CN115210265A - 蛋白酶加工的分子 - Google Patents

蛋白酶加工的分子 Download PDF

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CN115210265A
CN115210265A CN202180018397.5A CN202180018397A CN115210265A CN 115210265 A CN115210265 A CN 115210265A CN 202180018397 A CN202180018397 A CN 202180018397A CN 115210265 A CN115210265 A CN 115210265A
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T·兰格
E·雷奥
S·韦尔
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Sanofi Aventis France
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Abstract

本发明涉及包含至少一个蛋白酶可切割接头的蛋白质分子。本发明还涉及通过此类蛋白质分子的蛋白酶切割可获得的蛋白质分子及其用于疗法的用途。

Description

蛋白酶加工的分子
技术领域
本发明涉及包含至少一个蛋白酶可切割接头的蛋白质分子。本发明还涉及通过此类蛋白质分子的蛋白酶加工可获得的蛋白质分子以及其用于疗法的用途。
背景技术
天然存在的IgG抗体是二价和单特异性的。可以使用重组技术产生对多种不同抗原具有结合特异性的多特异性(例如，双特异性)抗体，并且所述多特异性抗体预计具有广泛的临床应用。众所周知，完整的IgG抗体分子是Y形分子，其包含四条多肽链：两条重链和两条轻链。每条轻链由两个结构域组成，N末端结构域被称为可变结构域或VL结构域(或区)并且C末端结构域被称为恒定结构域或CL结构域/区(恒定κ(Cκ)或恒定λ(Cλ)结构域)。每条重链由四个或五个结构域组成，具体取决于抗体的类别。N末端结构域被称为可变(或VH)结构域(或区)，其后是第一恒定(或CH1)结构域、铰链区、以及然后是第二和第三恒定(或CH2和CH3)结构域。在组装的抗体中，VL结构域和VH结构域缔合在一起形成抗原结合位点。而且，CL结构域和CH1结构域缔合在一起以保持一条重链与一条轻链缔合。通过CH2结构域和CH3结构域的相互作用以及两条重链上的铰链区之间的相互作用，两个重轻链异二聚体缔合在一起。
多特异性抗体的产生所伴随的主要问题之一是轻链错配。虽然两条不同重链的配对可以通过如“杵臼”的技术来解决，但还必要的是施加正确的轻链缔合。从结构的角度来看，这很可能是较大的挑战，因为应该在Fab片段界面内应用修饰，这需要更多修饰。在任何情况下，重链的异二聚化以及其与同源轻链的缔合需要相对大数量的不同设计以实现适当的链缔合，从而避免形成不需要的物质。此外，编码轻链和重链的来自不同载体/质粒的多特异性分子的表达需要所有质粒的翻译达到相同的程度，以提供正确链配对的最佳比率。
本发明通过范式的改变(从设计派生的多特异性形式到过程派生的多特异性形式)来解决轻链配对问题。在此，不需要抗体序列的修饰。
发明内容
在一方面，本发明涉及一种蛋白质，其包含至少一个具有下式的多肽链
PP1-PCL-PP2，
其中
PP1是第一多肽，
PP2是第二多肽，并且
PCL是蛋白酶可切割接头，所述蛋白酶可切割接头包含至少一个蛋白酶切割位点，所述至少一个蛋白酶切割位点包含氨基酸序列HRRX1X2RSVDE(SEQ ID NO:42)，其中X1和X2独立地选自氨基酸，优选天然存在的氨基酸。
在一个实施方案中，所述蛋白酶是弗林蛋白酶和/或弗林蛋白酶样蛋白酶。
在一个实施方案中，所述至少一个蛋白酶切割位点包含氨基酸序列HRRRKRSVDE(SEQ ID NO:43)或氨基酸序列HRRQQRSVDE(SEQ ID NO:44)。
在一个实施方案中，所述接头的蛋白酶切割导致所述蛋白质的活性改变。
在一个实施方案中，所述活性改变是与至少一种抗原的结合或增加的结合。
在一个实施方案中，蛋白酶切割在细胞内进行。
在一个实施方案中，所述蛋白酶可切割的接头包含两个蛋白酶切割位点，其中这两个蛋白酶切割位点分别位于所述蛋白酶可切割接头的N末端和C末端，其中这两个蛋白酶切割位点可以相同或不同。
在一个实施方案中，所述蛋白酶可切割接头具有10至80个氨基酸残基的长度。
在一个实施方案中，所述至少一个蛋白酶切割位点在至少2、至少3、至少4、至少5或至少10个连续的独立地选自甘氨酸和丝氨酸的氨基酸残基之前和/或之后。
在一个实施方案中，所述蛋白酶可切割接头进一步包含氨基酸序列，所述氨基酸序列选自(GS)n、(G4S)n(SEQ ID NO:51)、(HH)n、(EAAAK)n(SEQ ID NO:52)、(AP)nA(SEQ IDNO:53)、(KQGKQ)n(SEQ ID NO:54)及其组合，其中n是选自1至10的整数。
在一个实施方案中，所述蛋白酶可切割接头进一步包含氨基酸序列，所述氨基酸序列选自GSHHHHHHHHGGGGS(SEQ ID NO:45)、GGGGSEAAAKEAAAKGGGGS(SEQ ID NO:46)、APAPAPAPAPAPAPA(SEQ ID NO:47)、GSKQGKQKQGKQGS(SEQ ID NO:48)及其组合。
在一个实施方案中，PP1包含至少一个免疫球蛋白恒定区和/或至少一个免疫球蛋白可变区，和/或PP2包含至少一个免疫球蛋白恒定区和/或至少一个免疫球蛋白可变区。
在一个实施方案中，
PP1包含免疫球蛋白恒定区并且PP2包含免疫球蛋白可变区，
PP1包含免疫球蛋白可变区并且PP2包含免疫球蛋白恒定区，
PP1包含免疫球蛋白恒定区并且PP2包含免疫球蛋白恒定区，或
PP1包含免疫球蛋白可变区并且PP2包含免疫球蛋白可变区。
在一个实施方案中，所述蛋白质是抗体或抗体衍生物。
在一个实施方案中，所述蛋白质是单链抗体，优选多特异性单链抗体。
在一个实施方案中，所述蛋白质包含具有选自以下的式的多肽链：
(a)VL-CL-PCL-VH-CH1-铰链-CH2-CH3，
其中
VL是免疫球蛋白轻链可变区；
CL是免疫球蛋白轻链恒定区；
VH是免疫球蛋白重链可变区；
CH1是免疫球蛋白CH1重链恒定区；
铰链是免疫球蛋白铰链区；
CH2是免疫球蛋白CH2重链恒定区；
CH3是免疫球蛋白CH3重链恒定区；并且
PCL是所述蛋白酶可切割接头；
(b)VLa-CLa-PCL-VLb-CLb，
其中
VLa是第一免疫球蛋白轻链可变区；
VLb是第二免疫球蛋白轻链可变区；
CLa是第一免疫球蛋白轻链恒定区；
CLb是第二免疫球蛋白轻链恒定区；并且
PCL是所述蛋白酶可切割接头；以及
(c)VLa-CLa-PCL-VLb-CLb-L-铰链-CH2-CH3
其中
VLa是第一免疫球蛋白轻链可变区；
VLb是第二免疫球蛋白轻链可变区；
CLa是第一免疫球蛋白轻链恒定区；
CLb是第二免疫球蛋白轻链恒定区；
L是任选的接头；
铰链是免疫球蛋白铰链区；
CH2是免疫球蛋白CH2重链恒定区；
CH3是免疫球蛋白CH3重链恒定区；并且
PCL是所述蛋白酶可切割接头。
在一个实施方案中，所述蛋白质包含具有式VL-CL-PCL-VH-CH1-铰链-CH2-CH3的多肽链，其中VL与VH配对以形成抗原结合位点。
在一个实施方案中，在蛋白酶可切割接头的蛋白酶切割后，所述抗原结合位点变得有活性或展现出增加的活性。
在一个实施方案中，所述蛋白质包含具有式VL-CL-PCL-VH-CH1-铰链-CH2-CH3的多肽链的两个拷贝，其中这两个拷贝经由至少两个二硫键彼此缔合。
在一个实施方案中，所述蛋白质包含具有式VLa-CLa-PCL-VHa-CH1a-铰链-CH2-CH3的第一多肽链和具有式VLb-CLb-PCL-VHb-CH1b-铰链-CH2-CH3的第二多肽链，
其中
VLa是第一免疫球蛋白轻链可变区；
VLb是第二免疫球蛋白轻链可变区；
CLa是第一免疫球蛋白轻链恒定区；
CLb是第二免疫球蛋白轻链恒定区；
VHa是第一免疫球蛋白重链可变区；
VHb是第二免疫球蛋白重链可变区；
CH1a是第一免疫球蛋白CH1重链恒定区；并且
CH1b是第二免疫球蛋白CH1重链恒定区；
其中VLa与VHa配对以形成与抗原A结合的抗原结合位点，并且其中VLb与VHb配对以形成与抗原B结合的抗原结合位点；并且
其中第一多肽链经由至少两个二硫键并且任选地经由至少一种另外的Fc相互作用与第二多肽链缔合。
在一个实施方案中，在蛋白酶可切割接头的蛋白酶切割后，抗原结合位点A和/或抗原结合位点B变得有活性或展现出增加的活性。
在一个实施方案中，所述至少一种另外的Fc相互作用是至少一种杵臼(knob-into-hole)相互作用。
在一个实施方案中，所述蛋白质包含具有式VLb-L1-VLa-L2-CL的第一多肽链、具有式VHa-L3-VHb-L4-CH1-铰链-CH2-CH3的第二多肽链和具有式VLc-CLc-PCL-VHc-CH1c-铰链-CH2-CH3的第三多肽链，
其中
VLa是第一免疫球蛋白轻链可变区；
VLb是第二免疫球蛋白轻链可变区；
VLc是第三免疫球蛋白轻链可变区；
CL是免疫球蛋白轻链恒定区；
CLc是免疫球蛋白轻链恒定区；
VHa是第一免疫球蛋白重链可变区；
VHb是第二免疫球蛋白重链可变区；
VHc是第三免疫球蛋白重链可变区；
CH1是免疫球蛋白CH1重链恒定区；
CH1c是免疫球蛋白CH1重链恒定区；
铰链是免疫球蛋白铰链区；
CH2是免疫球蛋白CH2重链恒定区；并且
CH3是免疫球蛋白CH3重链恒定区；
L1、L2、L3和L4是接头；
其中VLa与VHa配对以形成与抗原A结合的抗原结合位点，其中VLb与VHb配对以形成与抗原B结合的抗原结合位点，并且其中VLc与VHc配对以形成与抗原C结合的抗原结合位点；并且
其中第二多肽链经由至少两个二硫键并且任选地经由至少一种另外的Fc相互作用与第三多肽链缔合。
在一个实施方案中，在蛋白酶可切割接头的蛋白酶切割后，抗原结合位点C变得有活性或展现出增加的活性。
在一个实施方案中，所述至少一种另外的Fc相互作用是至少一种杵臼相互作用。
在一个实施方案中，所述蛋白质包含具有式VLa-CLa-PCL-VLb-CLb的第一多肽链和具有式VHa-CH1a-L-VHb-CH1b-铰链-CH2-CH3的第二多肽链，
其中
VHa是第一免疫球蛋白重链可变区；
VHb是第二免疫球蛋白重链可变区；
CH1a是第一免疫球蛋白CH1重链恒定区；
CH1b是第二免疫球蛋白CH1重链恒定区；
L是任选的接头；
铰链是免疫球蛋白铰链区；
CH2是免疫球蛋白CH2重链恒定区；并且
CH3是免疫球蛋白CH3重链恒定区；
其中VLa与VHa配对以形成与抗原A结合的抗原结合位点，并且其中VLb与VHb配对以形成与抗原B结合的抗原结合位点。
在一个实施方案中，在蛋白酶可切割接头的蛋白酶切割后，抗原结合位点B变得有活性或展现出增加的活性。
在一个实施方案中，所述蛋白质包含第一多肽链的两个拷贝和第二多肽链的两个拷贝，其中第二多肽链的两个拷贝经由至少两个二硫键彼此缔合。
在一个实施方案中，所述蛋白质包含具有式VLb-CLb-PCL-VLa-CLa的第一多肽链、具有式VLc-CLc-PCL-VLd-CLd的第二多肽链、具有式VHa-CH1a-L-VHb-CH1b-铰链-CH2-CH3的第三多肽链、和具有式VHc-CH1c-L-VHd-CH1d-铰链-CH2-CH3的第四多肽链，
其中
VLa是第一免疫球蛋白轻链可变区；
VLb是第二免疫球蛋白轻链可变区；
VLc是第三免疫球蛋白轻链可变区；
VLd是第四免疫球蛋白轻链可变区；
CLa是第一免疫球蛋白轻链恒定区；
CLb是第二免疫球蛋白轻链恒定区；
CLc是第三免疫球蛋白轻链恒定区；
CLd是第四免疫球蛋白轻链恒定区；
VHa是第一免疫球蛋白重链可变区；
VHb是第二免疫球蛋白重链可变区；
VHc是第三免疫球蛋白重链可变区；
VHd是第四免疫球蛋白重链可变区；
CH1a是第一免疫球蛋白CH1重链恒定区；
CH1b是第二免疫球蛋白CH1重链恒定区；
CH1c是第三免疫球蛋白CH1重链恒定区；
CH1d是第四免疫球蛋白CH1重链恒定区；
L是任选的接头；
铰链是免疫球蛋白铰链区；
CH2是免疫球蛋白CH2重链恒定区；并且
CH3是免疫球蛋白CH3重链恒定区；
其中VLa与VHa配对以形成与抗原A结合的抗原结合位点，其中VLb与VHb配对以形成与抗原B结合的抗原结合位点，其中VLc与VHc配对以形成与抗原C结合的抗原结合位点，并且其中VLd与VHd配对以形成与抗原D结合的抗原结合位点；并且
其中第三多肽链经由至少两个二硫键并且任选地经由至少一种另外的Fc相互作用与第四多肽链缔合。
在一个实施方案中，在蛋白酶可切割接头的蛋白酶切割后，抗原结合位点B和/或抗原结合位点D变得有活性或展现出增加的活性。
在一个实施方案中，所述至少一种另外的Fc相互作用是至少一种杵臼相互作用。
在一个实施方案中，所述蛋白质包含具有式VLa-CLa-PCL-VLb-CLb-L-铰链-CH2-CH3的第一多肽链和具有式VHa-CH1a-L-VHb-CH1b-铰链-CH2-CH3的第二多肽链，
其中
VHa是第一免疫球蛋白重链可变区；
VHb是第二免疫球蛋白重链可变区；
CH1a是第一免疫球蛋白CH1重链恒定区；
CH1b是第二免疫球蛋白CH1重链恒定区；
L是任选的接头；
铰链是免疫球蛋白铰链区；
CH2是免疫球蛋白CH2重链恒定区；并且
CH3是免疫球蛋白CH3重链恒定区；
其中VLa与VHa配对以形成与抗原A结合的抗原结合位点，并且其中VLb与VHb配对以形成与抗原B结合的抗原结合位点；并且
其中第一多肽链经由至少两个二硫键并且任选地经由至少一种另外的Fc相互作用与第二多肽链缔合。
在一个实施方案中，在蛋白酶可切割接头的蛋白酶切割后，抗原结合位点B变得有活性或展现出增加的活性。
在一个实施方案中，所述至少一种另外的Fc相互作用是至少一种杵臼相互作用。
在另一方面，本发明涉及一种核酸或核酸组，其编码如上所定义的蛋白质。
在另一方面，本发明涉及一种载体或载体组，其包含如上所定义的核酸或核酸组。
在另一方面，本发明涉及一种宿主细胞，其包含如上所定义的蛋白质、如上所定义的核酸或核酸组、或如上所定义的载体或载体组。
在一个实施方案中，所述宿主细胞表达内源或外源弗林蛋白酶和/或弗林蛋白酶样蛋白酶。
在一个实施方案中，所述宿主细胞是哺乳动物细胞。
在一方面，本发明涉及一种生产蛋白质的方法，其包括以下步骤：
i)培养如上所定义的宿主细胞；以及
ii)将所述蛋白质与所述宿主细胞分离。
在另一方面，本发明涉及一种通过如上所定义的方法可获得的蛋白质。
在又另一方面，本发明涉及一种通过弗林蛋白酶和/或弗林蛋白酶样蛋白酶切割如上所定义的蛋白质可获得的蛋白质。
在一个实施方案中，如上所定义的蛋白质是治疗活性蛋白质。
在一方面，本发明涉及如上所定义的蛋白质，其用于疗法。
在另一方面，本发明涉及如上所定义的蛋白质在制造药剂中的用途。
在又另一方面，本发明涉及一种治疗或预防疾病或障碍的方法，其包括向有需要的受试者施用有效量的如上所定义的蛋白质。
在又另一方面，本发明涉及一种药物组合物或试剂盒，其包含如上所定义的蛋白质。
在另一方面，本发明涉及一种包含SEQ ID NO:42至44中任一项的氨基酸序列的肽；一种包含所述肽的蛋白质；一种编码所述肽或蛋白质的核酸；一种包含所述核酸的载体；或一种包含所述肽、蛋白质、核酸或载体的宿主细胞。
附图说明
图1：蛋白酶可切割的同型二聚体抗体形式。(A)单链(sC)单克隆IgG。CL(例如，Cκ)结构域在其C末端经由蛋白酶可切割接头(PCL)序列伸长到重链的VH结构域的N末端。(B)单轻链(sLC)二价双特异性串联-IgG。重链的VH-CH1(VHb)结构域在其N末端例如经由(G4S)3接头(SEQ ID NO:55)伸长到第二VH-CH1结构域(VHa)。轻链的VL-CL结构域(VLb)在其N末端使用PCL序列伸长到第二VL-CL结构域(VLa)。
图2：蛋白酶可切割的同型二聚体抗体形式。(A)单轻链(sLC)多特异性串联-IgG。重链的VH-CH1(VHb和VHd)结构域在N末端例如经由(G4S)3接头(SEQ ID NO:55)伸长到第二VH-CH1结构域(VHa和VHc)。轻链的VL-CL结构域(VLb和VLd)在N末端使用PCL序列伸长到第二VL-CL结构域(VLa和VLc)。(B)单链(sC)单价双特异性串联样-IgG。第一重链的VH-CH1结构域(VHb)在其N末端例如经由(G4S)3接头(SEQ ID NO:55)伸长到第二VH-CH1结构域(VHa)；第二重链的铰链区伸长到两个VL-CL-结构域，其中VLb和Fv a经由PCL序列连接。(C)含有三特异性CODV-IgG的单链(sC)Fab。抗体的一个臂包含与CODV-HC(VHa-接头-VHb-接头-CH1-铰链-CH2-CH3)缔合的CODV-LC(VLb-接头-VLa-接头-CL)，另一个臂包含单链(sC)Fab臂，其中VHc结构域在其N末端使用PCL序列伸长到VLc-CL结构域。(D)单链(sC)双特异性IgG。每条HC的VHa和VHb结构域在N末端使用PCL序列分别伸长到VLa-CL和VLb-CL结构域。所有异二聚体分子的重链优选包含一个或多个杵臼(KIH)突变，形成不对称抗体。
图3：来自HEK293-FS细胞的串联-抗IL4x抗IL13-huIgG1构建体的分析。图(A)示出了在使用分析型尺寸排阻色谱法进行亲和力和制备型SEC后串联-IgG产物(串联-IgG对照；sLC-PCL1-串联-IgG和sLC-PCL2-串联-IgG)的纯度。图(B)示出了使用分析型疏水性相互作用色谱法的抗体产物的同质性。图(C)示出了使用4％-12％Bis/Tris MOPS十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)，抗体的还原形式(分别为一条重链和两条轻链/一条单LC)和氧化形式(完整的串联-IgG)。
图4：来自HEK293-FS细胞的串联-抗IL4x抗IL13-huIgG1构建体的分析。图(A)示出了在使用分析型尺寸排阻色谱法进行亲和力和制备型SEC后串联-IgG产物(sLC-PCL1-串联-IgG、sLCΔPCL串联-IgG和sLC-PCL2-串联-IgG)的纯度。图(B)示出了使用分析型疏水性相互作用色谱法的抗体产物的同质性。图(C)示出了使用4％-12％Bis/Tris MOPS十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)，抗体的还原形式(分别为一条重链和两条轻链/一条单LC)和氧化形式(完整的串联-IgG)。
图5：在HEK293-FS细胞中与蛋白酶共表达的sLC串联-IgG的分析。图(A)示出了在使用分析型尺寸排阻色谱法进行亲和力和制备型SEC后串联-IgG产物(sLC-PCL1-串联-IgG、sLCΔPCL串联-IgG)的纯度。图(B)示出了使用分析型疏水性相互作用色谱法的抗体产物的同质性。图(C)示出了使用4％-12％Bis/Tris MOPS十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)，抗体的还原形式(分别为一条重链和两条轻链/一条单LC)和氧化形式(完整的串联-IgG)。图(D)示出了在与弗林蛋白酶和弗林蛋白酶-KDEL共表达后sLC串联-IgG-PCL1的在去糖基化后以及在还原条件下的LC-MS分析。
图6：在稳定的CHO 9E4细胞池中共表达和不共表达蛋白酶的情况下sLC串联-IgG的分析。使用4％-12％Bis/Tris MOPS十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)示出了在具有(左图)和没有(右图)蛋白酶共表达的稳定细胞池中表达的纯化抗体的氧化形式(完整的串联-IgG)和还原形式(分别为一条重链和两条轻链/一条单LC)。
图7：用SDS-PAGE分析在HEK293-FS和ExpiCHO细胞中表达的sC-IgG和串联样IgG图(A)示出了在HEK293-FS细胞中表达的两种抗体形式的还原形式(一条重链和一条轻链/一条sC(左)、一条重链和未加工/加工的sC(右))和氧化形式(完整的串联-IgG)，并且图(B)示出了在蛋白质A和SEC纯化后使用4％-12％Bis/Tris MOPS十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)的在ExpiCHO细胞中表达的对应构建体。
图8：在HEK293-FS细胞中与蛋白酶共表达的sC-IgG和串联样IgG的分析图(A)示出了在蛋白质A和SEC纯化后使用4％-12％Bis/Tris MOPS十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)，在HEK293-FS细胞中与PCSK家族成员共表达后sC-IgG的还原形式(一条重链和一条轻链/一条sC)和氧化形式，并且图(B)示出了串联样IgG的还原形式(一条重链和未加工/加工的sLC)和氧化形式(完整的串联样IgG)。
图9：在HEK293FS细胞中表达的三特异性CODV-sCFab-IgG的分析。图(A)示出了制备型SEC色谱图，并且图(B)示出了使用LabCHip电泳装置，在制备型SEC后的氧化形式(完整的三特异性CODV-Fab-IgG和还原形式(CODV重链和轻链以及单/加工的Fab重链和轻链)。
具体实施方式
在下文详细描述本发明之前，应理解，本发明不限于本文所述的特定方法、方案和试剂，因为这些可以变化。还应理解，本文所用的术语仅用于描述特定实施方案的目的，并且不旨在限制本发明的范围，本发明的范围将仅由随附权利要求限定。除非另外定义，否则本文所用的所有技术和科学术语都具有与本领域普通技术人员通常所理解的相同的含义。优选地，本文所述用的术语是如在“A multilingual glossary of biotechnologicalterms:(IUPAC Recommendations)”,Leuenberger,H.G.W,Nagel,B.和
Figure BDA0003827956940000111
H.编辑(1995),Helvetica Chimica Acta,CH-4010巴塞尔,瑞士)中所述定义的。
在本说明书的整个正文中引用了若干文献。将本文在上文或下文中引用的每个文献(包括所有专利、专利申请、科学出版物、制造商说明书、指导等)均通过引用以其整体特此并入。本文的任何内容都不应解释为承认本发明因在先发明而无权早于此类披露内容。
在下文中，将描述本发明的要素。这些要素是用具体实施方案来列出，然而，应理解，这些具体实施方案可以按任何方式和以任何数量组合以产生另外的实施方案。差异性描述的实施例和优选/特定实施方案不应解释为将本发明限制于仅明确描述的实施方案。本说明书应理解为支持并且涵盖组合明确描述的实施方案与任何数量的所公开和/或优选要素的实施方案。此外，除非上下文另外指示，否则应当考虑本申请的说明书公开本申请中所有所述要素的任何排列和组合。
贯穿本说明书以及其后的权利要求书，除非上下文另有要求，否则词语“包括(comprise)”以及变型诸如“包括(comprises)”或“包括(comprising)”应当被理解为暗指包含一个提及的整体或步骤或者整体或步骤的组，但不排除任何其他整体或步骤或者整体或步骤的组。除非上下文另有明确说明，否则如在本说明书及所附权利要求中所使用的，单数形式“一种/一个(a)”、“一种/一个(an)”和“所述”包括复数指示物。
在一方面，本发明涉及一种蛋白质，其包含至少一个具有下式的多肽链
PP1-PCL-PP2，
其中
PP1是第一多肽，
PP2是第二多肽，并且
PCL是蛋白酶可切割接头，所述蛋白酶可切割接头包含至少一个蛋白酶切割位点，所述至少一个蛋白酶切割位点包含氨基酸序列HRRX1X2RSVDE(SEQ ID NO:42)，其中X1和X2独立地选自氨基酸，优选天然存在的氨基酸。
如果所述蛋白质包含多于一条如上所定义的多肽链，则这些多肽链可以相同或不同。
根据本发明的术语“肽”是指包含两个或更多个、优选3或更多个、优选4或更多个、优选6或更多个、优选8或更多个、优选9或更多个、优选10或更多个、优选13或更多个、优选16更多个、优选21或更多个并且最多优选8、10、20、30、40或50个、特别是100个通过肽键共价连接的氨基酸的物质。术语“蛋白质”或“多肽”是指大的肽，优选是指具有多于100个氨基酸残基的肽，但是通常，术语“肽”、“多肽”和“蛋白质”是同义词并且在本文可互换使用。
在一个实施方案中，所述蛋白质是重组蛋白。在一个实施方案中，所述蛋白质是融合蛋白。
在本发明的上下文中，术语“重组”意指“通过基因工程制备的”。优选地，“重组对象”不是天然存在的。
如本文所用的应用于对象的术语“天然存在的”是指对象可以在自然界中发现的事实。例如，存在于生物体(包括病毒)中的可以从天然来源分离并且尚未经实验室的人员有意修饰的多肽或多核苷酸序列是天然存在的。
术语“融合蛋白”(或“嵌合蛋白”)通常是指通过以下方式产生的蛋白质：连接(特别是共价连接)两个或更多个不同的蛋白质和/或肽，从而产生具有源自每个原始蛋白质和/或肽的功能特性的单一蛋白质。
如本文所用的术语“接头”优选是指肽接头，即由经由肽键连接的氨基酸构成的接头。然而，根据本发明的接头还可以包括非肽组分，诸如非肽聚合物(例如PEG)。
根据本发明的肽接头可以具有任何长度，即包含任何数量的氨基酸残基。然而，它优选足够长以提供足够程度的柔韧性，使得连接(connected)/连接(linked)的部分可以例如彼此或与其他部分相互作用/配对并且允许适当的蛋白质折叠；而它优选足够短以在细胞中提供稳定性(例如蛋白水解稳定性)。合适的肽接头描述在例如Chen等人,Adv DrugDeliv Rev.2013,65(10):1357-69，将其以其整体并入本文。优选地，根据本发明的肽接头具有1至100个氨基酸的长度。
如果肽接头的氨基酸小并且没有阻碍氨基酸链旋转或弯曲的大体积侧链，则其柔韧性通常增加。因此，本发明的肽接头优选具有增加的小氨基酸含量，特别是甘氨酸、丙氨酸、丝氨酸、苏氨酸、亮氨酸和异亮氨酸的含量。优选地，肽接头的至少20％、30％、40％、50％、60％、70％、80％、90％或更多的氨基酸是这样的氨基酸。在特定的实施方案中，根据本发明的肽接头是富含甘氨酸-丝氨酸的接头，其中分别地，至少50％、优选至少60％、更优选至少70％、更优选至少80％、甚至更优选至少85％的氨基酸是甘氨酸残基或丝氨酸残基。根据本发明的肽接头也可以仅由甘氨酸残基和/或丝氨酸残基构成(分别称为甘氨酸接头、丝氨酸接头或甘氨酸-丝氨酸接头)。示例性肽接头包含氨基酸式(GlSm)n的序列，其中l是从1至4的整数，m是1或2，并且n是从1至12、优选1至10，例如2至10或4至10的整数。根据本发明的肽接头也可以包含其他序列元件(例如His标签)或由其组成。
在一些实施方案中，根据本发明的肽接头也可以是刚性肽接头。这样的刚性肽接头是本领域技术人员已知的，并且包括例如具有通式(XP)n的富含脯氨酸的肽接头，其中X表示任何氨基酸，优选丙氨酸、赖氨酸或谷氨酸，并且n是整数，优选从1至12的整数。该基序的具体例子是(AP)nA(SEQ ID NO:53)。刚性肽接头序列基序的另一个例子是(EAAAK)n(SEQID NO:52)，其中n是整数，优选从1至12的整数。
如本文所用的术语“蛋白酶可切割接头”(“PCL”)是指包含至少一个蛋白酶切割位点的接头。
根据本发明的“蛋白酶切割位点”(“PCS”；在本文中也称为蛋白酶识别位点)是作为酶(蛋白酶)的靶标的在蛋白质中的一种类型的酶促切割位点，其在蛋白质的翻译后起作用。在一个实施方案中，此类酶在从高尔基体腔转运至高尔基体反面房室期间起作用。在从细胞中分泌蛋白质之前细胞内加工酶(蛋白酶)切割多肽。
在一个实施方案中，所述蛋白酶是弗林蛋白酶和/或弗林蛋白酶样蛋白酶。术语“弗林蛋白酶”和“弗林蛋白酶样蛋白酶”是指对应于EC号3.4.21.75的酶。弗林蛋白酶是枯草杆菌蛋白酶样的前蛋白转化酶，其也称为PACE(配对的(P)碱性氨基酸(A)切割(C)酶(E))。弗林蛋白酶缺失无活性前体蛋白的部分，以将其转化为生物活性蛋白质。弗林蛋白酶和/或弗林蛋白酶样蛋白酶(其也可以称为弗林蛋白酶家族的成员)的例子包括但不限于PCSK1(也称为PC1/Pc3)、PCSK2(也称为PC2)、PCSK3(也称为弗林蛋白酶或PACE)、PCSK4(也称为PC4)、PCSK5(也称为PCS或PC6)、PCSK6(也称为PACE4)和PCSK7(也称为PC7/LPC、PC8、或SPC7)。
如本文所用的术语“氨基酸”或“氨基酸残基”是指天然存在的氨基酸、以与天然存在的氨基酸类似的方式起作用的非天然氨基酸、氨基酸类似物和氨基酸模拟物，全部呈其D和L立体异构体形式，如果其结构允许此类立体异构形式的话。氨基酸在本文中以其名称、其通常已知的三字母符号或由IUPAC-IUB生物化学命名委员会(IUPAC-IUB BiochemicalNomenclature Commission)推荐的单字母符号提及。
在与氨基酸结合使用时，术语“天然存在的”是指20种常规氨基酸(即，丙氨酸(A)、半胱氨酸(C)、天冬氨酸(D)、谷氨酸(E)、苯丙氨酸(F)、甘氨酸(G)、组氨酸(H)、异亮氨酸(I)、赖氨酸(K)、亮氨酸(L)、甲硫氨酸(M)、天冬酰胺(N)、脯氨酸(P)、谷氨酰胺(Q)、精氨酸(R)、丝氨酸(S)、苏氨酸(T)、缬氨酸(V)、色氨酸(W)和酪氨酸(Y))以及硒代半胱氨酸、吡咯赖氨酸和吡咯啉-羧基赖氨酸。
如本文所用的术语“非天然氨基酸”意在指代不是天然编码的或在任何生物体的遗传密码中发现的氨基酸。例如，它们可以是纯合成化合物。非天然氨基酸的例子包括但不限于羟脯氨酸、γ-羧基谷氨酸、O-磷酸丝氨酸、铃兰氨酸(azetidinecarboxylic acid)、2-氨基己二酸、3-氨基己二酸、β-丙氨酸、氨基丙酸、2-氨基丁酸、4-氨基丁酸、6-氨基己酸、2-氨基庚酸、2-氨基异丁酸、3-氨基异丁酸、2-氨基庚二酸、叔丁基甘氨酸、2,4-二氨基异丁酸、锁链素、2,2’-二氨基庚二酸、2,3-二氨基丙酸、N-乙基甘氨酸、N-甲基甘氨酸、N-乙基天冬酰胺、高脯氨酸、羟赖氨酸、别-羟赖氨酸、3-羟脯氨酸、4-羟脯氨酸、异锁链素、别-异亮氨酸、N-甲基丙氨酸、N-甲基甘氨酸、N-甲基异亮氨酸、N-甲基戊基甘氨酸、N-甲基缬氨酸、萘丙氨酸、正缬氨酸、正亮氨酸、鸟氨酸、D-鸟氨酸、D-精氨酸、对氨基苯丙氨酸、戊基甘氨酸、哌啶酸和硫代脯氨酸。
如本文所用的术语“氨基酸类似物”是指具有与天然存在的氨基酸相同的基础化学结构的化合物。氨基酸类似物包括天然氨基酸和非天然氨基酸，其经可逆或不可逆地化学封阻，或其C末端羧基、其N末端氨基和/或其侧链官能团经化学修饰。这样的类似物包括但不限于甲硫氨酸亚砜、甲硫氨酸砜、S-(羧甲基)-半胱氨酸、S-(羧甲基)-半胱氨酸亚砜、S-(羧甲基)-半胱氨酸砜、天冬氨酸-(β甲基酯)、N-乙基甘氨酸、丙氨酸甲酰胺、高丝氨酸、正亮氨酸和甲硫氨酸甲基锍。
如本文所用的术语“氨基酸模拟物”是指具有与氨基酸的通用化学结构不同的结构但以与天然存在的氨基酸类似的方式起作用的化学化合物。
在一个实施方案中，X1和X2独立地选自赖氨酸(K)、谷氨酰胺(Q)和精氨酸(R)。在一个实施方案中，X1X2选自KK、KQ、KR、QK、QQ、QR、RK、RQ和RR。
在一个实施方案中，所述至少一个蛋白酶切割位点包含氨基酸序列HRRRKRSVDE(SEQ ID NO:43)或氨基酸序列HRRQQRSVDE(SEQ ID NO:44)。
在一个实施方案中，所述接头的蛋白酶切割导致所述蛋白质的活性改变。
在一个实施方案中，所述活性改变是与至少一种抗原的结合或增加的结合。
根据本发明的术语“结合”优选涉及特异性结合。如果结合剂(诸如抗体或抗体衍生物)能够与所述预定靶标结合而其不能(基本上不能)与其他靶标结合，则它对预定靶标具有特异性。
根据本发明，如果结合剂(诸如抗体或抗体衍生物)对所述预定靶标具有显著的亲和力并且在标准测定中与所述预定靶标结合，则它能够与预定靶标结合。“亲和力”或“结合亲和力”通常通过平衡解离常数(KD)来测量。优选地，术语“显著的亲和力”是指以10-5M或更低、10-6M或更低、10-7M或更低、10-8M或更低、10-9M或更低、10-10M或更低、10-11M或更低、或10-12M或更低的解离常数(KD)与预定靶标结合。
