctggac aggctcctag aggtctgatt ggaggcacta acaagagagc tccagggact 180
cctgccaggt tcagcggatc tctgatcggt gggaaggcag ctctgacaat cactggagtg 240
caacccgagg atgaggctat ctacttctgt gctctctggt actcaaatct gtgggtgttc 300
ggaggtggaa caaagctgga gatcaagcgg tcttcagcta gcaccaaggg cccatcggtc 360
ttccccctgg caccctcctc caagagcacc tctgggggca cagcggccct gggctgcctg 420
gtcaaggact acttccccga accggtgacg gtgtcgtgga actcaggcgc cctgaccagc 480
ggcgtgcaca ccttcccggc tgtcctacag tcctcaggac tctactccct cagcagcgtg 540
gtgaccgtgc cctccagcag cttgggcacc cagacctaca tctgcaacgt gaatcacaag 600
cccagcaaca ccaaggtgga caagaaagtt 630
<210> 163
<211> 464
<212> PRT
<213> 人工序列 Crossmab-CD3双特异性抗体CD3重链
<400> 163
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr
20 25 30
Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp
50 55 60
Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr
65 70 75 80
Ala Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr
85 90 95
Tyr Cys Val Arg His Gly Asn Phe Gly Thr Ser Tyr Val Ser Trp Phe
100 105 110
Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Val
115 120 125
Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys
130 135 140
Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg
145 150 155 160
Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn
165 170 175
Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser
180 185 190
Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys
195 200 205
Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr
210 215 220
Lys Ser Phe Asn Arg Gly Glu Cys Glu Pro Lys Ser Cys Asp Lys Thr
225 230 235 240
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser
245 250 255
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
260 265 270
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
275 280 285
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
290 295 300
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
305 310 315 320
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
325 330 335
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
340 345 350
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
355 360 365
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
370 375 380
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
385 390 395 400
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Lys Lys Lys Leu Asp
405 410 415
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
420 425 430
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
435 440 445
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
450 455 460
<210> 164
<211> 1392
<212> DNA
<213> 人工序列 Crossmab-CD3双特异性抗体CD3重链
<400> 164
gaagtgcaac tggttgaaag cggtggagga ctggttcagc ctggaggctc cctcaaactc 60
agttgcgccg cttccggttt tacattcaac acttatgcca tgaattgggt gcgccaggct 120
cccggaaaag gcctggaatg ggtggcacgg atcaggagca aatacaacaa ttacgccaca 180