如果药剂对靶标没有显著的亲和力并且在标准测定中不与靶标显著结合、特别是可检测地不结合，则它不能(基本上不能)与所述靶标结合。优选地，如果以最高2、优选10、更优选20、特别是50或100μg/ml或更高的浓度存在，则所述药剂可检测地不结合至所述靶标。优选地，如果药剂以比与所述药剂能够结合的预定靶标的结合的KD高至少10倍、100倍、103倍、104倍、105倍、或106倍的KD与所述靶标结合，则它对所述靶标没有显著的亲和力。例如，如果药剂与所述药剂能够结合的靶标的结合的KD是10-7M，则与药剂对其无显著亲和力的靶标的结合的KD将是至少10-6M、10-5M、10-4M、10-3M、10-2M、或10-1M。
药剂与靶标的结合可以使用任何合适的方法通过实验确定；参见例如，Berzofsky等人,“Antibody-Antigen Interactions”In Fundamental Immunology,Paul,W.E.编辑,Raven Press纽约,纽约州(1984),Kuby,Janis Immunology,W.H.Freeman and Company纽约,纽约州(1992)以及本文所述的方法。亲和力可以容易地使用常规技术来确定，诸如通过平衡透析；通过使用BIAcore2000仪器使用由制造商概述的通用程序；通过使用放射标记的靶抗原的放射免疫测定；或通过技术人员已知的另一种方法。亲和力数据可以例如通过Scatchard等人,Ann N.Y.Acad.ScL,51:660(1949)的方法进行分析。如果在不同条件(例如，盐浓度、pH)下测量，则测量的特定抗体-抗原相互作用的亲和力可能变化。因此，优选用抗体和抗原的标准化溶液以及标准化缓冲液进行亲和力和其他抗原结合参数(例如，KD、IC50)的测量。
术语“增加的结合”可以指与蛋白酶切割之前的结合相比增加至少50％、至少100％、至少200％、至少300％、至少400％或至少500％的结合。
在一个实施方案中，蛋白酶切割发生在细胞内，例如在如本文所定义的宿主细胞内。因此，蛋白酶可切割接头中所含的蛋白酶切割位点也可以称为细胞内加工位点。根据本发明的蛋白酶可切割接头也可以称为体内可切割接头。
优选地，包含根据本发明的至少一个蛋白酶切割位点的根据本发明的蛋白酶可切割接头在不共表达蛋白酶(例如，弗林蛋白酶和/或弗林蛋白酶样蛋白酶)、特别是外源蛋白酶的情况下在细胞内/体内被切割/加工。如本文所用的术语“共表达”意在指代人工共表达，例如通过遗传操纵宿主细胞以表达蛋白酶，例如经由用编码所述蛋白酶的核酸转染宿主细胞。
在一个实施方案中，所述蛋白酶可切割接头包含两个蛋白酶切割位点，其中这两个蛋白酶切割位点分别位于蛋白酶可切割接头的N末端和C末端，其中这两个蛋白酶切割位点可以相同或不同。
在一个实施方案中，所述蛋白酶可切割接头具有10至80个氨基酸残基、优选20至80个氨基酸、更优选20至70个氨基酸、更优选30至70个氨基酸、甚至更优选30至60个氨基酸的长度。
在一个实施方案中，所述至少一个蛋白酶切割位点在至少2、至少3、至少4、至少5或至少10个连续的独立地选自甘氨酸和丝氨酸的氨基酸残基之前和/或之后。
在一个实施方案中，所述蛋白酶可切割接头进一步包含选自(GS)n、(G4S)n(SEQ IDNO:51)、(HH)n、(EAAAK)n(SEQ ID NO:52)、(AP)nA(SEQ ID NO:53)、(KQGKQ)n(SEQ ID NO:54)及其组合的氨基酸序列，其中n是选自1至12、优选1至10的整数。在一个实施方案中，所述蛋白酶可切割接头包含氨基酸序列(G4S)n(SEQ ID NO:51)，其中n是选自2至10、优选4至10的整数。
在一个实施方案中，所述蛋白酶可切割的另外接头包含选自GSHHHHHHHHGGGGS(SEQ ID NO:45)、GGGGSEAAAKEAAAKGGGGS(SEQ ID NO:46)、APAPAPAPAPAPAPA(SEQ ID NO:47)、GSKQGKQKQGKQGS(SEQ ID NO:48)及其组合的氨基酸序列。
在一个实施方案中，所述蛋白酶可切割接头(PCL)具有结构PCSA-L-PCSB，其中PCSA和PCSB是如本文所定义的蛋白酶切割位点，可以是相同或不同，并且其中L是包含选自如上所定义的SEQ ID NO:45至48和51至54、(GS)n、(HH)n及其组合的氨基酸序列或由其组成的接头。
在一个实施方案中，PP1包含至少一个免疫球蛋白恒定区和/或至少一个免疫球蛋白可变区，和/或PP2包含至少一个免疫球蛋白恒定区和/或至少一个免疫球蛋白可变区。
在一个实施方案中，所述蛋白质是抗体或抗体衍生物。
在一个实施方案中，所述蛋白质是单链抗体、优选多特异性(例如，双特异性、三特异性、四特异性、五特异性、六特异性等)单链抗体。如本文所用的术语“单链抗体”还指包含多于一条(例如，两条)单链的抗体或抗体衍生物，所述多于一条单链优选经由至少一个共价键或非共价键(例如，两个二硫键)彼此缔合。
术语“抗体”(或“免疫球蛋白”)是指糖蛋白，所述糖蛋白通过二硫键互相连接的至少两条重(H)链和两条轻(L)链。术语“抗体”包括单克隆抗体、重组抗体、人抗体、人源化抗体、嵌合抗体以及任何前述项的组合。每条重链由重链可变区(VH)和重链恒定区(CH)构成。每条轻链由轻链可变区(VL)和轻链恒定区(CL)构成。可变区和恒定区在本文中也分别称为可变结构域和恒定结构域。VH区和VL区可以进一步细分为具有高变性的区域，称为互补决定区(CDR)，散布有更保守的区域，称为框架区(FR)。每个VH和VL由三个CDR和四个FR组成，按照以下顺序从氨基末端到羧基末端排列：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。VH的CDR称为HCDR1、HCDR2和HCDR3，VL的CDR称为LCDR1、LCDR2和LCDR3。重链和轻链的可变区含有与抗原相互作用的结合结构域。抗体的恒定区包含重链恒定区(CH)和轻链恒定区(CL)，其中CH可以进一步细分为恒定结构域CH1、铰链区以及恒定结构域CH2和CH3(按照以下顺序从氨基末端到羧基末端排列：CH1、CH2、CH3)。抗体的恒定区可以介导免疫球蛋白与宿主组织或因子的结合，所述宿主组织或因子包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(Clq)。在一个实施方案中，本文所提及的CL区/结构域是Cκ区/结构域。
抗体可以源自不同物种，包括但不限于小鼠、大鼠、兔、豚鼠和人。
本文所述的抗体包括IgA(诸如IgA1或IgA2)、IgG1、IgG2、IgG3、IgG4、IgE、IgM和IgD抗体。在各种实施方案中，抗体是IgG1抗体，更特别是IgG1κ或IgG1λ同种型(即，IgG1，κ、λ)、IgG2a抗体(例如，IgG2a，κ、λ)、IgG2b抗体(例如，IgG2b，κ、λ)、IgG3抗体(例如，IgG3，κ、λ)或IgG4抗体(例如，IgG4，κ、λ)。在一个实施方案中，抗体或抗体衍生物分别是IgG1抗体或IgG1抗体衍生物。
如本文所用，“同种型”是指由重链恒定区基因编码的抗体类别(例如，IgM和IgG1)。“同种型转换”是指抗体的类别或同种型从一种Ig类别变为另一种Ig类别的现象。
如本文所用，术语“单克隆抗体”是指具有单一分子组成的抗体分子制剂。单克隆抗体显示出单一的结合特异性和亲和力。在一个实施方案中，单克隆抗体是由杂交瘤产生的，所述杂交瘤包括与永生细胞融合的获自非人动物(例如，小鼠)的B细胞。
如本文所用的术语“铰链”或“铰链区”是指IgG和IgA、特别是IgG(即，IgG1、IgG2、IgG3或IgG4，尤其是IgG1)免疫球蛋白类别的重链的中心部分中的柔性氨基酸拉伸物，所述柔性氨基酸拉伸物通过二硫键连接这两条链。
如本文所用的术语“抗体衍生物”是指至少包含结构域的分子，所述结构域是指定包含的，但不具有抗体(诸如IgA、IgD、IgE、IgG、IgM、IgY或IgW)的整体结构，尽管仍然能够结合靶分子。所述衍生物可以是但不限于其功能(即靶标结合，特别是特异性靶标结合)抗体片段(诸如Fab2)或此类衍生物的组合(例如，二价Fab)。它还涉及其中已经添加其他抗体结构域(诸如其他可变结构域)的抗体。在一个实施方案中，术语“抗体衍生物”是指如本文所述的单链抗体。因此，术语“抗体衍生物”还包括多特异性(例如，双特异性、三特异性、四特异性、五特异性、六特异性等)和多价(例如，二价、三价、四价等)抗体。
在一个实施方案中，
PP1包含免疫球蛋白恒定区并且PP2包含免疫球蛋白可变区，
PP1包含免疫球蛋白可变区并且PP2包含免疫球蛋白恒定区，
PP1包含免疫球蛋白恒定区并且PP2包含免疫球蛋白恒定区，或
PP1包含免疫球蛋白可变区并且PP2包含免疫球蛋白可变区。
在一个实施方案中，所述蛋白质选自单链(sC)单克隆IgG(例如，如Spies等人,JMol Imm.2015,67:95-106中所述)、单轻链(sLC)二价双特异性串联-IgG(例如，如WO 2009/052081A2中所述)、单轻链(sLC)多特异性串联-IgG(例如，如Wu等人,Nat.Biotechnol.2007,25:1290-1297中所述)、单链(sC)单价双特异性串联样-IgG(例如，如Brinkmann&Kontermann,MAbs2017,9:182-212中所述)、三特异性CODV-sC-Fab-IgG(例如，如WO2017/180913A2和/或Xu等人,Science 2017,358(6359):85-90中所述)和单链(sC)双特异性IgG(例如，如Fitzgerald等人,Mol Cancer Ther.2013,13:410-25中所述)。
在一个实施方案中，所述蛋白质包含具有选自以下的式的多肽链：
(a)VL-CL-PCL-VH-CH1-铰链-CH2-CH3，
其中
VL是免疫球蛋白轻链可变区；
CL是免疫球蛋白轻链恒定区；
VH是免疫球蛋白重链可变区；
CH1是免疫球蛋白CH1重链恒定区；
铰链是免疫球蛋白铰链区；
CH2是免疫球蛋白CH2重链恒定区；
CH3是免疫球蛋白CH3重链恒定区；并且
PCL是所述蛋白酶可切割接头；
(b)VLa-CLa-PCL-VLb-CLb，
其中
VLa是第一免疫球蛋白轻链可变区；
VLb是第二免疫球蛋白轻链可变区；
CLa是第一免疫球蛋白轻链恒定区；
CLb是第二免疫球蛋白轻链恒定区；并且
PCL是所述蛋白酶可切割接头；以及
(c)VLa-CLa-PCL-VLb-CLb-L-铰链-CH2-CH3
其中
VLa是第一免疫球蛋白轻链可变区；
VLb是第二免疫球蛋白轻链可变区；
CLa是第一免疫球蛋白轻链恒定区；
CLb是第二免疫球蛋白轻链恒定区；
L是任选的接头；
铰链是免疫球蛋白铰链区；
CH2是免疫球蛋白CH2重链恒定区；
CH3是免疫球蛋白CH3重链恒定区；并且
PCL是所述蛋白酶可切割接头。
当与本文公开的式结合使用时，符号(-)意在指代共价键，特别是肽键，或一个或多个氨基酸残基，例如，如本文所述的肽接头。
在一个实施方案中，L是如本文所定义的肽接头，优选具有5至25个氨基酸残基、优选10至20个氨基酸残基、优选12至18个氨基酸残基、更优选13至17个氨基酸残基、甚至更优选14至16个氨基酸残基，例如15个氨基酸残基的长度。在一个实施方案中，L是富含甘氨酸-丝氨酸的接头，其中至少50％、优选至少60％、更优选至少70％、更优选至少80％、甚至更优选至少85％的氨基酸分别是甘氨酸残基或丝氨酸残基。在一个实施方案中，L是甘氨酸接头、丝氨酸接头或甘氨酸-丝氨酸接头。在一个实施方案中，L具有序列(G4S)n(SEQ ID NO:51)，其中n是选自1至5，例如1至3或2至4的整数。在一个实施方案中，L具有序列(G4S)3(SEQID NO:55)。
在一个实施方案中，所述蛋白质包含具有式VL-CL-PCL-VH-CH1-铰链-CH2-CH3的多肽链，其中VL与VH配对以形成抗原结合位点。
在一个实施方案中，在蛋白酶可切割接头的蛋白酶切割后，所述抗原结合位点变得有活性或展现出增加的活性。
优选地，如本文所用的术语“与......配对”是指免疫球蛋白恒定区或免疫球蛋白可变区的二聚化，如其在正常(例如天然存在的)免疫球蛋白、特别是IgG中发生的那样。
如本文所用的术语“抗原结合位点”(也称为“互补位”)是指抗体的识别并且结合抗原的部分。
在一个实施方案中，所述蛋白质包含具有式VL-CL-PCL-VH-CH1-铰链-CH2-CH3的多肽链的两个拷贝，其中这两个拷贝经由至少两个二硫键彼此缔合。
在一个实施方案中，所述蛋白质包含具有式VLa-CLa-PCL-VHa-CH1a-铰链-CH2-CH3的第一多肽链和具有式VLb-CLb-PCL-VHb-CH1b-铰链-CH2-CH3的第二多肽链，
其中
VLa是第一免疫球蛋白轻链可变区；
VLb是第二免疫球蛋白轻链可变区；
CLa是第一免疫球蛋白轻链恒定区；
CLb是第二免疫球蛋白轻链恒定区；
VHa是第一免疫球蛋白重链可变区；
VHb是第二免疫球蛋白重链可变区；
CH1a是第一免疫球蛋白CH1重链恒定区；并且
CH1b是第二免疫球蛋白CH1重链恒定区；
其中VLa与VHa配对以形成与抗原A结合的抗原结合位点，并且其中VLb与VHb配对以形成与抗原B结合的抗原结合位点；并且
其中第一多肽链经由至少两个二硫键并且任选地经由至少一种另外的Fc相互作用与第二多肽链缔合。
在一个实施方案中，在蛋白酶可切割接头的蛋白酶切割后，抗原结合位点A和/或抗原结合位点B变得有活性或展现出增加的活性。
除了本文提及的至少两个二硫键之外，如本文所用的术语“另外的Fc相互作用”还指两个Fc分子(例如，两个异源Fc分子)之间，特别是两个Fc分子的CH3结构域之间的相互作用。另外的Fc相互作用的例子包括但不限于杵臼相互作用(对于细节，参见下文)、疏水性相互作用(例如，由于特定突变的引入而引起的；参见例如，Von Kreudenstein等人,MAbs2013,5(5):646-654)、静电相互作用(例如，由于静电转向而引起的；参见例如，Gunasekaran等人,J Biol Chem 2010,285(25):19637-19646)、由于IgG和IgA的交替CH3区段而引起的相互作用(SEED技术；参见例如，Davis等人,Protein Eng Des Sel 2010,23(4):195-202)、由于异二聚体分子的融合而引起的相互作用，诸如在CH3结构域的C末端的可切割亮氨酸拉链(LUZ-Y技术；参见例如，Wranik等人,J Biol Chem 2012,287(52):43331-43339)。
在一个实施方案中，所述至少一种另外的Fc相互作用是至少一种杵臼相互作用(例如，两种杵臼相互作用)。
如本文所用的术语“杵臼相互作用”是指基于杵臼氨基酸改变的相互作用。这样的改变表示在用于重(H)免疫球蛋白链的异二聚化(例如，在双特异性抗体的产生中)的抗体工程中众所周知的合理设计策略，如例如Merchant等人,Nat.Biotechnol.1998,16:677-681中所述。工程改造氨基酸改变以便在第一免疫球蛋白链或第一抗体的恒定区(例如，在CH3上)上形成杵以及在第二免疫球蛋白链或第二抗体的恒定区(例如，在CH3上)上形成臼。杵由属于“非常大”IMGT体积的氨基酸类别的氨基酸(例如，酪氨酸，Y)表示，而臼由属于“小”IMGT体积类别的氨基酸(例如，苏氨酸；T)表示-对于20种常规氨基酸的IMGT类别，参见Pommié等人,J.Mol.Recognit.2004,17:17-32。根据本发明的特别优选的杵臼氨基酸改变包括但不限于在Spies等人,J Mol Imm.2015,67:95-106中所述的那些。
在一个实施方案中，所述蛋白质包含具有式VLb-L1-VLa-L2-CL的第一多肽链、具有式VHa-L3-VHb-L4-CH1-铰链-CH2-CH3的第二多肽链和具有式VLc-CLc-PCL-VHc-CH1c-铰链-CH2-CH3的第三多肽链，
其中
VLa是第一免疫球蛋白轻链可变区；
VLb是第二免疫球蛋白轻链可变区；
VLc是第三免疫球蛋白轻链可变区；
CL是免疫球蛋白轻链恒定区；
CLc是免疫球蛋白轻链恒定区；
VHa是第一免疫球蛋白重链可变区；
VHb是第二免疫球蛋白重链可变区；
VHc是第三免疫球蛋白重链可变区；
CH1是免疫球蛋白CH1重链恒定区；
CH1c是免疫球蛋白CH1重链恒定区；
铰链是免疫球蛋白铰链区；
CH2是免疫球蛋白CH2重链恒定区；并且
CH3是免疫球蛋白CH3重链恒定区；
L1、L2、L3和L4是接头；
其中VLa与VHa配对以形成与抗原A结合的抗原结合位点，其中VLb与VHb配对以形成与抗原B结合的抗原结合位点，并且其中VLc与VHc配对以形成与抗原C结合的抗原结合位点；并且
其中第二多肽链经由至少两个二硫键并且任选地经由至少一种另外的Fc相互作用与第三多肽链缔合。
在一个实施方案中，在蛋白酶可切割接头的蛋白酶切割后，抗原结合位点C变得有活性或展现出增加的活性。
在一个实施方案中，所述至少一种另外的Fc相互作用是至少一种杵臼相互作用(例如，两种杵臼相互作用)。
在一个实施方案中，L1、L2、L3和L4独立地选自如本文所定义的肽接头，优选具有在1至25之间，优选2至25、更优选5至20、更优选10至20个氨基酸残基的长度。在一个实施方案中，L1、L2、L3和L4是富含甘氨酸-丝氨酸的接头，其中至少50％、优选至少60％、更优选至少70％、更优选至少80％、甚至更优选至少85％的氨基酸分别是甘氨酸残基或丝氨酸残基。在一个实施方案中，L1、L2、L3和L4分别是甘氨酸接头、丝氨酸接头或甘氨酸-丝氨酸接头。在一个实施方案中，L1、L2、L3和L4具有序列(G4S)n(SEQ ID NO:51)，其中n是独立地选自1至5，例如1至3或2至4的整数。
在一个实施方案中，所述蛋白质包含具有式VLa-CLa-PCL-VLb-CLb的第一多肽链和具有式VHa-CH1a-L-VHb-CH1b-铰链-CH2-CH3的第二多肽链，
其中
VHa是第一免疫球蛋白重链可变区；
VHb是第二免疫球蛋白重链可变区；
CH1a是第一免疫球蛋白CH1重链恒定区；
CH1b是第二免疫球蛋白CH1重链恒定区；
L是任选的接头；
铰链是免疫球蛋白铰链区；
CH2是免疫球蛋白CH2重链恒定区；并且
CH3是免疫球蛋白CH3重链恒定区；
其中VLa与VHa配对以形成与抗原A结合的抗原结合位点，并且其中VLb与VHb配对以形成与抗原B结合的抗原结合位点。
在一个实施方案中，在蛋白酶可切割接头的蛋白酶切割后，抗原结合位点B变得有活性或展现出增加的活性。
在一个实施方案中，L是如本文所定义的肽接头。在一个实施方案中，L具有5至25个氨基酸残基、优选10至20个氨基酸残基的长度。
在一个实施方案中，所述蛋白质包含第一多肽链的两个拷贝和第二多肽链的两个拷贝，其中第二多肽链的两个拷贝经由至少两个二硫键彼此缔合。
在一个实施方案中，所述蛋白质包含具有式VLa-CLa-PCL-VLb-CLb的第一多肽链、具有式VLc-CLc-PCL-VLd-CLd的第二多肽链、具有式VHa-CH1a-L-VHb-CH1b-铰链-CH2-CH3的第三多肽链、和具有式VHc-CH1c-L-VHd-CH1d-铰链-CH2-CH3的第四多肽链，
其中
VLa是第一免疫球蛋白轻链可变区；
VLb是第二免疫球蛋白轻链可变区；
VLc是第三免疫球蛋白轻链可变区；
VLd是第四免疫球蛋白轻链可变区；
CLa是第一免疫球蛋白轻链恒定区；
CLb是第二免疫球蛋白轻链恒定区；
CLc是第三免疫球蛋白轻链恒定区；
CLd是第四免疫球蛋白轻链恒定区；
VHa是第一免疫球蛋白重链可变区；
VHb是第二免疫球蛋白重链可变区；
VHc是第三免疫球蛋白重链可变区；
VHd是第四免疫球蛋白重链可变区；
CH1a是第一免疫球蛋白CH1重链恒定区；
CH1b是第二免疫球蛋白CH1重链恒定区；
CH1c是第三免疫球蛋白CH1重链恒定区；
CH1d是第四免疫球蛋白CH1重链恒定区；
L是任选的接头；
铰链是免疫球蛋白铰链区；
CH2是免疫球蛋白CH2重链恒定区；并且
CH3是免疫球蛋白CH3重链恒定区；
其中VLa与VHa配对以形成与抗原A结合的抗原结合位点，其中VLb与VHb配对以形成与抗原B结合的抗原结合位点，其中VLc与VHc配对以形成与抗原C结合的抗原结合位点，并且其中VLd与VHd配对以形成与抗原D结合的抗原结合位点；并且
其中第三多肽链经由至少两个二硫键并且任选地经由至少一种另外的Fc相互作用与第四多肽链缔合。
在一个实施方案中，在蛋白酶可切割接头的蛋白酶切割后，抗原结合位点B和/或抗原结合位点D变得有活性或展现出增加的活性。
在一个实施方案中，所述至少一种另外的Fc相互作用是至少一种杵臼相互作用(例如，两种杵臼相互作用)。
在一个实施方案中，L是如本文所定义的肽接头。在一个实施方案中，L具有5至25个氨基酸残基、优选10至20个氨基酸残基的长度。
在一个实施方案中，所述蛋白质包含具有式VLa-CLa-PCL-VLb-CLb-L-铰链-CH2-CH3的第一多肽链和具有式VHa-CH1a-L-VHb-CH1b-铰链-CH2-CH3的第二多肽链，
其中
VHa是第一免疫球蛋白重链可变区；
VHb是第二免疫球蛋白重链可变区；
CH1a是第一免疫球蛋白CH1重链恒定区；
CH1b是第二免疫球蛋白CH1重链恒定区；
L是任选的接头；
铰链是免疫球蛋白铰链区；
CH2是免疫球蛋白CH2重链恒定区；并且
CH3是免疫球蛋白CH3重链恒定区；
其中VLa与VHa配对以形成与抗原A结合的抗原结合位点，并且其中VLb与VHb配对以形成与抗原B结合的抗原结合位点；并且
其中第一多肽链经由至少两个二硫键并且任选地经由至少一种另外的Fc相互作用与第二多肽链缔合。
在一个实施方案中，在蛋白酶可切割接头的蛋白酶切割后，抗原结合位点B变得有活性或展现出增加的活性。
在一个实施方案中，所述至少一种另外的Fc相互作用是至少一种杵臼相互作用(例如，两种杵臼相互作用)。
在一个实施方案中，L是如本文所定义的肽接头。在一个实施方案中，L具有5至25个氨基酸残基、优选10至20个氨基酸残基的长度。
在另一方面，本发明涉及一种编码如本文所定义的蛋白质的核酸或核酸组。
根据本发明，“核酸”(或“核酸分子”)优选是脱氧核糖核酸(DNA)或核糖核酸(RNA)。根据本发明，核酸分子可以呈分子的形式，所述分子是单链或双链并且是线性或共价闭合的以形成环。
术语“DNA”涉及包含脱氧核糖核苷酸残基并且优选完全或基本上由脱氧核糖核苷酸残基构成的分子。“脱氧核糖核苷酸”是指在β-D-呋喃核糖基的2’-位置缺少羟基的核苷酸。术语“DNA”包括分离的DNA，诸如部分或完全纯化的DNA、基本上纯的DNA、合成DNA、和重组产生的DNA，并且包括经修饰的DNA，其与天然存在的DNA的不同之处在于一个或多个核苷酸的添加、缺失、取代和/或改变。此类改变可以包括添加非核苷酸材料，诸如添加到DNA的一个或多个末端或添加到内部，例如在DNA的一个或多个核苷酸处添加。DNA分子中的核苷酸还可以包括非标准核苷酸，诸如非天然存在的核苷酸或化学合成的核苷酸。这些经改变的DNA可以被称为类似物或天然存在的DNA的类似物。在与核苷酸结合使用时，术语“天然存在的”是指碱基腺嘌呤(A)、胞嘧啶(C)、鸟嘌呤(G)、胸腺嘧啶(T)和尿嘧啶(U)。
术语“RNA”涉及包含核糖核苷酸残基并且优选完全或基本上由核糖核苷酸残基构成的分子。“核糖核苷酸”是指在β-D-呋喃核糖基的2’-位置具有羟基的核苷酸。术语“RNA”包括分离的RNA，诸如部分或完全纯化的RNA、基本上纯的RNA、合成RNA、和重组产生的RNA，并且包括经修饰的RNA，其与天然存在的RNA的不同之处在于一个或多个核苷酸的添加、缺失、取代和/或改变。此类改变可以包括添加非核苷酸材料，诸如添加到RNA的一个或多个末端或添加到内部，例如在RNA的一个或多个核苷酸处添加。RNA分子中的核苷酸还可以包含非标准核苷酸，诸如非天然存在的核苷酸或化学合成的核苷酸或脱氧核苷酸。这些经改变的RNA可以被称为类似物或天然存在的RNA的类似物。根据本发明，“RNA”是指单链RNA或双链RNA。在一个实施方案中，RNA是mRNA，例如体外转录的RNA(IVT RNA)或合成RNA。RNA还可以被修饰，例如具有增加RNA稳定性(例如，半衰期)的一个或多个修饰。此类修饰是本领域技术人员已知的，并且包括例如5’-帽或5’帽类似物。
在另一方面，本发明涉及一种载体或载体组，其包含如本文所定义的核酸或核酸组。
如本文所用的术语“载体”包括技术人员已知的所有载体，包括质粒载体、粘粒载体、噬菌体载体(诸如λ噬菌体)、病毒载体(诸如腺病毒或杆状病毒载体)或人工染色体载体(诸如细菌人工染色体(BAC)、酵母人工染色体(YAC)或P1人工染色体(PAC))。所述载体包括表达以及克隆载体。表达载体包括质粒以及病毒载体，并且通常含有所希望的编码序列和在特定宿主生物体(例如，细菌、酵母、植物、昆虫或哺乳动物)中或在体外表达系统中表达可操作地连接的编码序列所需的适当DNA序列。克隆载体通常用于工程化和扩增某一所希望的DNA片段，并且可能缺少表达所希望的DNA片段所需的功能序列。
可替代地，可以将根据本发明的核酸分子整合到基因组(例如，宿主细胞的基因组)中。将特定核酸分子整合到基因组中的手段和方法是本领域技术人员已知的。
在另一方面，本发明涉及一种宿主细胞，其包含如本文所定义的蛋白质、如本文所定义的核酸或核酸组、或如本文所定义的载体或载体组。
术语“细胞”或“宿主细胞”优选涉及完整细胞，即具有完整膜并且尚未释放其正常细胞内组分(诸如酶、细胞器或遗传材料)的细胞。完整细胞优选是活细胞，即能够执行其正常代谢功能的活细胞。优选地，根据本发明，所述术语涉及可以被外源核酸转染的任何细胞。优选地，在被外源核酸转染并且转移至接受者时，细胞可以在接受者体内表达所述核酸。术语“细胞”包括细菌细胞；其他有用的细胞是酵母细胞、真菌细胞或哺乳动物细胞。合适的细菌细胞包括来自以下的细胞：革兰氏阴性细菌菌株，诸如大肠杆菌(Escherichiacoli)、变形杆菌属(Proteus)和假单胞菌属(Pseudomonas)的菌株；以及革兰氏阳性细菌菌株，诸如芽孢杆菌属(Bacillus)、链霉菌属(Streptomyces)、葡萄球菌属(Staphylococcus)和乳球菌属(Lactococcus)的菌株。合适的真菌细胞包括来自木霉属(Trichoderma)、链孢霉属(Neurospora)和曲霉属(Aspergillus)物种的细胞。合适的酵母细胞包括来自以下物种的细胞：酵母属(Saccharomyces)(例如，酿酒酵母(Saccharomyces cerevisiae))、裂殖酵母属(Schizosaccharomyces)(例如，粟酒裂殖酵母(Schizosaccharomyces pombe))、毕赤酵母属(Pichia)(例如，巴斯德毕赤酵母(Pichia pastoris)和甲醇毕赤酵母(Pichiamethanolica))、和汉逊酵母属(Hansenula)。合适的哺乳动物细胞包括例如CHO细胞、BHK细胞、HeLa细胞、COS细胞、HEK 293(例如，HEK 293-FS)等。然而，也可以使用两栖动物细胞、昆虫细胞、植物细胞和在本领域中用于表达异源蛋白质的任何其他细胞。“细胞”或“宿主细胞”可以是分离的或者组织或生物体(特别是“非人生物体”)的一部分。如本文所用的术语“非人生物体”意在包括非人灵长类动物或其他动物，特别是哺乳动物，诸如牛、马、猪、绵羊、山羊、狗、猫、兔或啮齿类动物(诸如小鼠、大鼠、豚鼠和仓鼠)。
在一个实施方案中，所述宿主细胞表达内源或外源弗林蛋白酶和/或弗林蛋白酶样蛋白酶。
在一个实施方案中，所述宿主细胞是哺乳动物细胞。在一个实施方案中，哺乳动物细胞是CHO细胞，优选选自CHO 9E4、ExpiCHO、CHO DG44和CHO K1。
在一方面，本发明涉及一种生产蛋白质(例如，如本文所定义的蛋白质)的方法，其包括以下步骤：
i)培养如本文所定义的宿主细胞；以及
ii)将所述蛋白质与所述宿主细胞分离。
在一个实施方案中，在允许表达所述蛋白质的条件下培养所述宿主细胞。
在另一方面，本发明涉及一种通过如本文所定义的方法可获得(或获得)的蛋白质。
在又另一方面，本发明涉及一种通过弗林蛋白酶和/或弗林蛋白酶样蛋白酶切割如本文所定义的蛋白质可获得(或获得)的蛋白质。
在一个实施方案中，如本文所定义的蛋白质是治疗活性蛋白质。
如本文所用的术语“治疗活性蛋白质”是指适用于疗法的蛋白质，即可以用于治疗疾病或障碍的蛋白质。在一个实施方案中，治疗活性蛋白质是与治疗相关抗原结合的抗体或抗体衍生物。
对于如本文所述的所有抗体和抗体衍生物，治疗相关抗原例如针对每种特异性独立地选自IL-4、IL-13、PD-1、4.1BB、OX40和GITR。对于如本文所述的所有双特异性抗体和抗体衍生物，抗原对例如选自以下抗原对：IL-4和IL-13、PD-1和OX40、PD-1和GITR、和PD-1和4.1BB。对于如本文所述的所有三特异性抗体或抗体衍生物，两种抗原例如选自以下抗原对：IL-4和IL-13、PD-1和OX40、PD-1和GITR、和PD-1和4.1BB。第三抗原和第四抗原可以是选自以上列表的另一种抗原。如本文所述，在三特异性抗体或抗体衍生物的一个实施方案中，抗原是PD-1、GITR和OX40。
在一方面，本发明涉及一种如本文所定义的蛋白质，其用于疗法。
在另一方面，本发明涉及如本文所定义的蛋白质在制造药剂中的用途。
在又另一方面，本发明涉及一种治疗疾病或障碍的方法，其包括向有需要的受试者施用有效量的如本文所定义的蛋白质。
如本文所用的术语“药剂”是指用于疗法(即，用于治疗疾病或障碍)的物质/组合物。
“治疗”意在向受试者施用化合物或组合物或者化合物或组合物的组合，以便预防或消除疾病或障碍；阻止或减慢受试者的疾病或障碍；抑制或减慢受试者的新生疾病或障碍的发展；降低目前患有或先前曾患有疾病或障碍的受试者的症状和/或复发的频率或严重程度；和/或延长(即，增加)受试者的寿命。
特别地，术语“治疗(treating/treatment)疾病或障碍”包括治愈疾病或障碍或其症状，缩短疾病或障碍或其症状的持续时间，改善、预防、减慢或抑制疾病或障碍或其症状的进展或恶化，或预防或延迟疾病或障碍或其症状的发作。
根据本发明，术语“疾病”是指任何病理状态，特别是癌症、传染性疾病、炎性疾病、自身免疫性障碍、和移植排斥。
如本文所用，术语“癌症”包括以异常调节的细胞生长、增殖、分化、粘附和/或迁移为特征的疾病。“癌细胞”意指这样的异常细胞，其通过快速不受控制的细胞增殖而生长并且在启动新生长的刺激停止后继续生长。根据本发明的术语“癌症”包括白血病、精原细胞瘤、黑素瘤、畸胎瘤、淋巴瘤、神经母细胞瘤、神经胶质瘤、直肠癌、子宫内膜癌、肾癌、肾上腺癌、甲状腺癌、血液癌、皮肤癌、脑癌、子宫颈癌、肠癌(intestinal cancer)、肝癌、结肠癌、胃癌、肠癌(intestine cancer)、头颈癌、胃肠道癌、淋巴结癌、食道癌、结直肠癌、胰腺癌、耳鼻喉(ENT)癌、乳腺癌、前列腺癌、子宫癌、卵巢癌和肺癌及其转移。其例子是肺癌、乳房癌、前列腺癌、结肠癌、肾细胞癌、子宫颈癌、或上述癌症类型或肿瘤的转移。
根据本发明的术语“癌症”还包括癌症转移。“转移”意指癌细胞从其原始部位向身体的另一部分的扩散。转移的形成是非常复杂的过程，并且取决于恶性细胞从原发肿瘤的分离、细胞外基质的侵袭、内皮基底膜渗透进入体腔和血管并且然后经血液运输后浸润靶器官。最后，在靶位点上新肿瘤(即，继发性肿瘤或转移性肿瘤)的生长取决于血管生成。即使在去除原发肿瘤之后，肿瘤转移也经常发生，因为肿瘤细胞或组分可能保留并且发展出转移潜力。在一个实施方案中，根据本发明的术语“转移”涉及“远处转移”，其涉及远离原发肿瘤和区域淋巴结系统的转移。
术语“传染性疾病”是指可以从个体到个体传播或从生物体到生物体传播并且是由微生物因子引起的任何疾病(例如，普通感冒)。传染性疾病的例子包括病毒性传染性疾病，诸如艾滋病(HIV)、甲型、乙型或丙型肝炎、疱疹、带状疱疹(水痘)、德国麻疹(风疹病毒)、黄热病、登革热等、黄热病毒、流感病毒、出血性传染性疾病(马尔堡病毒或埃博拉病毒)、和严重急性呼吸系统综合征(SARS)；细菌性传染性疾病，诸如军团菌病(军团菌属(Legionella))、性传播疾病(例如，衣原体或淋病)、胃溃疡(螺杆菌属(Helicobacter))、霍乱(弧菌属(Vibrio))、肺结核、白喉、通过大肠杆菌(E.coli)、葡萄球菌属(Staphylococci)、沙门氏菌属(Salmonella)或链球菌属(Streptococci)(破伤风(tetanus))的感染；通过原生动物病原体的感染，诸如疟疾、昏睡病、利什曼病；弓形虫病，即通过疟原虫属(Plasmodium)、锥虫属(Trypanosoma)、利什曼原虫属(Leishmania)和弓形体属(Toxoplasma)的感染；或真菌感染，其例如是由新隐球菌(Cryptococcusneoformans)、荚膜组织胞浆菌(Histoplasma capsulatum)、粗球类芽生菌(Coccidioidesimmitis)、皮炎芽生菌(Blastomyces dermatitidis)或白色念珠菌(Candida albicans)引起的。
术语“炎性疾病”是指以组织、特别是结缔组织中的高水平炎症或这些组织的变性为特征或与之相关的任何疾病。慢性炎性疾病是以持续性炎症为特征的医学病症。(慢性)炎性疾病的例子包括乳糜泻、血管炎、狼疮、慢性阻塞性肺疾病(COPD)、肠易激病、动脉粥样硬化、关节炎、强直性脊柱炎、克罗恩病、结肠炎、慢性活动性肝炎、皮炎和银屑病。
术语“自身免疫性障碍”是指身体对其自身组织的某些成分产生免疫原性(即，免疫系统)反应的任何疾病/障碍。换句话说，免疫系统失去了其将体内的某些组织或系统识别为自身的能力并且如同它是异物一样靶向和攻击它。自身免疫性疾病可以分为其中主要一个器官受影响的那些疾病(例如，溶血性贫血和抗免疫甲状腺炎)以及其中自身免疫性疾病过程扩散在许多组织中的那些疾病(例如，系统性红斑狼疮)。例如，多发性硬化症被认为是由T细胞攻击脑和脊髓的神经纤维周围的鞘引起的。这会导致协调丧失、虚弱和视力模糊。自身免疫性疾病是本领域已知的，并且包括例如桥本氏甲状腺炎、格雷夫氏病、狼疮、多发性硬化症、风湿性关节炎、溶血性贫血、抗免疫甲状腺炎、系统性红斑狼疮、乳糜泻、克罗恩病、结肠炎、糖尿病、硬皮病、银屑病等。
术语“移植排斥”是指接受者的免疫系统对移植的组织或器官的排斥，这最终可能破坏移植的组织或器官。
如本文所用的术语“有效量”特别是指“治疗有效量”，其量单独地或与其他剂量一起实现所希望的治疗反应或所希望的治疗效果，优选不引起不可接受的副作用。在治疗特定疾病或特定病症的情况下，所希望的反应优选涉及对病程的抑制。这包括减慢疾病的进展，并且特别是中断或逆转疾病的进展。在疾病或病症的治疗中所希望的反应也可以是延迟所述疾病或所述病症的发作或者预防所述疾病或所述病症的发作。本文所述的蛋白质的有效量将取决于有待治疗的病症、疾病的严重性、受试者的个别参数(包括年龄、生理状况、体型和体重)、治疗的持续时间、伴随疗法(如果存在)的类型、具体施用途径和类似因素。因此，本文所述的药剂的施用剂量可以取决于若干此类参数。在初始剂量引起的受试者反应不足的情况下，可以使用较高剂量(或通过不同的更局部性的施用途径实现的有效较高的剂量)。
根据本发明，术语“受试者”意指用于治疗的受试者，特别是患病受试者(也被称为“患者”)，包括人、非人灵长类动物或其他动物，特别是哺乳动物，诸如牛、马、猪、绵羊、山羊、狗、猫、兔或啮齿类动物(诸如小鼠、大鼠、豚鼠和仓鼠)。在一个实施方案中，受试者/患者是人类。
在又另一方面，本发明涉及一种药物组合物或试剂盒，其包含如上所定义的蛋白质。在一个实施方案中，药物组合物进一步包含药学上可接受的载体。
本发明的药物组合物可以选择用于肠胃外递送。可替代地，组合物可以选择用于吸入或用于经消化道递送，诸如口服。此类药学上可接受的组合物的制备在本领域的技术范围内。
如本文所用的术语“药学上可接受的载体”或“生理学上可接受的载体”是指适合用于实现或增强抗体样结合蛋白的递送的一种或多种配制材料。药物组合物中的主要载体的性质可以是水性或非水性的。例如，用于注射的合适媒介物或载体可以是水、生理盐水溶液或人工脑脊液，可能补充有用于肠胃外施用的组合物中常用的其他材料。中性缓冲盐水或与血清白蛋白混合的盐水是其他示例性媒介物。其他示例性药物组合物包含约pH 7.0-8.5的Tris缓冲液或约pH 4.0-5.5的乙酸盐缓冲液，它们可以进一步包括山梨醇或合适的取代物。在本发明的一个实施方案中，抗体样结合蛋白组合物可以通过将具有希望纯度的所选组合物与任选的配制剂以冻干饼或水溶液形式混合来制备用于储存。此外，可以使用适当赋形剂(诸如蔗糖)将抗体样结合蛋白配制为冻干物。
药物组合物可以含有用于修饰、维持或保持例如组合物的pH、摩尔渗透压浓度、粘度、澄清度、颜色、等渗性、气味、无菌度、稳定性、溶解或释放速率、吸附或渗透的配制材料。合适的配制材料包括但不限于氨基酸(诸如甘氨酸、谷氨酰胺、天冬酰胺、精氨酸或赖氨酸)、抗微生物剂、抗氧化剂(诸如抗坏血酸、亚硫酸钠或亚硫酸氢钠)、缓冲液(诸如硼酸盐、碳酸氢盐、Tris-HCl、柠檬酸盐、磷酸盐或其他有机酸)、增量剂(诸如甘露醇或甘氨酸)、螯合剂(诸如乙二胺四乙酸(EDTA))、络合剂(诸如咖啡因、聚乙烯吡咯烷酮、β-环糊精或羟丙基-β-环糊精)、填充剂、单糖、二糖和其他碳水化合物(诸如葡萄糖、甘露糖或糊精)、蛋白质(诸如血清白蛋白、明胶或免疫球蛋白)、着色剂、调味剂和稀释剂、乳化剂、亲水聚合物(诸如聚乙烯吡咯烷酮)、低分子量多肽、成盐抗衡离子(诸如钠)、防腐剂(诸如苯扎氯铵、苯甲酸、水杨酸、硫柳汞、苯乙醇、对羟基苯甲酸甲酯、对羟基苯甲酸丙酯、氯己定、山梨酸或过氧化氢)、溶剂(诸如丙三醇、丙二醇或聚乙二醇)、糖醇(诸如甘露醇或山梨醇)、悬浮剂、表面活性剂或润湿剂(诸如普朗尼克；PEG；脱水山梨糖醇酯；聚山梨酯，诸如聚山梨酯20或聚山梨酯80；曲拉通(triton)；氨丁三醇；卵磷脂；胆固醇或泰洛沙泊)、稳定性增强剂(诸如蔗糖或山梨醇)、张力调节剂(诸如碱金属卤化物，优选氯化钠或氯化钾-或甘露醇山梨醇)、递送媒介物、稀释剂、赋形剂和/或药用佐剂(参见例如，REMINGTON’s PHARMACEUTICALSCIENCES(第18版,A.R.Gennaro编辑,Mack Publishing Company 1990)及其后续版本)。
如本文所用的术语“部件试剂盒(简写：试剂盒)”是指包含一个或多个容器以及任选地数据载体的制品。所述一个或多个容器可以填充有上文所提到的一种或多种药剂(试剂)。在试剂盒中可以包含另外的容器，所述另外的容器含有例如稀释剂、缓冲液和其他试剂。所述数据载体可以是非电子数据载体，例如图形数据载体，诸如信息传单、信息表、条码或存取码；或电子数据载体，诸如光盘(CD)、数字多功能光盘(DVD)、微芯片或另一种基于半导体的电子数据载体。存取码可以允许访问数据库，例如互联网数据库、集中式数据库或分布式数据库。所述数据载体可以包含本发明药剂的使用说明书，所述药剂例如如本文所述的蛋白质和药物组合物以及相关药剂，诸如核酸分子和宿主细胞。
在另一方面，本发明涉及一种蛋白质，其包含具有式VLb-L1-VLa-L2-CL的第一多肽链、具有式VHa-L3-VHb-L4-CH1-铰链-CH2-CH3的第二多肽链和具有式VLc-CLc-PCL-VHc-CH1c-铰链-CH2-CH3的第三多肽链，
其中
VLa是第一免疫球蛋白轻链可变区；
VLb是第二免疫球蛋白轻链可变区；
VLc是第三免疫球蛋白轻链可变区；
CL是免疫球蛋白轻链恒定区；
CLc是免疫球蛋白轻链恒定区；
VHa是第一免疫球蛋白重链可变区；
VHb是第二免疫球蛋白重链可变区；
VHc是第三免疫球蛋白重链可变区；
CH1是免疫球蛋白CH1重链恒定区；
CH1c是免疫球蛋白CH1重链恒定区；
铰链是免疫球蛋白铰链区；
CH2是免疫球蛋白CH2重链恒定区；并且
CH3是免疫球蛋白CH3重链恒定区；
PCL是蛋白酶可切割接头；
L1、L2、L3和L4是接头；
其中VLa与VHa配对以形成与抗原A结合的抗原结合位点，其中VLb与VHb配对以形成与抗原B结合的抗原结合位点，并且其中VLc与VHc配对以形成与抗原C结合的抗原结合位点；并且
其中第二多肽链经由至少两个二硫键并且任选地经由至少一种另外的Fc相互作用与第三多肽链缔合。
在一个实施方案中，在蛋白酶可切割接头的蛋白酶切割后，抗原结合位点C变得有活性或展现出增加的活性。
在一个实施方案中，所述蛋白酶可切割接头是如本文所定义的蛋白酶可切割接头。
在一个实施方案中，所述至少一种另外的Fc相互作用是至少一种杵臼相互作用(例如，两种杵臼相互作用)。
在一个实施方案中，L1、L2、L3和L4独立地选自如本文所定义的肽接头，优选具有在1至25之间，优选2至25、更优选5至20、更优选10至20个氨基酸残基的长度。在一个实施方案中，L1、L2、L3和L4是富含甘氨酸-丝氨酸的接头，其中至少50％、优选至少60％、更优选至少70％、更优选至少80％、甚至更优选至少85％的氨基酸分别是甘氨酸残基或丝氨酸残基。在一个实施方案中，L1、L2、L3和L4分别是甘氨酸接头、丝氨酸接头或甘氨酸-丝氨酸接头。在一个实施方案中，L1、L2、L3和L4具有序列(G4S)n(SEQ ID NO:51)，其中n是独立地选自1至5，例如1至3或2至4的整数。
在如本文所述的本发明的任何方面，本发明还包括SEQ ID NO:42至44中任一项的氨基酸序列的功能(即，蛋白酶可切割的)变体(及其用途)。在一个实施方案中，变体在/从/向SEQ ID NO:42至44中任一项的氨基酸序列中包含最多3、2或1个氨基酸取代、优选保守氨基酸取代，最多3、2或1个氨基酸残基的缺失(例如，在N末端和/或C末端)和/或最多3、2或1个氨基酸残基的添加(例如，添加到N末端和/或C末端)。保守氨基酸取代涉及氨基酸被相同氨基酸家族的另一种氨基酸(即，在其侧链上相关的氨基酸(例如，在电荷和/或大小方面))取代。天然存在的氨基酸通常分为四个家族：酸性(天冬氨酸盐、谷氨酸盐)、碱性(赖氨酸、精氨酸、组氨酸)、非极性(丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸、色氨酸)、和不带电荷的极性(甘氨酸、天冬酰胺、谷氨酰胺、半胱氨酸、丝氨酸、苏氨酸、酪氨酸)氨基酸。苯丙氨酸、色氨酸和酪氨酸有时被共同分类为芳族氨基酸。保守取代是本领域中熟知的(参见例如Creighton(1984)Proteins.W.H.Freeman and Company)。在一个实施方案中，变体包含以下氨基酸取代中的一个或多个(例如，最多3、2或1个)：R→K、S→T/A(即，S→T或S→A)、V→A/I/L(即，V→A或V→I或V→L)、D→E、E→D。在一个实施方案中，变体(例如，SEQ ID NO:42的变体)包含以下氨基酸取代中的一个或多个(例如，最多3、2或1个)：S→T/A、V→A/I/L、D→E、E→D。
现在参考以下实施例进一步描述本发明，所述实施例旨在说明而不是限制本发明的范围。
实施例
实施例1：材料和方法
在HEK 293-FS、ExpiCHO细胞和稳定的CHO 9E4细胞池中蛋白酶可切割的单特异性和双特异性分子的表达
对于单克隆抗体，表达质粒编码单链，所述单链包含经由蛋白酶可切割接头连接的重链和轻链(抗IL4-hIgG1)；对于双特异性串联-IgG抗体，表达质粒编码重链或单轻链(用蛋白酶可切割接头连接的2个Fab片段)；对于双特异性串联样抗体，表达质粒编码重链或重链-轻链连接的构建体。对于蛋白酶，表达质粒编码具有另外的KDEL信号肽的弗林蛋白酶、PCSK5、PCSK6、PCSK7或弗林蛋白酶的人前肽序列。所有表达质粒均在大肠杆菌DH5a中繁殖。使用Qiagen EndoFree Plasmid Mega试剂盒从大肠杆菌中制备用于转染的质粒。
使用聚乙烯亚胺转染试剂用所指示的单链或LC和HC质粒转染在F17无血清悬浮培养物(Invitrogen)中生长的HEK 293-FS细胞。使用合适的转染试剂，用所指示的质粒转染在F17无血清悬浮培养物(Invitrogen)中生长的ExpiCHO细胞。
在37℃下培养7天之后，通过离心去除细胞，并且使上清液通过0.22μm过滤器以去除颗粒。
对于HEK293-FS或ExpiCHO细胞的共转染，将抗体构建体的质粒与编码蛋白酶的前肽序列的单独表达质粒以1:1的摩尔比混合。
对于稳定的CHO 9E4细胞池的产生，将CHO 9E4细胞在CD CHO无血清培养基(Invitrogen)中培养，并且用编码单轻链和重链的基于转座子的质粒使用MaxCyte STX仪器和缓冲液试剂(MaxCyte Inc.)进行电穿孔。对于蛋白酶共表达，质粒含量的30％是基于转座子的蛋白酶编码质粒。在含有MSX选择标记的CD CHO培养基中选择稳定的细胞池，持续6天。对于抗体的表达，将细胞在选择压力下在含有FeedB(Invitrogen)的OPTi CHO培养基(Invitrogen)中在37℃下培养13天，并且然后通过离心分离上清液并且使其通过0.22μm过滤器以去除颗粒。
对于纯化，将抗体捕获在MabSelect SuRe柱(目录号：11-0034-93，GEHealthcare)上，并且用0.1M柠檬酸盐缓冲液pH 3.0洗脱。通过添加1:6(v/v)Trizma pH8.0(Sigma Aldrich)直接中和洗脱级分。在通过尺寸排阻色谱(SEC)使用Superdex200 16/60(GE)精制蛋白质以及最终的超滤浓缩步骤之后，将蛋白质用于进一步表征。
十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)和毛细管电泳
对于SDS-PAGE，将2μg蛋白质样品与NuPage LDS样品缓冲液(ThermoFisherScientific)混合；对于还原条件，将2μg蛋白质与含有DTT(二硫苏糖醇)的NuPage LDS样品缓冲液混合，并且在95℃下加热5分钟。将样品和彩色蛋白质标准宽范围梯(New EnglandBiolabs)加载到4％-12％Bis/Tris凝胶(Invitrogen)上，并且在NuPage MOPS SDS运行缓冲液(ThermoFisher Scientific)中以200V运行45分钟。将凝胶用Instant BlueCoomassie蛋白质染色剂(Expedeon)染色。对于毛细管电泳，根据蛋白质制造商的方案，使用Protein Clear HR测定试剂盒和LabChip Touch SP2仪器(Perkin Elmer)在还原和氧化条件下制备蛋白质样品。
分析型尺寸排阻色谱法(SEC)
使用BioSECcurity仪器(PSS Polymer)用AdvanceBio 300柱(4.6mm x300mm)和AdvanceBio 300保护柱(Agilent Technologies)在25℃下进行分析型SEC。以0.5ml/min的流速使用2x浓缩的D-PBS缓冲液(Thermo Fisher Scientific)运行分析，其中在280nm处检测。将10μl蛋白质样品(1mg/ml)应用到柱上。使用WinGPC软件v8.1(PSS Polymer)进行数据评估。对于分子量的估计，用蛋白质校准标准混合物(Agilent Technologies)校准SEC柱。
分析型疏水性相互作用色谱法(HIC)
使用配备有TSKgel丁基-NPR柱(2.5μm，4.6x 35mm)(Tosoh Bioscience)的LC10HPLC仪器(Shimadzu)或Vanquish HPLC仪器(Thermo Fisher Scientific)在25℃下进行分析型HIC。以1ml/min的流速运行分析，其中在280nm处检测。将5μg未稀释的蛋白质样品应用到柱上。梯度洗脱是在7min内从15％B至85％B，随后经1min到100％B，然后经1min到15％B，并且然后在15％B下3分钟平衡。缓冲液A由1.5M硫酸铵、25mM磷酸钠(pH 7.0)构成。缓冲液B由25mM磷酸钠(pH 7.0)构成。使用LabSolutions软件v5.85(Shimadzu)或Chromeleon 7软件(Thermo Fisher Scientific)进行数据评估。
质谱法(MS)
通过LC-质谱法(LC-MS)分析异二聚体构建体的蛋白质完整性和潜在错配。将蛋白质样品用12.5μg蛋白质(在用0.5μl PNGaseF(无甘油，New England Biolabs)处理的LC-MS级水(Thermo Scientific)中稀释至0.17mg/ml)在37℃下去糖基，持续16小时。使用ThermoFisher Orbitrap Lumos LC/MS仪器进行LC-MS分析。使用MabPac RP HPLC柱(分析型4μm粒度，2.1x100mm)(Thermo Scientific)以300μL/min进行反相(RP)色谱法。洗脱剂是LC水、0.1％甲酸(A)以及90％乙腈、10％LC水、0.1％甲酸(B)。将2μg蛋白质注射到柱上，并且在12分钟内用从0％至95％B的线性梯度洗脱。使用Expressionist软件13.0.3(Genedata)进行数据分析。使用GPMAW软件10.32b1版(Lighthouse data)基于蛋白质的氨基酸序列计算分子质量。
表面等离子体共振(SPR)
使用表面等离子体共振(SPR)用BIAcore 3000仪器(GE Healthcare)和HBS-EP缓冲液(GE Healthcare)测量抗原与抗体构建体的结合。作为抗原，使用人IL4(IL004，Millipore)和人IL13(IL012，Millipore)。在研究级CM5芯片(GE Life Sciences)上使用标准程序经由伯胺基团(11000RU)固定抗人Fc捕获抗体(人抗体捕获试剂盒，GE LifeSciences)。以导致30RU的最大分析物结合的经调节的RU值以10μl/min的流速捕获配体。将所测试的抗体构建体用作分析物，并且以100nM浓度注射240秒，其中解离时间为300秒，流速为30μL/min。对于人IL4和IL13，分别使用0.1nM至3nM和0.8nM至25nM的稀释系列。通过用捕获试剂盒所提供的再生缓冲液的2min注射使芯片表面再生。用空白芯片表面和HBS-EP缓冲液空白对传感图进行双重参照。使用BIAevaluation软件v4.1进行数据分析。
实施例2：在HEK293-FS细胞中蛋白酶可切割的串联-IgG的表达和体内加工
在编码重链(HC)和单轻链(sLC)的两条质粒的短暂转染后在HEK293-FS细胞中表达双特异性串联-IgG(图1B；表1)构建体，其中VLa-Cκ和VLb-Cκ链经由不同的蛋白酶可切割接头序列连接。作为对照，使用其中VLa-Cκ和VLb-Cκ表达为两条独立链(串联-IgG对照)的串联-(抗IL4x抗IL13)-huIgG1。sLC串联-IgG显示出与对照串联-IgG可比较的表达和纯化特性(图3A-B；在表1中以粗体描绘的选定构建体)。
表1：来自HEK293-FS培养物的具有蛋白酶可切割接头的串联-IgG构建体的表达和纯化结果。
Figure BDA0003827956940000391
具有蛋白酶可切割接头的串联-IgG构建体的体内加工的在还原条件下的SDS-PAGE分析揭示，含有具有经修饰的蛋白酶识别/切割位点HRRRKRSVDE(PCS2；SEQ ID NO:43；“PCL2”是指含有PCS2的PCL)的接头序列的sLC显示出如针对串联-IgG对照所看到的两条独立的轻链(图3C)。在经修饰的蛋白酶识别/切割位点HRRQQRSVDE(PCS2a；SEQ ID NO:44；“PCL2a”是指含有PCS2a的PCL)的情况下获得了相似的结果。相比之下，含有最小蛋白酶识别序列(PCS1；SEQ ID NO:49；“PCL1”是指含有PCS1的PCL)、不同的接头序列或缺失的识别序列(ΔPCS/PCL)的所有构建体在HEK293-FS细胞中表达期间未被体内加工，并且显示出在还原条件下单LC和HC的相同谱(图3C；对于sLC PCL1串联-IgG所显示的示例谱)。
通过LC-质谱法(LC-MS)进一步分析串联-IgG构建体的蛋白质完整性和体内加工。在还原和非还原条件下，在去糖基化后测量样品。基于蛋白质的氨基酸序列计算分子质量(表2)。LC-MS分析证实了含有PCS2识别位点的串联-IgG构建体中接头序列的完全体内加工。相比之下，在具有缺失的(ΔPCS/PCL)或最小PCS(PCS1)识别位点的串联-IgG的蛋白质样品中未检测到加工。此外，由两条单独的LC组成的对照串联-IgG显示出LC与HC的错配(3*LC1+1*LC2+2*HC)，在sLC加工成LC1和LC2后在sLC串联-IgG-PCL2中未观察到所述错配。因此，sLC串联-IgG构建体减少/避免了此双特异性IgG-形式中的LC-HC错配。此外，在完全加工的样品中，观察到包含蛋白酶识别位点RRKR的C末端带电荷的氨基酸被羧基末端蛋白酶进一步加工(RRKR损失)。相比之下，通过Edman-测序不能观察到其他N末端加工(数据未示出)。
表2：在去糖基化的还原或非还原条件下HEK293-FS表达的串联-IgG的LC-MS结果。
Figure BDA0003827956940000401
Figure BDA0003827956940000411
实施例3：在ExpiCHO细胞中蛋白酶可切割的串联-IgG的表达和体内加工
对于在ExpiCHO细胞中串联-IgG的短暂表达，所选择的sLC构建体示出在表3中。具有不同蛋白酶识别位点的sLC串联-IgG显示出与对照串联-IgG可比较的表达和纯化特征(图4A-B)。然而，与短暂转染和在HEK293-FS细胞中的表达相比，表达水平更低。
表3：来自ExpiCHO培养物的具有蛋白酶可切割接头的串联-IgG构建体的表达和纯化结果。
Figure BDA0003827956940000412
Figure BDA0003827956940000421
串联-IgG的体内加工的在还原条件下的SDS-PAGE分析揭示，含有具有经修饰的蛋白酶识别位点HRRRKRSVDE(PCS2；SEQ ID NO:43)的接头序列的sLC显示出两条独立的轻链(图4C)。含有最小蛋白酶识别序列(PCS1；SEQ ID NO:49)或缺失的识别序列(ΔPCS/PCL)的构建体未被在ExpiCHO细胞中内源表达的蛋白酶体内加工。因此，在ExpiCHO细胞中蛋白酶可切割的sLC串联-IgG的表达证实了来自HEK293-FS培养物的结果。
LC-MS分析证实了在ExpiCHO细胞中完全加工的sLC-PCL2串联-IgG的SDS-PAGE结果以及羧基末端蛋白酶对C末端带电荷的氨基酸的进一步加工谱(RRKR-损失)。
表4：在去糖基化的还原条件下ExpiCHO表达的串联IgG的LC-MS结果。
Figure BDA0003827956940000422
实施例4：蛋白酶和蛋白酶可切割的构建体在HEK293-FS细胞中的共转染
将在sLC的接头序列中含有PCS1和ΔPCS识别位点的sLC的串联-IgG构建体与PCSK家族的蛋白酶共转染到HEK293-FS细胞(表5)中，以通过蛋白酶的过表达诱导体内加工。sLC串联-IgG构建体显示出与单转染抗体可比较的表达和纯化特征(图5A和B)。表达产率的降低是由于在共转染中较低的质粒量(与编码单独的蛋白酶的质粒1:1摩尔比)。在SDS-PAGE和LC-MS中体内加工的分析证实了在重组表达的人弗林蛋白酶和具有KDEL-保留序列的人弗林蛋白酶的情况下含有PCS1识别位点的sLC串联-IgG的加工(图5C和D)。相比之下，其他蛋白酶家族成员的过表达未导致sLC串联-IgG的加工。对于sC-IgG和串联样IgG构建体，获得了可比较的结果(图7A和B)。
表5：在与PCSK家族的蛋白酶共表达后，来自HEK293-FS培养物的具有蛋白酶可切割接头的串联-IgG构建体的表达和纯化结果。
Figure BDA0003827956940000431
Figure BDA0003827956940000441
对于稳定的CHO细胞池的产生，将编码在sLC的接头序列中含有PCS1、PCS2和ΔPCS识别位点的sLC的串联-IgG构建体的基于转座子的质粒以及串联-IgG对照(2个LC质粒)用于具有和没有蛋白酶共表达的CHO 9E4细胞的电穿孔。sLC串联-IgG构建体显示出与串联-IgG对照可比较的表达和纯化特征。在SDS-PAGE中体内加工的分析证实了在没有蛋白酶共表达的情况下表达的样品中的含有PCS2识别位点的sLC串联-IgG的加工，以及对于具有PCS1和PCS2识别位点的串联-IgG，在重组表达的人弗林蛋白酶的情况下加工的sLC(图6)。
实施例5：评估具有蛋白酶可切割接头的双特异性sLC串联-IgG的抗原结合
通过表面等离振子共振(SPR)测量在HEK293-FS或ExpiCHO细胞中表达的不同双特异性串联-IgG构建体的结合以及由与弗林蛋白酶变体的共表达而在体内加工的串联-IgG。将与两种人抗原IL4和IL13的结合与单独的单克隆抗体的结合特征进行比较。单克隆抗IL4和抗IL13mAb以及双特异性串联-IgG的结合特征示出在表6中。测量所有串联-IgG的与IL4的特异性结合，并且是与抗IL4对照抗体的特异性结合可比较的。相比之下，对于体内加工的串联-IgG以及包含两个LC的串联-IgG对照，仅可检测到与IL13的特异性结合。未加工的sLC串联-IgG显示没有结合。因此，功能IL13结合位点需要将在串联-IgG的内部位置的VLb(针对IL13的VL)与在外部位置的IL4结合Fab的Cκ结构域连接的接头序列的加工。因此，体内加工可以经由内源表达的蛋白酶或经由共转染的弗林蛋白酶变体介导。
表6：通过SPR的结合动力学：具有双特异性串联-IgG的抗IL4和抗IL13mAb的比较
Figure BDA0003827956940000442
Figure BDA0003827956940000451
实施例6：在HEK293-FS和ExpiCHO细胞中具有蛋白酶可切割接头的sC-IgG和串联样IgG的表达和纯化
表6和表7分别示出了在短暂转染后来自HEK293-FS和ExpiCHO培养物的具有蛋白酶可切割接头序列的sC-IgG和串联样IgG构建体的表达和纯化数据。
在蛋白质A纯化和制备型SEC后，sC-IgG显示出高纯度，单体峰分数为83％-99％。对于sC-IgG，蛋白质产率随着(G4S)n(SEQ ID NO:51)接头序列的长度递减而下降。
与sC-IgG相比，串联样IgG形式仅显示出低的蛋白纯度(23％-75％单体峰分数)，具有高的聚集潜力，如在制备型SEC中在高分子量物质中所看到的。
表7：来自HEK293-FS培养物的具有蛋白酶可切割接头的sc-IgG和串联样IgG构建体的表达和纯化结果。
Figure BDA0003827956940000461
表8：来自ExpiCHO培养物的具有蛋白酶可切割接头的sc-IgG和串联样IgG构建体的表达和纯化结果。
Figure BDA0003827956940000471
在两种细胞系中，仅具有PCS2识别位点的sC-IgG和串联样IgG在蛋白质表达期间被内源蛋白酶切割成相应的链片段(图7A和B)，如对于sLC串联-IgG形式所看到的。
实施例7：蛋白酶和蛋白酶可切割的构建体在HEK293-FS细胞中的共转染
将接头序列中含有PCS1和ΔPCS识别位点的sLC的单链Ig和串联样IgG构建体与PCSK家族的蛋白酶共转染在HEK293-FS细胞中，以通过蛋白酶的过表达诱导体内加工。在SDS-PAGE中体内加工的分析证实了在重组表达的人弗林蛋白酶和具有KDEL保留序列的人弗林蛋白酶的情况下含有PCS1识别位点的sC-IgG和串联样IgG的诱导加工(图8A和B)。此外，PACE4的过表达导致部分加工的构建体。
实施例8：在HEK293-FS细胞中蛋白酶可切割的CODV-scFab-IgG的表达和体内加工
在短暂转染编码CODV HC、CODV LC和单链(sC)的三种质粒后，三特异性CODV-scFab-IgG(图2C；表8)构建体在HEK293-FS细胞中表达，其中VLc-Cκ和VHc-CH1-CH2-CH3链经由不同的蛋白酶可切割接头序列连接。作为对照，使用具有表达为两条独立链的Fab-LC和HC的三特异性CODV-Fab-IgG(经典的CODV-Fab-IgG对照)。单链Fab CODV显示出与对照可比较的表达和纯化特征，并且在SEC中的聚集峰减少(表9，图9A)。
表9：来自HEK293-FS培养物的具有蛋白酶可切割接头的三特异性CODV-Fab-IgG构建体的表达和纯化结果。
Figure BDA0003827956940000481
在还原条件下的电泳分析显示，在HEK293-FS细胞中表达后，含有PCS2(SEQ IDNO:43)或PCS2a(SEQ ID NO:44)序列的Fab臂的单链被加工，导致Fab-LC部分和Fab-HC部分(图9B)。
表10：序列/构建体的列表。
Figure BDA0003827956940000482
Figure BDA0003827956940000491
Figure BDA0003827956940000501
Figure BDA0003827956940000511
Figure BDA0003827956940000521
Figure BDA0003827956940000531
Figure BDA0003827956940000541
Figure BDA0003827956940000551
Figure BDA0003827956940000561
Figure BDA0003827956940000571
Figure BDA0003827956940000581
Figure BDA0003827956940000591
Figure BDA0003827956940000601
Figure BDA0003827956940000611
Figure BDA0003827956940000621
Figure BDA0003827956940000631
Figure BDA0003827956940000641
Figure BDA0003827956940000651
Figure BDA0003827956940000661
Figure BDA0003827956940000671
Figure BDA0003827956940000681
Figure BDA0003827956940000691
Figure BDA0003827956940000701
Figure BDA0003827956940000711
Figure BDA0003827956940000721
Figure BDA0003827956940000731
Figure BDA0003827956940000741
Figure BDA0003827956940000751
Figure BDA0003827956940000761
Figure BDA0003827956940000771
Figure BDA0003827956940000781
Figure BDA0003827956940000791
Figure BDA0003827956940000801
Figure BDA0003827956940000811
Figure BDA0003827956940000821
序列表
<110> 赛诺菲
<120> 蛋白酶加工的分子
<130> 589-312 PCT
<150> EP 20305233.7
<151> 2020-03-05
<160> 75
<170> PatentIn version 3.5
<210> 1
<211> 724
<212> PRT
<213> 人工序列
<220>
<223> 抗IL4-VL-huIGKC-[PCS1]-<(G4S)10>-[PCS1]-抗IL4-VH-huIGHG1
<400> 1
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<210> 2
<211> 714
<212> PRT
<213> 人工序列
<220>
<223> 抗IL4-VL-huIGKC-[PCS1]-<(G4S)8>-[PCS1]-抗IL4-VH-huIGHG1
<400> 2
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<210> 3
<211> 704
<212> PRT
<213> 人工序列
<220>
<223> 抗IL4-VL-huIGKC-[PCS1]-<(G4S)6>-[PCS1]-抗IL4-VH-huIGHG1
<400> 3
Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Val Ser Val Gly
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Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
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Gly Gly Ser Gly Gly Gly Gly Ser Arg Arg Lys Arg Gln Val Gln Leu
245 250 255
Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser Val Lys Ile