tattacgctg actccgttaa agacagattc acaatttcca gagatgattc caagaatact 240
gcttatctcc agatgaacaa cctgaagaca gaggatacag ctatgtacta ttgcgtgcgg 300
catggcaatt tcggaacttc ctatgtgagt tggtttgcct actggggaca gggaactctc 360
gttactgtca gctccgcaag tgtcgcagct ccatccgtgt tcatctttcc accctccgac 420
gagcaactga aaagtggcac agcctccgtc gtctgtctgc tgaacaattt ctaccctcgg 480
gaggccaagg tccagtggaa ggtggacaac gctctccaga gtggaaatag ccaagagtcc 540
gttactgagc aggactctaa ggattcaacc tacagcctct ccagcaccct gactctgagc 600
aaggcagatt atgagaagca caaagtctac gcatgtgaag tgactcatca gggactcagc 660
tcacccgtta ccaagtcttt caacagggga gagtgtgagc ccaaatcttg tgacaaaact 720
cacacatgcc caccgtgccc agcacctgaa gccgcagggg gaccgtcagt cttcctcttc 780
cccccaaaac ccaaggacac cctcatgatc tcccggaccc ctgaggtcac atgcgtggtg 840
gtggacgtga gccacgaaga ccctgaggtc aagttcaact ggtacgtgga cggcgtggag 900
gtgcataatg ccaagacaaa gccgcgggag gagcagtaca acagcacgta ccgtgtggtc 960
agcgtcctca ccgtcctgca ccaggactgg ctgaatggca aggagtacaa gtgcaaggtc 1020
tccaacaaag ccctcccagc ccccatcgag aaaaccatct ccaaagccaa agggcagccc 1080
cgagaaccac aggtgtacac cctgccccca tcccgggagg agatgaccaa gaaccaggtc 1140
agcctgacct gcctggtcaa aggcttctat cccagcgaca tcgccgtgga gtgggagagc 1200
aatgggcagc cggagaacaa ctacaagacc acgaagaaga agctggactc cgacggctcc 1260
ttcttcctct acagcaagct caccgtggac aagagcaggt ggcagcaggg gaacgtcttc 1320
tcatgctccg tgatgcatga ggctctgcac aaccactaca cgcagaagag cctctccctg 1380
tctccgggta aa 1392
<210> 165
<211> 212
<212> PRT
<213> 人工序列 Crossmab-GPC3双特异性抗体GPC3轻链
<400> 165
Asp Ile Val Leu Thr Gln Ser Pro Leu Ser Leu Thr Val Thr Pro Gly
1 5 10 15
Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Glu Ser Leu Val His Ser
20 25 30
Asn Gly Asn Thr Tyr Leu Ser Trp Leu Gln Gln Arg Pro Gly Gln Pro
35 40 45
Pro Arg Leu Leu Ile Tyr Lys Ile Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Val Gln Val
85 90 95
Ser Ser Phe Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105 110
Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser
115 120 125
Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys
130 135 140
Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
145 150 155 160
Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
165 170 175
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr
180 185 190
Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val
195 200 205
Asp Lys Lys Val
210
<210> 166
<211> 636
<212> DNA
<213> 人工序列 Crossmab-GPC3双特异性抗体GPC3轻链
<400> 166
gatattgtgc tgacgcagtc tccactctca ctgaccgtca cccctggaga gccggcctcc 60
atctcctgca ggtctagtga aagcctcgta cacagtaatg gaaacaccta cttgagttgg 120
cttcagcaga ggccaggcca gcctccaaga ctcctaattt ataagatttc taaccggttc 180
tctggggtcc cagacagatt cagtggcagt ggggcaggga cagatttcac actgaaaatc 240
agcagggtgg aagctgagga tgtcggggtt tattactgcg ttcaggtgtc gtcgtttccg 300
tacacttttg gccaggggac caaggtggag atcaaatctt cagctagcac caagggccca 360