260 265 270
Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr Trp Ile His Trp
275 280 285
Ile Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Met Ile Asp
290 295 300
Pro Ser Asp Gly Glu Thr Arg Leu Asn Gln Arg Phe Gln Gly Arg Ala
305 310 315 320
Thr Leu Thr Val Asp Glu Ser Thr Ser Thr Ala Tyr Met Gln Leu Arg
325 330 335
Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Thr Arg Leu Lys
340 345 350
Glu Tyr Gly Asn Tyr Asp Ser Phe Tyr Phe Asp Val Trp Gly Ala Gly
355 360 365
Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
370 375 380
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
385 390 395 400
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
405 410 415
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
420 425 430
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
435 440 445
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
450 455 460
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys
465 470 475 480
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
485 490 495
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
500 505 510
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
515 520 525
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
530 535 540
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
545 550 555 560
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
565 570 575
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
580 585 590
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
595 600 605
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
610 615 620
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
625 630 635 640
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
645 650 655
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
660 665 670
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
675 680 685
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
690 695 700
<210> 4
<211> 694
<212> PRT
<213> 人工序列
<220>
<223> 抗IL4-VL-huIGKC-[PCS1]-<(G4S)4>-[PCS1]-抗IL4-VH-huIGHG1
<400> 4
Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Val Ser Val Gly
1 5 10 15
Asp Thr Ile Thr Leu Thr Cys His Ala Ser Gln Asn Ile Asp Val Trp
20 25 30
Leu Ser Trp Phe Gln Gln Lys Pro Gly Asn Ile Pro Lys Leu Leu Ile
35 40 45
Tyr Lys Ala Ser Asn Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Gly Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Ala His Ser Tyr Pro Phe
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys Arg Arg Lys Arg Gly Gly Gly Gly Ser Gly
210 215 220
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg Arg
225 230 235 240
Lys Arg Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro
245 250 255
Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr
260 265 270
Ser Tyr Trp Ile His Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Glu
275 280 285
Trp Ile Gly Met Ile Asp Pro Ser Asp Gly Glu Thr Arg Leu Asn Gln
290 295 300
Arg Phe Gln Gly Arg Ala Thr Leu Thr Val Asp Glu Ser Thr Ser Thr
305 310 315 320
Ala Tyr Met Gln Leu Arg Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr
325 330 335
Tyr Cys Thr Arg Leu Lys Glu Tyr Gly Asn Tyr Asp Ser Phe Tyr Phe
340 345 350
Asp Val Trp Gly Ala Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr
355 360 365
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
370 375 380
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
385 390 395 400
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
405 410 415
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
420 425 430
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
435 440 445
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
450 455 460
Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
465 470 475 480
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
485 490 495
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
500 505 510
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
515 520 525
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
530 535 540
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
545 550 555 560
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
565 570 575
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
580 585 590
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
595 600 605
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
610 615 620
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
625 630 635 640
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
645 650 655
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
660 665 670
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
675 680 685
Leu Ser Leu Ser Pro Gly
690
<210> 5
<211> 684
<212> PRT
<213> 人工序列
<220>
<223> 抗IL4-VL-huIGKC-[PCS1]-<(G4S)2>-[PCS1]-抗IL4-VH-huIGHG1
<400> 5
Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Val Ser Val Gly
1 5 10 15
Asp Thr Ile Thr Leu Thr Cys His Ala Ser Gln Asn Ile Asp Val Trp
20 25 30
Leu Ser Trp Phe Gln Gln Lys Pro Gly Asn Ile Pro Lys Leu Leu Ile
35 40 45
Tyr Lys Ala Ser Asn Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Gly Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Ala His Ser Tyr Pro Phe
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys Arg Arg Lys Arg Gly Gly Gly Gly Ser Gly
210 215 220
Gly Gly Gly Ser Arg Arg Lys Arg Gln Val Gln Leu Gln Gln Ser Gly
225 230 235 240
Pro Glu Leu Val Lys Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala
245 250 255
Ser Gly Tyr Ser Phe Thr Ser Tyr Trp Ile His Trp Ile Lys Gln Arg
260 265 270
Pro Gly Gln Gly Leu Glu Trp Ile Gly Met Ile Asp Pro Ser Asp Gly
275 280 285
Glu Thr Arg Leu Asn Gln Arg Phe Gln Gly Arg Ala Thr Leu Thr Val
290 295 300
Asp Glu Ser Thr Ser Thr Ala Tyr Met Gln Leu Arg Ser Pro Thr Ser
305 310 315 320
Glu Asp Ser Ala Val Tyr Tyr Cys Thr Arg Leu Lys Glu Tyr Gly Asn
325 330 335
Tyr Asp Ser Phe Tyr Phe Asp Val Trp Gly Ala Gly Thr Leu Val Thr
340 345 350
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
355 360 365
Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val
370 375 380
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
385 390 395 400
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
405 410 415
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
420 425 430
Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys
435 440 445
Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys
450 455 460
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
465 470 475 480
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
485 490 495
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
500 505 510
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
515 520 525
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
530 535 540
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
545 550 555 560
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
565 570 575
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
580 585 590
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
595 600 605
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
610 615 620
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
625 630 635 640
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
645 650 655
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
660 665 670
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
675 680
<210> 6
<211> 724
<212> PRT
<213> 人工序列
<220>
<223> 抗IL4-VL-huIGKC-[deltaPCS]-<(G4S)10>-[deltaPCS]-抗IL4-VH-huIGHG1
<400> 6
Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Val Ser Val Gly
1 5 10 15
Asp Thr Ile Thr Leu Thr Cys His Ala Ser Gln Asn Ile Asp Val Trp
20 25 30
Leu Ser Trp Phe Gln Gln Lys Pro Gly Asn Ile Pro Lys Leu Leu Ile
35 40 45
Tyr Lys Ala Ser Asn Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Gly Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Ala His Ser Tyr Pro Phe
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys Arg Gln Gln Arg Gly Gly Gly Gly Ser Gly
210 215 220
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
225 230 235 240
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
245 250 255
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg Gln Gln Arg
260 265 270
Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
275 280 285
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr
290 295 300
Trp Ile His Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
305 310 315 320
Gly Met Ile Asp Pro Ser Asp Gly Glu Thr Arg Leu Asn Gln Arg Phe
325 330 335
Gln Gly Arg Ala Thr Leu Thr Val Asp Glu Ser Thr Ser Thr Ala Tyr
340 345 350
Met Gln Leu Arg Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
355 360 365
Thr Arg Leu Lys Glu Tyr Gly Asn Tyr Asp Ser Phe Tyr Phe Asp Val
370 375 380
Trp Gly Ala Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly
385 390 395 400
Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
405 410 415
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
420 425 430
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
435 440 445
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
450 455 460
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
465 470 475 480
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
485 490 495
Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
500 505 510
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
515 520 525
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
530 535 540
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
545 550 555 560
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
565 570 575
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
580 585 590
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
595 600 605
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
610 615 620
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
625 630 635 640
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
645 650 655
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
660 665 670
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
675 680 685
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
690 695 700
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
705 710 715 720
Leu Ser Pro Gly
<210> 7
<211> 722
<212> PRT
<213> 人工序列
<220>
<223> 抗IL4-VL-huIGKC-[PCS1]-<(G4S)4>-His(8)-<(G4S)4>-[PCS1]-抗IL4-VH-huIGHG1
<400> 7
Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Val Ser Val Gly
1 5 10 15
Asp Thr Ile Thr Leu Thr Cys His Ala Ser Gln Asn Ile Asp Val Trp
20 25 30
Leu Ser Trp Phe Gln Gln Lys Pro Gly Asn Ile Pro Lys Leu Leu Ile
35 40 45
Tyr Lys Ala Ser Asn Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Gly Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Ala His Ser Tyr Pro Phe
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys Arg Arg Lys Arg Gly Gly Gly Gly Ser Gly
210 215 220
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser His His
225 230 235 240
His His His His His His Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
245 250 255
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg Arg Lys Arg Gln Val
260 265 270
Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser Val
275 280 285
Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr Trp Ile
290 295 300
His Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Met
305 310 315 320
Ile Asp Pro Ser Asp Gly Glu Thr Arg Leu Asn Gln Arg Phe Gln Gly
325 330 335
Arg Ala Thr Leu Thr Val Asp Glu Ser Thr Ser Thr Ala Tyr Met Gln
340 345 350
Leu Arg Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Thr Arg
355 360 365
Leu Lys Glu Tyr Gly Asn Tyr Asp Ser Phe Tyr Phe Asp Val Trp Gly
370 375 380
Ala Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
385 390 395 400
Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
405 410 415
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
420 425 430
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
435 440 445
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
450 455 460
Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
465 470 475 480
Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
485 490 495
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
500 505 510
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
515 520 525
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
530 535 540
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
545 550 555 560
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
565 570 575
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
580 585 590
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
595 600 605
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
610 615 620
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
625 630 635 640
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
645 650 655
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
660 665 670
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
675 680 685
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
690 695 700
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
705 710 715 720
Pro Gly
<210> 8
<211> 722
<212> PRT
<213> 人工序列
<220>
<223> 抗IL4-VL-huIGKC-[deltaPCS]-<(G4S)4>-His(8)-<(G4S)4>-[deltaPCS]-抗IL4-VH-huIGHG1
<400> 8
Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Val Ser Val Gly
1 5 10 15
Asp Thr Ile Thr Leu Thr Cys His Ala Ser Gln Asn Ile Asp Val Trp
20 25 30
Leu Ser Trp Phe Gln Gln Lys Pro Gly Asn Ile Pro Lys Leu Leu Ile
35 40 45
Tyr Lys Ala Ser Asn Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Gly Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Ala His Ser Tyr Pro Phe
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys Arg Gln Gln Arg Gly Gly Gly Gly Ser Gly
210 215 220
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser His His
225 230 235 240
His His His His His His Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
245 250 255
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg Gln Gln Arg Gln Val
260 265 270
Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser Val
275 280 285
Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr Trp Ile
290 295 300
His Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Met
305 310 315 320
Ile Asp Pro Ser Asp Gly Glu Thr Arg Leu Asn Gln Arg Phe Gln Gly
325 330 335