tcggtcttcc ccctggcacc ctcctccaag agcacctctg ggggcacagc ggccctgggc 420
tgcctggtca aggactactt ccccgaaccg gtgacggtgt cgtggaactc aggcgccctg 480
accagcggcg tgcacacctt cccggctgtc ctacagtcct caggactcta ctccctcagc 540
agcgtggtga ccgtgccctc cagcagcttg ggcacccaga cctacatctg caacgtgaat 600
cacaagccca gcaacaccaa ggtggacaag aaagtt 636
<210> 167
<211> 454
<212> PRT
<213> 人工序列 Crossmab-GPC3双特异性抗体GPC3重链
<400> 167
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Glu Met His Trp Val Lys Gln Thr Pro Gly Lys Gly Leu Lys Trp Ile
35 40 45
Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Ala Thr Leu Thr Ala Asp Thr Ser Thr Asp Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ser Ala Ser Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro
115 120 125
Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu
130 135 140
Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn
145 150 155 160
Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser
165 170 175
Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala
180 185 190
Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly
195 200 205
Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Glu Pro
210 215 220
Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
225 230 235 240
Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
245 250 255
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
260 265 270
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
275 280 285
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
290 295 300
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
305 310 315 320
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
325 330 335
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
340 345 350
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn
355 360 365
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
370 375 380
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
385 390 395 400
Asp Asp Asp Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
405 410 415
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
420 425 430
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
435 440 445
Ser Leu Ser Pro Gly Lys
450
<210> 168
<211> 1362
<212> DNA
<213> 人工序列 Crossmab-GPC3双特异性抗体GPC3重链
<400> 168
caagtgcaac tcgttcaatc aggagctgag gtgaagaaac ctggagcatc tgtcaagctc 60
tcctgtaagg cctccggata cacctttacc gattacgaga tgcactgggt gaagcagact 120
cctggaaagg gtctgaagtg gatcggcgcc ctggacccta agaccggaga caccgcttat 180
gcccagaagt ttcagggaag agccactctg accgctgaca catcaaccga taccgcttac 240
atggagctca gctccctgcg cagtgaggat tctgctgtgt actattgtac acgcttttat 300
agctacacct actggggaca gggcactctc gtgaccgtct cctccgcaag tgtcgcagct 360
ccatccgtgt tcatctttcc accctccgac gagcaactga aaagtggcac agcctccgtc 420
gtctgtctgc tgaacaattt ctaccctcgg gaggccaagg tccagtggaa ggtggacaac 480
gctctccaga gtggaaatag ccaagagtcc gttactgagc aggactctaa ggattcaacc 540
tacagcctct ccagcaccct gactctgagc aaggcagatt atgagaagca caaagtctac 600
gcatgtgaag tgactcatca gggactcagc tcacccgtta ccaagtcttt caacagggga 660
gagtgtgagc ccaaatcttg tgacaaaact cacacatgcc caccgtgccc agcacctgaa 720
gccgcagggg gaccgtcagt cttcctcttc cccccaaaac ccaaggacac cctcatgatc 780
tcccggaccc ctgaggtcac atgcgtggtg gtggacgtga gccacgaaga ccctgaggtc 840
aagttcaact ggtacgtgga cggcgtggag gtgcataatg ccaagacaaa gccgcgggag 900
gagcagtaca acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg 960
ctgaatggca aggagtacaa gtgcaaggtc tccaacaaag ccctcccagc ccccatcgag 1020
aaaaccatct ccaaagccaa agggcagccc cgagaaccac aggtgtacac cctgccccca 1080
tcccgggagg agatgaccaa gaaccaggtc agcctgacct gcctggtcaa aggcttctat 1140
cccagcgaca tcgccgtgga gtgggagagc aatgggcagc cggagaacaa ctacaagacc 1200
gacgatgacg tgctggactc cgacggctcc ttcttcctct acagcaagct caccgtggac 1260
aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg tgatgcatga ggctctgcac 1320
aaccactaca cgcagaagag cctctccctg tctccgggta aa 1362

Claims (18)

1.抗体，其特征在于，所述抗体的Fab区在重链恒定区CH1及轻链恒定区(CL)引入以下氨基酸突变后，较野生型相比，可促进相应的重/轻链正确配对，使其更好的结合相应抗原，其中，CH1突变序列及CL突变序列如以下组合：
a)CH1突变SEQ ID NO:1及CL突变SEQ ID NO:3
b)CH1突变SEQ ID NO:5及CL突变SEQ ID NO:7
c)CH1突变SEQ ID NO:9及CL突变SEQ ID NO:11
d)CH1突变SEQ ID NO:13及CL突变SEQ ID NO:15
e)CH1突变SEQ ID NO:17及CL突变SEQ ID NO:19
f)CH1突变SEQ ID NO:21及CL突变SEQ ID NO:23
g)CH1突变SEQ ID NO:25及CL突变SEQ ID NO:27
h)CH1突变SEQ ID NO:29及CL突变SEQ ID NO:31
i)CH1突变SEQ ID NO:33及CL突变SEQ ID NO:35
j)CH1突变SEQ ID NO:37及CL突变SEQ ID NO:39
k)CH1突变SEQ ID NO:41及CL突变SEQ ID NO:43
l)CH1突变SEQ ID NO:45及CL突变SEQ ID NO:47
m)CH1突变SEQ ID NO:49及CL突变SEQ ID NO:51
n)CH1突变SEQ ID NO:53及CL突变SEQ ID NO:55
o)CH1突变SEQ ID NO:57及CL突变SEQ ID NO:59
p)CH1突变SEQ ID NO:61及CL突变SEQ ID NO:63
q)CH1突变SEQ ID NO:65及CL突变SEQ ID NO:67
r)CH1突变SEQ ID NO:69及CL突变SEQ ID NO:71
s)CH1突变SEQ ID NO:73及CL突变SEQ ID NO:75
t)CH1突变SEQ ID NO:77及CL突变SEQ ID NO:79
u)CH1突变SEQ ID NO:81及CL突变SEQ ID NO:83
v)CH1突变SEQ ID NO:85及CL突变SEQ ID NO:87
w)CH1突变SEQ ID NO:89及CL突变SEQ ID NO:91
x)CH1突变SEQ ID NO:93及CL突变SEQ ID NO:95
y)CH1突变SEQ ID NO:97及CL突变SEQ ID NO:99
z)CH1突变SEQ ID NO:101及CL突变SEQ ID NO:103
aa)CH1突变SEQ ID NO:105及CL突变SEQ ID NO:107
bb)CH1突变SEQ ID NO:109及CL突变SEQ ID NO:111
cc)CH1突变SEQ ID NO:113及CL突变SEQ ID NO:115。
2.双特异性抗体，其特征在于，具有两个的Fab区的抗体；其中所述抗体是具有第一重链和第一轻链，以及第二重链和第二轻链的双特异性抗体；其中所述第一重链和第一轻链形成第一Fab区，所述第二重链和第二轻链形成第二Fab区；为促进第一Fab区及第二Fab区相应的重/轻链正确配对，第一Fab区及第二Fab区同时含有如权利要求1所述的CH1突变序列及CL突变序列的不同组合，这些突变的组合如下：
i.第一Fab区含有CH1突变SEQ ID NO:77及CL突变SEQ ID NO:79，第二Fab区含有CH1突变SEQ ID NO:33及CL突变SEQ ID NO:35；
ii.第一Fab区含有CH1突变SEQ ID NO:113及CL突变SEQ ID NO:115，第二Fab区含有CH1突变SEQ ID NO:53及CL突变SEQ ID NO:55；
iii.第一Fab区含有CH1突变SEQ ID NO:77及CL突变SEQ ID NO:79，第二Fab区含有CH1突变SEQ ID NO:53及CL突变SEQ ID NO:55；
iv.第一Fab区含有CH1突变SEQ ID NO:113及CL突变SEQ ID NO:115，第二Fab区含有CH1突变SEQ ID NO:33及CL突变SEQ ID NO:35；
v.第一Fab区含有CH1突变SEQ ID NO113:及CL突变SEQ ID NO115:，第二Fab区含有CH1突变SEQ ID NO85:及CL突变SEQ ID NO:87；
vi.第一Fab区含有CH1突变SEQ ID NO:65及CL突变SEQ ID NO:67，第二Fab区含有CH1突变SEQ ID NO:33及CL突变SEQ ID NO:35；
vii.第一Fab区含有CH1突变SEQ ID NO:9及CL突变SEQ ID NO:11，第二Fab区含有CH1突变SEQ ID NO:33及CL突变SEQ ID NO:35；
viii.第一Fab区含有CH1突变SEQ ID NO:25及CL突变SEQ ID NO:27，第二Fab区含有CH1突变SEQ ID NO:53及CL突变SEQ ID NO:55；
ix.第一Fab区含有CH1突变SEQ ID NO:113及CL突变SEQ ID NO:115，第二Fab区含有CH1突变SEQ ID NO:53及CL突变SEQ ID NO:55；
x.第一Fab区含有CH1突变SEQ ID NO:77及CL突变SEQ ID NO:79，第二Fab区含有CH1突变SEQ ID NO:41及CL突变SEQ ID NO:43。
3.根据权利要求2所述的双特异性抗体，其特征在于，所述第一Fab区的轻链恒定区CL还引入突变178D，第二Fab区的重链恒定区CH1还引入突变188D；
或所述第一Fab区的轻链恒定区CL还引入突变178K，第二Fab区的重链恒定区CH1还引入突变192K；
所述抗体的第一Fab区和第二Fab区引入的突变相同或不同。
4.根据权利要求2所述的双特异性抗体，其特征在于，所述双特异性抗体具有识别第一抗原的第一Fab区以及识别第二抗原的第二Fab区组成；第一Fab区与第二Fab区组识别不同种类的抗原，或识别同一抗原的不同表位。
5.根据权利要2所述的双特异性抗体为选自以下中的任意一种：所述双特异性抗体的选自以下氨基酸序列中的任意一种：
所述第一重链具有SEQ ID NO:129所示的氨基酸序列，且所述第一轻链具有SEQ IDNO:127所示的氨基酸序列；所述第二重链具有SEQ ID NO:133所示的氨基酸序列，且所述第二轻链具有SEQ ID NO:131所示的氨基酸序列；
或所述第一重链具有SEQ ID NO:145所示的氨基酸序列，且所述第一轻链具有SEQ IDNO:143所示的氨基酸序列；所述第二重链具有SEQ ID NO:149所示的氨基酸序列，且所述第二轻链具有SEQ ID NO:147所示的氨基酸序列；
或所述第一重链具有SEQ ID NO:137所示的氨基酸序列，且所述第一轻链具有SEQ IDNO:135所示的氨基酸序列；所述第二重链具有SEQ ID NO:141所示的氨基酸序列，且所述第二轻链具有SEQ ID NO:139所示的氨基酸序列；
或所述第一重链具有SEQ ID NO:153所示的氨基酸序列，且所述第一轻链具有SEQ IDNO:151所示的氨基酸序列；所述第二重链具有SEQ ID NO:157所示的氨基酸序列，且所述第二轻链具有SEQ ID NO:155所示的氨基酸序列。
6.根据权利要求2-5任一项所述的双特异性抗体，其特征在于，所述双特异性抗体来源于IgG、IgA、IgM、IgE或IgD。
7.根据权利要求6所述的双特异性抗体，其特征在于所述双特异性抗体来源于IgG1、IgG2、IgG3或IgG4。
8.核酸分子，其特征在于，所述核酸分子编码权利要求1所述的抗体和/或权利要求2-7任一项所述的双特异性抗体。
9.表达载体，其特征在于，所述表达载体含有权利要求8所述的核酸分子。
10.重组细胞，其特征在于，所述重组细胞表达权利要求1所述的抗体和/或权利要求2-7任一项所述的双特异性抗体。
11.根据权利要求10所述的重组细胞，其特征在于，所述重组细胞的基因组中整合有权利要求8所述的核酸分子。
12.根据权利要求10所述的重组细胞，其特征在于，所述重组细胞中含有权利要求9所述的表达载体。
13.权利要求1所述的抗体的制备方法，其特征在于，包括以下步骤：
将编码所述抗体的核酸分子连入质粒，转入感受态细胞中，随后挑选单克隆细胞进行筛选；提取筛选的阳性克隆中的表达载体，转入受体细胞中，筛选得到重组细胞；
培养所述重组细胞，收集培养上清，分离纯化，得到所述抗体。
14.抗体偶联物，其特征在于，所述抗体偶联物包括权利要求1所述的抗体和/或权利要求2-7任一项所述的双特异性抗体，以及偶联的标记物。
15.根据权利要求14所述的抗体偶联物，其特征在于，所述标记物包括细胞毒素、放射性同位素、荧光标记物、发光物、显色物质或酶中的任意一种或至少两种的组合。
16.药物组合物，其特征在于，所述药物组合物含有权利要求1所述的抗体、权利要求2-7任一项所述的双特异性抗体或权利要求14或15所述的抗体偶联物中的任意一种或至少两种的组合。
17.根据权利要求16所述的药物组合物，其特征在于，所述药物组合物还包括药学上可接受的载体、表面活性剂、稀释剂或赋形剂中的任意一种或至少两种的组合。
18.权利要求1所述的抗体、权利要求2-7任一项所述的双特异性抗体、权利要求14所述的抗体偶联物或权利要求15或16所述的药物组合物中的任意一种或至少两种的组合在制备疾病治疗药物中的应用；
所述疾病包括癌症。
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* Cited by examiner, † Cited by third party
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EP2543680A1 (en) * 2011-07-07 2013-01-09 Centre National de la Recherche Scientifique Multispecific mutated antibody Fab fragments
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Design and Validation of a Novel Generic Platform for the Production of Tetravalent IgG1-like Bispecific Antibodies;Jose´e Golay,et al.;The Journal of Immunology;第196卷(第7期);第3201-3202页全部 *

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