Arg Ala Thr Leu Thr Val Asp Glu Ser Thr Ser Thr Ala Tyr Met Gln
340 345 350
Leu Arg Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Thr Arg
355 360 365
Leu Lys Glu Tyr Gly Asn Tyr Asp Ser Phe Tyr Phe Asp Val Trp Gly
370 375 380
Ala Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
385 390 395 400
Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
405 410 415
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
420 425 430
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
435 440 445
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
450 455 460
Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
465 470 475 480
Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
485 490 495
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
500 505 510
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
515 520 525
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
530 535 540
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
545 550 555 560
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
565 570 575
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
580 585 590
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
595 600 605
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
610 615 620
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
625 630 635 640
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
645 650 655
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
660 665 670
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
675 680 685
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
690 695 700
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
705 710 715 720
Pro Gly
<210> 9
<211> 724
<212> PRT
<213> 人工序列
<220>
<223> 抗IL4-VL-huIGKC-[PCS2]-<(G4S)4>-His(8)-<(G4S)4>-[PCS2]-抗IL4-VH-huIGHG1
<400> 9
Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Val Ser Val Gly
1 5 10 15
Asp Thr Ile Thr Leu Thr Cys His Ala Ser Gln Asn Ile Asp Val Trp
20 25 30
Leu Ser Trp Phe Gln Gln Lys Pro Gly Asn Ile Pro Lys Leu Leu Ile
35 40 45
Tyr Lys Ala Ser Asn Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Gly Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Ala His Ser Tyr Pro Phe
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys His Arg Arg Arg Lys Arg Ser Val Asp Glu
210 215 220
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser His
225 230 235 240
His His His His His His His Gly Gly Gly Gly Ser Gly Gly Gly Gly
245 250 255
Ser Gly Gly Gly Gly Ser His Arg Arg Arg Lys Arg Ser Val Asp Glu
260 265 270
Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
275 280 285
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr
290 295 300
Trp Ile His Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
305 310 315 320
Gly Met Ile Asp Pro Ser Asp Gly Glu Thr Arg Leu Asn Gln Arg Phe
325 330 335
Gln Gly Arg Ala Thr Leu Thr Val Asp Glu Ser Thr Ser Thr Ala Tyr
340 345 350
Met Gln Leu Arg Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
355 360 365
Thr Arg Leu Lys Glu Tyr Gly Asn Tyr Asp Ser Phe Tyr Phe Asp Val
370 375 380
Trp Gly Ala Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly
385 390 395 400
Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
405 410 415
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
420 425 430
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
435 440 445
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
450 455 460
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
465 470 475 480
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
485 490 495
Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
500 505 510
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
515 520 525
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
530 535 540
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
545 550 555 560
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
565 570 575
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
580 585 590
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
595 600 605
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
610 615 620
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
625 630 635 640
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
645 650 655
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
660 665 670
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
675 680 685
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
690 695 700
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
705 710 715 720
Leu Ser Pro Gly
<210> 10
<211> 724
<212> PRT
<213> 人工序列
<220>
<223> 抗IL4-VL-huIGKC-[PCS3]-<(G4S)4>-His(8)-<(G4S)4>-[PCS3]-抗IL4-VH-huIGHG1
<400> 10
Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Val Ser Val Gly
1 5 10 15
Asp Thr Ile Thr Leu Thr Cys His Ala Ser Gln Asn Ile Asp Val Trp
20 25 30
Leu Ser Trp Phe Gln Gln Lys Pro Gly Asn Ile Pro Lys Leu Leu Ile
35 40 45
Tyr Lys Ala Ser Asn Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Gly Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Ala His Ser Tyr Pro Phe
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys Pro Met Lys Lys Arg Arg Ala Gly Val Pro
210 215 220
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser His
225 230 235 240
His His His His His His His Gly Gly Gly Gly Ser Gly Gly Gly Gly
245 250 255
Ser Gly Gly Gly Gly Ser Pro Met Lys Lys Arg Arg Ala Gly Val Pro
260 265 270
Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
275 280 285
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr
290 295 300
Trp Ile His Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
305 310 315 320
Gly Met Ile Asp Pro Ser Asp Gly Glu Thr Arg Leu Asn Gln Arg Phe
325 330 335
Gln Gly Arg Ala Thr Leu Thr Val Asp Glu Ser Thr Ser Thr Ala Tyr
340 345 350
Met Gln Leu Arg Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
355 360 365
Thr Arg Leu Lys Glu Tyr Gly Asn Tyr Asp Ser Phe Tyr Phe Asp Val
370 375 380
Trp Gly Ala Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly
385 390 395 400
Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
405 410 415
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
420 425 430
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
435 440 445
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
450 455 460
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
465 470 475 480
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
485 490 495
Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
500 505 510
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
515 520 525
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
530 535 540
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
545 550 555 560
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
565 570 575
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
580 585 590
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
595 600 605
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
610 615 620
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
625 630 635 640
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
645 650 655
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
660 665 670
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
675 680 685
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
690 695 700
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
705 710 715 720
Leu Ser Pro Gly
<210> 11
<211> 214
<212> PRT
<213> 人工序列
<220>
<223> 抗IL4-VL-huIGKC
<400> 11
Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Val Ser Val Gly
1 5 10 15
Asp Thr Ile Thr Leu Thr Cys His Ala Ser Gln Asn Ile Asp Val Trp
20 25 30
Leu Ser Trp Phe Gln Gln Lys Pro Gly Asn Ile Pro Lys Leu Leu Ile
35 40 45
Tyr Lys Ala Ser Asn Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Gly Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Ala His Ser Tyr Pro Phe
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 12
<211> 218
<212> PRT
<213> 人工序列
<220>
<223> 抗IL13-IGKC
<400> 12
Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Ser Tyr
20 25 30
Gly Gln Ser Tyr Met His Trp Tyr Gln Gln Lys Ala Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asp
65 70 75 80
Pro Val Gln Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Asn Ala
85 90 95
Glu Asp Ser Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
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> 13
<211> 693
<212> PRT
<213> 人工序列
<220>
<223> 抗IL4-VH-huIGH-G11-huIGH-H1-<(G4S)3>-抗IL13-VH-huIGHG1
<400> 13
Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr
20 25 30
Trp Ile His Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Met Ile Asp Pro Ser Asp Gly Glu Thr Arg Leu Asn Gln Arg Phe
50 55 60
Gln Gly Arg Ala Thr Leu Thr Val Asp Glu Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Arg Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Thr Arg Leu Lys Glu Tyr Gly Asn Tyr Asp Ser Phe Tyr Phe Asp Val
100 105 110
Trp Gly Ala Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly
115 120 125
Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
130 135 140
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
145 150 155 160
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
165 170 175
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
180 185 190
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
195 200 205
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
210 215 220
Ser Cys Asp Lys Thr His Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly
225 230 235 240
Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Lys Glu Ser Gly Pro Gly
245 250 255
Leu Val Ala Pro Gly Gly Ser Leu Ser Ile Thr Cys Thr Val Ser Gly
260 265 270
Phe Ser Leu Thr Asp Ser Ser Ile Asn Trp Val Arg Gln Pro Pro Gly
275 280 285
Lys Gly Leu Glu Trp Leu Gly Met Ile Trp Gly Asp Gly Arg Ile Asp
290 295 300
Tyr Ala Asp Ala Leu Lys Ser Arg Leu Ser Ile Ser Lys Asp Ser Ser
305 310 315 320
Lys Ser Gln Val Phe Leu Glu Met Thr Ser Leu Arg Thr Asp Asp Thr
325 330 335
Ala Thr Tyr Tyr Cys Ala Arg Asp Gly Tyr Phe Pro Tyr Ala Met Asp
340 345 350
Phe Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys
355 360 365
Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly
370 375 380
Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
385 390 395 400
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
405 410 415
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
420 425 430
Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
435 440 445
Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro
450 455 460
Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
465 470 475 480
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
485 490 495
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
500 505 510
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
515 520 525
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
530 535 540
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
545 550 555 560
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
565 570 575
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
580 585 590
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
595 600 605
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
610 615 620
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
625 630 635 640
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
645 650 655
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
660 665 670
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
675 680 685
Ser Leu Ser Pro Gly
690
<210> 14
<211> 446
<212> PRT
<213> 人工序列
<220>
<223> 抗IL4-VL-huIGKC-<G4S>-[PCS1]-<G4S>-抗IL13-VL-huIGKC
<400> 14
Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Val Ser Val Gly
1 5 10 15
Asp Thr Ile Thr Leu Thr Cys His Ala Ser Gln Asn Ile Asp Val Trp
20 25 30
Leu Ser Trp Phe Gln Gln Lys Pro Gly Asn Ile Pro Lys Leu Leu Ile
35 40 45
Tyr Lys Ala Ser Asn Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Gly Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Ala His Ser Tyr Pro Phe
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser Arg Arg Lys Arg Gly
210 215 220
Gly Gly Gly Ser Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala
225 230 235 240
Val Ser Leu Gly Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser
245 250 255
Val Asp Ser Tyr Gly Gln Ser Tyr Met His Trp Tyr Gln Gln Lys Ala
260 265 270
Gly Gln Pro Pro Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser
275 280 285
Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr
290 295 300
Leu Thr Ile Asp Pro Val Gln Ala Glu Asp Ala Ala Thr Tyr Tyr Cys
305 310 315 320
Gln Gln Asn Ala Glu Asp Ser Arg Thr Phe Gly Gly Gly Thr Lys Leu
325 330 335
Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro
340 345 350
Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu
355 360 365
Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn
370 375 380
Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser
385 390 395 400
Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala
405 410 415
Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly
420 425 430
Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
435 440 445
<210> 15
<211> 455
<212> PRT
<213> 人工序列
<220>
<223> 抗IL4-VL-huIGKC-[PCS1]-<(G4S)3>-[PCS1]-抗IL13-VL-huIGKC
<400> 15
Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Val Ser Val Gly
1 5 10 15
Asp Thr Ile Thr Leu Thr Cys His Ala Ser Gln Asn Ile Asp Val Trp
20 25 30
Leu Ser Trp Phe Gln Gln Lys Pro Gly Asn Ile Pro Lys Leu Leu Ile
35 40 45
Tyr Lys Ala Ser Asn Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Gly Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Ala His Ser Tyr Pro Phe
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys Arg Arg Lys Arg Gly Gly Gly Gly Ser Gly
210 215 220
Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg Arg Lys Arg Asp Ile Val
225 230 235 240
Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly Gln Arg Ala
245 250 255
Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Ser Tyr Gly Gln Ser
260 265 270
Tyr Met His Trp Tyr Gln Gln Lys Ala Gly Gln Pro Pro Lys Leu Leu
275 280 285
Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala Arg Phe Ser
290 295 300
Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asp Pro Val Gln
305 310 315 320
Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Asn Ala Glu Asp Ser
325 330 335
Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala
340 345 350
Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser
355 360 365
Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu
370 375 380
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser
385 390 395 400
Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu
405 410 415
Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val
420 425 430
Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys
435 440 445
Ser Phe Asn Arg Gly Glu Cys
450 455
<210> 16
<211> 455
<212> PRT
<213> 人工序列
<220>
<223> 抗IL4-VL-huIGKC-[deltaPCS]-<(G4S)3>-[deltaPCS]-抗IL13-VL-huIGKC
<400> 16
Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Val Ser Val Gly
1 5 10 15
Asp Thr Ile Thr Leu Thr Cys His Ala Ser Gln Asn Ile Asp Val Trp
20 25 30
Leu Ser Trp Phe Gln Gln Lys Pro Gly Asn Ile Pro Lys Leu Leu Ile
35 40 45
Tyr Lys Ala Ser Asn Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Gly Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Ala His Ser Tyr Pro Phe
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys Arg Gln Gln Arg Gly Gly Gly Gly Ser Gly
210 215 220
Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg Gln Gln Arg Asp Ile Val
225 230 235 240
Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly Gln Arg Ala
245 250 255
Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Ser Tyr Gly Gln Ser
260 265 270
Tyr Met His Trp Tyr Gln Gln Lys Ala Gly Gln Pro Pro Lys Leu Leu
275 280 285
Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala Arg Phe Ser
290 295 300
Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asp Pro Val Gln
305 310 315 320
Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Asn Ala Glu Asp Ser
325 330 335
Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala
340 345 350
Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser
355 360 365
Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu
370 375 380
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser
385 390 395 400
Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu
405 410 415
Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val
420 425 430
Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys
435 440 445
Ser Phe Asn Arg Gly Glu Cys
450 455
<210> 17
<211> 455
<212> PRT
<213> 人工序列
<220>
<223> 抗IL4-VL-huIGKC-[PCS1]-<GS>-His(8)-<G4S>-[PCS1]-抗IL13-VL-huIGKC
<400> 17
Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Val Ser Val Gly
1 5 10 15
Asp Thr Ile Thr Leu Thr Cys His Ala Ser Gln Asn Ile Asp Val Trp
20 25 30
Leu Ser Trp Phe Gln Gln Lys Pro Gly Asn Ile Pro Lys Leu Leu Ile
35 40 45
Tyr Lys Ala Ser Asn Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Gly Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Ala His Ser Tyr Pro Phe
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys Arg Arg Lys Arg Gly Ser His His His His
210 215 220
His His His His Gly Gly Gly Gly Ser Arg Arg Lys Arg Asp Ile Val
225 230 235 240
Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly Gln Arg Ala
245 250 255
Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Ser Tyr Gly Gln Ser
260 265 270
Tyr Met His Trp Tyr Gln Gln Lys Ala Gly Gln Pro Pro Lys Leu Leu
275 280 285
Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala Arg Phe Ser
290 295 300
Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asp Pro Val Gln
305 310 315 320
Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Asn Ala Glu Asp Ser
325 330 335
Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala
340 345 350
Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser
355 360 365
Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu
370 375 380
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser
385 390 395 400
Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu
405 410 415
Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val
420 425 430
Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys
435 440 445
Ser Phe Asn Arg Gly Glu Cys
450 455
<210> 18
<211> 455
<212> PRT
<213> 人工序列
<220>
<223> 抗IL4-VL-huIGKC-[ΔPCS]-<GS>-His(8)-<G4S>-[ΔPCS]-抗IL13-VL-huIGKC
<400> 18
Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Val Ser Val Gly
1 5 10 15
Asp Thr Ile Thr Leu Thr Cys His Ala Ser Gln Asn Ile Asp Val Trp
20 25 30
Leu Ser Trp Phe Gln Gln Lys Pro Gly Asn Ile Pro Lys Leu Leu Ile
35 40 45
Tyr Lys Ala Ser Asn Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Gly Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Ala His Ser Tyr Pro Phe
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys Arg Gln Gln Arg Gly Ser His His His His
210 215 220
His His His His Gly Gly Gly Gly Ser Arg Gln Gln Arg Asp Ile Val
225 230 235 240
Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly Gln Arg Ala
245 250 255
Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Ser Tyr Gly Gln Ser
260 265 270
Tyr Met His Trp Tyr Gln Gln Lys Ala Gly Gln Pro Pro Lys Leu Leu
275 280 285
Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala Arg Phe Ser
290 295 300
Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asp Pro Val Gln
305 310 315 320
Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Asn Ala Glu Asp Ser
325 330 335
Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala
340 345 350
Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser
355 360 365
Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu
370 375 380
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser
385 390 395 400
Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu
405 410 415
Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val
420 425 430
Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys
435 440 445
Ser Phe Asn Arg Gly Glu Cys
450 455
<210> 19
<211> 457
<212> PRT
<213> 人工序列
<220>
<223> 抗IL4-VL-huIGKC-[PCS2]-<(G4S)3>-[PCS2]-抗IL13-VL-huIGKC
<400> 19
Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Val Ser Val Gly
1 5 10 15
Asp Thr Ile Thr Leu Thr Cys His Ala Ser Gln Asn Ile Asp Val Trp
20 25 30
Leu Ser Trp Phe Gln Gln Lys Pro Gly Asn Ile Pro Lys Leu Leu Ile
35 40 45
Tyr Lys Ala Ser Asn Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Gly Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Ala His Ser Tyr Pro Phe
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys His Arg Arg Arg Lys Arg Ser Val Asp Glu
210 215 220
Gly Gly Gly Gly Ser His Arg Arg Arg Lys Arg Ser Val Asp Glu Asp
225 230 235 240
Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly Gln
245 250 255
Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Ser Tyr Gly
260 265 270
Gln Ser Tyr Met His Trp Tyr Gln Gln Lys Ala Gly Gln Pro Pro Lys
275 280 285
Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala Arg
290 295 300
Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asp Pro
305 310 315 320
Val Gln Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Asn Ala Glu
325 330 335
Asp Ser Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr
340 345 350
Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu
355 360 365
Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro
370 375 380
Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly
385 390 395 400
Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr
405 410 415
Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His
420 425 430
Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val
435 440 445
Thr Lys Ser Phe Asn Arg Gly Glu Cys
450 455
<210> 20
<211> 457
<212> PRT
<213> 人工序列
<220>
<223> 抗IL4-VL-huIGKC-[PCS3]-<(G4S)>-[PCS3]-抗IL13-VL-huIGKC
<400> 20
Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Val Ser Val Gly
1 5 10 15
Asp Thr Ile Thr Leu Thr Cys His Ala Ser Gln Asn Ile Asp Val Trp
20 25 30
Leu Ser Trp Phe Gln Gln Lys Pro Gly Asn Ile Pro Lys Leu Leu Ile
35 40 45
Tyr Lys Ala Ser Asn Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Gly Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Ala His Ser Tyr Pro Phe
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys Pro Met Lys Lys Arg Arg Ala Gly Val Pro
210 215 220
Gly Gly Gly Gly Ser Pro Met Lys Lys Arg Arg Ala Gly Val Pro Asp
225 230 235 240
Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly Gln
245 250 255
Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Ser Tyr Gly
260 265 270
Gln Ser Tyr Met His Trp Tyr Gln Gln Lys Ala Gly Gln Pro Pro Lys
275 280 285
Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala Arg
290 295 300
Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asp Pro
305 310 315 320
Val Gln Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Asn Ala Glu
325 330 335
Asp Ser Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr
340 345 350
Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu
355 360 365
Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro
370 375 380
Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly
385 390 395 400
Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr
405 410 415
Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His
420 425 430
Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val
435 440 445
Thr Lys Ser Phe Asn Arg Gly Glu Cys
450 455
<210> 21
<211> 460
<212> PRT
<213> 人工序列
<220>
<223> 抗IL4-VL-huIGKC-[PCS1]-<G4S>-<(EAAAK)2>-<G4S>-[PCS1]-抗IL13-VL-huIGKC
<400> 21
Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Val Ser Val Gly
1 5 10 15
Asp Thr Ile Thr Leu Thr Cys His Ala Ser Gln Asn Ile Asp Val Trp
20 25 30
Leu Ser Trp Phe Gln Gln Lys Pro Gly Asn Ile Pro Lys Leu Leu Ile
35 40 45
Tyr Lys Ala Ser Asn Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Gly Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Ala His Ser Tyr Pro Phe
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys Arg Arg Lys Arg Gly Gly Gly Gly Ser Glu
210 215 220
Ala Ala Ala Lys Glu Ala Ala Ala Lys Gly Gly Gly Gly Ser Arg Arg
225 230 235 240
Lys Arg Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser
245 250 255
Leu Gly Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp
260 265 270
Ser Tyr Gly Gln Ser Tyr Met His Trp Tyr Gln Gln Lys Ala Gly Gln
275 280 285
Pro Pro Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val
290 295 300
Pro Ala Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr
305 310 315 320
Ile Asp Pro Val Gln Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln
325 330 335
Asn Ala Glu Asp Ser Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
340 345 350
Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
355 360 365
Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
370 375 380
Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
385 390 395 400
Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
405 410 415
Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
420 425 430
Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
435 440 445
Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
450 455 460
<210> 22
<211> 698
<212> PRT
<213> 人工序列
<220>
<223> 抗IL4-VH-huIGH-G11-huIGH-H1-<G4S>-<(EAAAK)2>-<G4S>-抗IL13-VH-huIGHG1
<400> 22
Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr
20 25 30
Trp Ile His Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Met Ile Asp Pro Ser Asp Gly Glu Thr Arg Leu Asn Gln Arg Phe
50 55 60
Gln Gly Arg Ala Thr Leu Thr Val Asp Glu Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Arg Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Thr Arg Leu Lys Glu Tyr Gly Asn Tyr Asp Ser Phe Tyr Phe Asp Val
100 105 110
Trp Gly Ala Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly
115 120 125
Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
130 135 140
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
145 150 155 160
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
165 170 175
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
180 185 190
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
195 200 205
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
210 215 220
Ser Cys Asp Lys Thr His Thr Gly Gly Gly Gly Ser Glu Ala Ala Ala
225 230 235 240
Lys Glu Ala Ala Ala Lys Gly Gly Gly Gly Ser Glu Val Gln Leu Lys
245 250 255
Glu Ser Gly Pro Gly Leu Val Ala Pro Gly Gly Ser Leu Ser Ile Thr
260 265 270
Cys Thr Val Ser Gly Phe Ser Leu Thr Asp Ser Ser Ile Asn Trp Val
275 280 285
Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu Gly Met Ile Trp Gly
290 295 300
Asp Gly Arg Ile Asp Tyr Ala Asp Ala Leu Lys Ser Arg Leu Ser Ile
305 310 315 320
Ser Lys Asp Ser Ser Lys Ser Gln Val Phe Leu Glu Met Thr Ser Leu
325 330 335
Arg Thr Asp Asp Thr Ala Thr Tyr Tyr Cys Ala Arg Asp Gly Tyr Phe
340 345 350
Pro Tyr Ala Met Asp Phe Trp Gly Gln Gly Thr Ser Val Thr Val Ser
355 360 365
Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser
370 375 380
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
385 390 395 400
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
405 410 415
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
420 425 430
Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln
435 440 445
Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
450 455 460
Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro
465 470 475 480
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
485 490 495
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
500 505 510
Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
515 520 525
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
530 535 540
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
545 550 555 560
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
565 570 575
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
580 585 590
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
595 600 605
Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
610 615 620
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
625 630 635 640
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
645 650 655
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
660 665 670
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
675 680 685
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
690 695
<210> 23
<211> 455
<212> PRT
<213> 人工序列
<220>
<223> 抗IL4-VL-huIGKC-[PCS1]-<(AP)7A>-[PCS1]-抗IL13-VL-huIGKC
<400> 23
Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Val Ser Val Gly
1 5 10 15
Asp Thr Ile Thr Leu Thr Cys His Ala Ser Gln Asn Ile Asp Val Trp
20 25 30
Leu Ser Trp Phe Gln Gln Lys Pro Gly Asn Ile Pro Lys Leu Leu Ile
35 40 45
Tyr Lys Ala Ser Asn Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Gly Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Ala His Ser Tyr Pro Phe
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys Arg Arg Lys Arg Ala Pro Ala Pro Ala Pro
210 215 220
Ala Pro Ala Pro Ala Pro Ala Pro Ala Arg Arg Lys Arg Asp Ile Val
225 230 235 240
Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly Gln Arg Ala
245 250 255
Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Ser Tyr Gly Gln Ser
260 265 270
Tyr Met His Trp Tyr Gln Gln Lys Ala Gly Gln Pro Pro Lys Leu Leu
275 280 285
Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala Arg Phe Ser
290 295 300
Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asp Pro Val Gln
305 310 315 320
Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Asn Ala Glu Asp Ser
325 330 335
Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala
340 345 350
Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser
355 360 365
Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu
370 375 380
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser
385 390 395 400
Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu
405 410 415
Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val
420 425 430
Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys
435 440 445
Ser Phe Asn Arg Gly Glu Cys
450 455
<210> 24
<211> 454
<212> PRT
<213> 人工序列
<220>
<223> 抗IL4-VL-huIGKC-[PCS1]-<GS>-<(KQGKQ)2>-<GS>-[PCS1]-抗IL13-VL-huIGKC
<400> 24
Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Val Ser Val Gly
1 5 10 15
Asp Thr Ile Thr Leu Thr Cys His Ala Ser Gln Asn Ile Asp Val Trp
20 25 30
Leu Ser Trp Phe Gln Gln Lys Pro Gly Asn Ile Pro Lys Leu Leu Ile
35 40 45
Tyr Lys Ala Ser Asn Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Gly Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Ala His Ser Tyr Pro Phe
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys Arg Arg Lys Arg Gly Ser Lys Gln Gly Lys
210 215 220
Gln Lys Gln Gly Lys Gln Gly Ser Arg Arg Lys Arg Asp Ile Val Leu
225 230 235 240
Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly Gln Arg Ala Thr
245 250 255
Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Ser Tyr Gly Gln Ser Tyr
260 265 270
Met His Trp Tyr Gln Gln Lys Ala Gly Gln Pro Pro Lys Leu Leu Ile
275 280 285
Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala Arg Phe Ser Gly
290 295 300
Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asp Pro Val Gln Ala
305 310 315 320
Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Asn Ala Glu Asp Ser Arg
325 330 335
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
340 345 350
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
355 360 365
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
370 375 380
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
385 390 395 400
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
405 410 415
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
420 425 430
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
435 440 445
Phe Asn Arg Gly Glu Cys
450
<210> 25
<211> 686
<212> PRT
<213> 人工序列
<220>
<223> 抗IL4-VL-huIGKC-[PCS1]-<(G4S)3>-[PCS1]-抗IL13-VL-huIGKC-<G4S>-Fc-huIgG1(臼-RF)
<400> 25
Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Val Ser Val Gly
1 5 10 15
Asp Thr Ile Thr Leu Thr Cys His Ala Ser Gln Asn Ile Asp Val Trp
20 25 30
Leu Ser Trp Phe Gln Gln Lys Pro Gly Asn Ile Pro Lys Leu Leu Ile
35 40 45
Tyr Lys Ala Ser Asn Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Gly Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Ala His Ser Tyr Pro Phe
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys Arg Arg Lys Arg Gly Gly Gly Gly Ser Gly
210 215 220
Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg Arg Lys Arg Asp Ile Val
225 230 235 240
Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly Gln Arg Ala
245 250 255
Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Ser Tyr Gly Gln Ser
260 265 270
Tyr Met His Trp Tyr Gln Gln Lys Ala Gly Gln Pro Pro Lys Leu Leu
275 280 285
Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala Arg Phe Ser
290 295 300
Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asp Pro Val Gln
305 310 315 320
Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Asn Ala Glu Asp Ser
325 330 335
Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala
340 345 350
Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser
355 360 365
Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu
370 375 380
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser
385 390 395 400
Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu
405 410 415
Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val
420 425 430
Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys
435 440 445
Ser Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser Asp Lys Thr His
450 455 460
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
465 470 475 480
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
485 490 495
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
500 505 510
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
515 520 525
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
530 535 540
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
545 550 555 560
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
565 570 575
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro
580 585 590
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala
595 600 605
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
610 615 620
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
625 630 635 640
Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg
645 650 655
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
660 665 670
His Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
675 680 685
<210> 26
<211> 693
<212> PRT
<213> 人工序列
<220>
<223> 抗IL4-VH-huIGH-G11-huIGH-H1-<(G4S)3>-抗IL13-VH-huIGHG1(杵)
<400> 26
Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr
20 25 30
Trp Ile His Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Met Ile Asp Pro Ser Asp Gly Glu Thr Arg Leu Asn Gln Arg Phe
50 55 60
Gln Gly Arg Ala Thr Leu Thr Val Asp Glu Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Arg Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Thr Arg Leu Lys Glu Tyr Gly Asn Tyr Asp Ser Phe Tyr Phe Asp Val
100 105 110
Trp Gly Ala Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly
115 120 125
Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
130 135 140
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
145 150 155 160
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
165 170 175
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
180 185 190
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
195 200 205
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
210 215 220
Ser Cys Asp Lys Thr His Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly
225 230 235 240
Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Lys Glu Ser Gly Pro Gly
245 250 255
Leu Val Ala Pro Gly Gly Ser Leu Ser Ile Thr Cys Thr Val Ser Gly
260 265 270
Phe Ser Leu Thr Asp Ser Ser Ile Asn Trp Val Arg Gln Pro Pro Gly
275 280 285
Lys Gly Leu Glu Trp Leu Gly Met Ile Trp Gly Asp Gly Arg Ile Asp
290 295 300
Tyr Ala Asp Ala Leu Lys Ser Arg Leu Ser Ile Ser Lys Asp Ser Ser
305 310 315 320
Lys Ser Gln Val Phe Leu Glu Met Thr Ser Leu Arg Thr Asp Asp Thr
325 330 335
Ala Thr Tyr Tyr Cys Ala Arg Asp Gly Tyr Phe Pro Tyr Ala Met Asp
340 345 350
Phe Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys
355 360 365
Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly
370 375 380
Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
385 390 395 400
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
405 410 415
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
420 425 430
Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
435 440 445
Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro
450 455 460
Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
465 470 475 480
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
485 490 495
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
500 505 510
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
515 520 525
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
530 535 540
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
545 550 555 560
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
565 570 575
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
580 585 590
Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn
595 600 605
Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
610 615 620
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
625 630 635 640
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
645 650 655
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
660 665 670
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
675 680 685
Ser Leu Ser Pro Gly
690
<210> 27
<211> 677
<212> PRT
<213> 人工序列
<220>
<223> 抗IL4-VL-huIGKC-<G4S>-[PCS1]-<G4S>-抗IL13-VL-huIGKC-<G4S>-Fc-huIgG1(臼-RF)
<400> 27
Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Val Ser Val Gly
1 5 10 15
Asp Thr Ile Thr Leu Thr Cys His Ala Ser Gln Asn Ile Asp Val Trp
20 25 30
Leu Ser Trp Phe Gln Gln Lys Pro Gly Asn Ile Pro Lys Leu Leu Ile
35 40 45
Tyr Lys Ala Ser Asn Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Gly Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Ala His Ser Tyr Pro Phe
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser Arg Arg Lys Arg Gly
210 215 220
Gly Gly Gly Ser Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala
225 230 235 240
Val Ser Leu Gly Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser
245 250 255
Val Asp Ser Tyr Gly Gln Ser Tyr Met His Trp Tyr Gln Gln Lys Ala
260 265 270
Gly Gln Pro Pro Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser
275 280 285
Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr
290 295 300
Leu Thr Ile Asp Pro Val Gln Ala Glu Asp Ala Ala Thr Tyr Tyr Cys
305 310 315 320
Gln Gln Asn Ala Glu Asp Ser Arg Thr Phe Gly Gly Gly Thr Lys Leu
325 330 335
Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro
340 345 350
Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu
355 360 365
Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn
370 375 380
Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser
385 390 395 400
Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala
405 410 415
Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly
420 425 430
Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Gly Gly
435 440 445
Gly Gly Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
450 455 460
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
465 470 475 480
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
485 490 495
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
500 505 510
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
515 520 525
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
530 535 540
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
545 550 555 560
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
565 570 575
Pro Gln Val Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
580 585 590
Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile
595 600 605
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
610 615 620
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys
625 630 635 640
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
645 650 655
Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln Lys Ser Leu
660 665 670
Ser Leu Ser Pro Gly
675
<210> 28
<211> 686
<212> PRT
<213> 人工序列
<220>
<223> 抗IL4-VL-huIGKC-[deltaPCS]-<(G4S)3>-[deltaPCS]-抗IL13-VL-huIGKC-<G4S>-Fc-huIgG1(臼-RF)
<400> 28
Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Val Ser Val Gly
1 5 10 15
Asp Thr Ile Thr Leu Thr Cys His Ala Ser Gln Asn Ile Asp Val Trp
20 25 30
Leu Ser Trp Phe Gln Gln Lys Pro Gly Asn Ile Pro Lys Leu Leu Ile
35 40 45
Tyr Lys Ala Ser Asn Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Gly Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Ala His Ser Tyr Pro Phe
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys Arg Gln Gln Arg Gly Gly Gly Gly Ser Gly
210 215 220
Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg Gln Gln Arg Asp Ile Val
225 230 235 240
Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly Gln Arg Ala
245 250 255
Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Ser Tyr Gly Gln Ser
260 265 270
Tyr Met His Trp Tyr Gln Gln Lys Ala Gly Gln Pro Pro Lys Leu Leu
275 280 285
Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala Arg Phe Ser
290 295 300
Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asp Pro Val Gln
305 310 315 320
Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Asn Ala Glu Asp Ser
325 330 335
Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala
340 345 350
Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser
355 360 365
Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu
370 375 380
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser
385 390 395 400
Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu
405 410 415
Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val
420 425 430
Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys
435 440 445
Ser Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser Asp Lys Thr His
450 455 460
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
465 470 475 480
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
485 490 495
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
500 505 510
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
515 520 525
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
530 535 540
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
545 550 555 560
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
565 570 575
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro
580 585 590
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala
595 600 605
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
610 615 620
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
625 630 635 640
Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg
645 650 655
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
660 665 670
His Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
675 680 685
<210> 29
<211> 686
<212> PRT
<213> 人工序列
<220>
<223> 抗IL4-VL-huIGKC-[PCS1]-<GS>-His(8)-<G4S>-[PCS1]-抗IL13-VL-huIGKC-<G4S>-Fc-huIgG1(臼-RF)
<400> 29
Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Val Ser Val Gly
1 5 10 15
Asp Thr Ile Thr Leu Thr Cys His Ala Ser Gln Asn Ile Asp Val Trp
20 25 30
Leu Ser Trp Phe Gln Gln Lys Pro Gly Asn Ile Pro Lys Leu Leu Ile
35 40 45
Tyr Lys Ala Ser Asn Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Gly Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Ala His Ser Tyr Pro Phe
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys Arg Arg Lys Arg Gly Ser His His His His
210 215 220
His His His His Gly Gly Gly Gly Ser Arg Arg Lys Arg Asp Ile Val
225 230 235 240
Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly Gln Arg Ala
245 250 255
Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Ser Tyr Gly Gln Ser
260 265 270
Tyr Met His Trp Tyr Gln Gln Lys Ala Gly Gln Pro Pro Lys Leu Leu
275 280 285
Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala Arg Phe Ser
290 295 300
Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asp Pro Val Gln
305 310 315 320
Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Asn Ala Glu Asp Ser
325 330 335
Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala
340 345 350
Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser
355 360 365
Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu
370 375 380
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser
385 390 395 400
Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu
405 410 415
Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val
420 425 430
Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys
435 440 445
Ser Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser Asp Lys Thr His
450 455 460
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
465 470 475 480
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
485 490 495
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
500 505 510
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
515 520 525
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
530 535 540
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
545 550 555 560
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
565 570 575
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro
580 585 590
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala
595 600 605
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
610 615 620
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
625 630 635 640
Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg
645 650 655
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
660 665 670
His Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
675 680 685
<210> 30
<211> 686
<212> PRT
<213> 人工序列
<220>
<223> 抗IL4-VL-huIGKC-[deltaPCS]-<GS>-His(8)-<G4S>-[deltaPCS]-抗IL13-VL-huIGKC-<G4S>-Fc-huIgG1(臼-RF)
<400> 30
Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Val Ser Val Gly
1 5 10 15
Asp Thr Ile Thr Leu Thr Cys His Ala Ser Gln Asn Ile Asp Val Trp
20 25 30
Leu Ser Trp Phe Gln Gln Lys Pro Gly Asn Ile Pro Lys Leu Leu Ile
35 40 45
Tyr Lys Ala Ser Asn Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Gly Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Ala His Ser Tyr Pro Phe
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys Arg Gln Gln Arg Gly Ser His His His His
210 215 220
His His His His Gly Gly Gly Gly Ser Arg Gln Gln Arg Asp Ile Val
225 230 235 240
Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly Gln Arg Ala
245 250 255
Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Ser Tyr Gly Gln Ser
260 265 270
Tyr Met His Trp Tyr Gln Gln Lys Ala Gly Gln Pro Pro Lys Leu Leu
275 280 285
Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala Arg Phe Ser
290 295 300
Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asp Pro Val Gln
305 310 315 320
Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Asn Ala Glu Asp Ser
325 330 335
Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala
340 345 350
Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser
355 360 365
Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu
370 375 380
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser
385 390 395 400
Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu
405 410 415
Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val
420 425 430
Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys
435 440 445
Ser Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser Asp Lys Thr His
450 455 460
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
465 470 475 480
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
485 490 495
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
500 505 510
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
515 520 525
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
530 535 540
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
545 550 555 560
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
565 570 575
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro
580 585 590
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala
595 600 605
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
610 615 620
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
625 630 635 640
Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg
645 650 655
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
660 665 670
His Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
675 680 685
<210> 31
<211> 688
<212> PRT
<213> 人工序列
<220>
<223> 抗IL4-VL-huIGKC-[PCS2]-<(G4S)3>-[PCS2]-抗IL13-VL-huIGKC-<G4S>-Fc-huIgG1(臼-RF)
<400> 31
Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Val Ser Val Gly
1 5 10 15
Asp Thr Ile Thr Leu Thr Cys His Ala Ser Gln Asn Ile Asp Val Trp
20 25 30
Leu Ser Trp Phe Gln Gln Lys Pro Gly Asn Ile Pro Lys Leu Leu Ile
35 40 45
Tyr Lys Ala Ser Asn Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Gly Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Ala His Ser Tyr Pro Phe
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys His Arg Arg Arg Lys Arg Ser Val Asp Glu
210 215 220
Gly Gly Gly Gly Ser His Arg Arg Arg Lys Arg Ser Val Asp Glu Asp
225 230 235 240
Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly Gln
245 250 255
Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Ser Tyr Gly
260 265 270
Gln Ser Tyr Met His Trp Tyr Gln Gln Lys Ala Gly Gln Pro Pro Lys
275 280 285
Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala Arg
290 295 300
Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asp Pro
305 310 315 320
Val Gln Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Asn Ala Glu
325 330 335
Asp Ser Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr
340 345 350
Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu
355 360 365
Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro
370 375 380
Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly
385 390 395 400
Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr
405 410 415
Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His
420 425 430
Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val
435 440 445
Thr Lys Ser Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser Asp Lys
450 455 460
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
465 470 475 480
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
485 490 495
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
500 505 510
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
515 520 525
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
530 535 540
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
545 550 555 560
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
565 570 575
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr
580 585 590
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser
595 600 605
Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
610 615 620
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
625 630 635 640
Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys
645 650 655
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
660 665 670
Ala Leu His Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
675 680 685
<210> 32
<211> 688
<212> PRT
<213> 人工序列
<220>
<223> 抗IL4-VL-huIGKC-[PCS3]-<(G4S)3>-[PCS3]-抗IL13-VL-huIGKC-<G4S>-Fc-huIgG1(臼-RF)
<400> 32
Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Val Ser Val Gly
1 5 10 15
Asp Thr Ile Thr Leu Thr Cys His Ala Ser Gln Asn Ile Asp Val Trp
20 25 30
Leu Ser Trp Phe Gln Gln Lys Pro Gly Asn Ile Pro Lys Leu Leu Ile
35 40 45
Tyr Lys Ala Ser Asn Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Gly Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Ala His Ser Tyr Pro Phe
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys Pro Met Lys Lys Arg Arg Ala Gly Val Pro
210 215 220
Gly Gly Gly Gly Ser Pro Met Lys Lys Arg Arg Ala Gly Val Pro Asp
225 230 235 240
Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly Gln
245 250 255
Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Ser Tyr Gly
260 265 270
Gln Ser Tyr Met His Trp Tyr Gln Gln Lys Ala Gly Gln Pro Pro Lys
275 280 285
Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala Arg
290 295 300
Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asp Pro
305 310 315 320
Val Gln Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Asn Ala Glu
325 330 335
Asp Ser Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr
340 345 350
Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu
355 360 365
Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro
370 375 380
Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly
385 390 395 400
Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr
405 410 415
Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His
420 425 430
Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val
435 440 445
Thr Lys Ser Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser Asp Lys
450 455 460
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
465 470 475 480
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
485 490 495
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
500 505 510
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
515 520 525
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
530 535 540
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
545 550 555 560
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
565 570 575
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr
580 585 590
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser
595 600 605
Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
610 615 620
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
625 630 635 640
Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys
645 650 655
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
660 665 670
Ala Leu His Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
675 680 685
<210> 33
<211> 691
<212> PRT
<213> 人工序列
<220>
<223> 抗IL4-VL-huIGKC-[PCS1]-<G4S>-<(EAAAK)2>-<G4S>-[PCS1]-抗IL13-VL-huIGKC-<G4S>-Fc-huIgG1(臼-RF)
<400> 33
Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Val Ser Val Gly
1 5 10 15
Asp Thr Ile Thr Leu Thr Cys His Ala Ser Gln Asn Ile Asp Val Trp
20 25 30
Leu Ser Trp Phe Gln Gln Lys Pro Gly Asn Ile Pro Lys Leu Leu Ile
35 40 45
Tyr Lys Ala Ser Asn Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Gly Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Ala His Ser Tyr Pro Phe
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys Arg Arg Lys Arg Gly Gly Gly Gly Ser Glu
210 215 220
Ala Ala Ala Lys Glu Ala Ala Ala Lys Gly Gly Gly Gly Ser Arg Arg
225 230 235 240
Lys Arg Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser
245 250 255
Leu Gly Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp
260 265 270
Ser Tyr Gly Gln Ser Tyr Met His Trp Tyr Gln Gln Lys Ala Gly Gln
275 280 285
Pro Pro Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val
290 295 300
Pro Ala Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr
305 310 315 320
Ile Asp Pro Val Gln Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln
325 330 335
Asn Ala Glu Asp Ser Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
340 345 350
Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
355 360 365
Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
370 375 380
Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
385 390 395 400
Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
405 410 415
Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
420 425 430
Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
435 440 445
Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly
450 455 460
Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
465 470 475 480
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
485 490 495
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
500 505 510
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
515 520 525
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
530 535 540
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
545 550 555 560
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
565 570 575
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
580 585 590
Val Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
595 600 605
Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
610 615 620
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
625 630 635 640
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr
645 650 655
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
660 665 670
Met His Glu Ala Leu His Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu
675 680 685
Ser Pro Gly
690
<210> 34
<211> 698
<212> PRT
<213> 人工序列
<220>
<223> 抗IL4-VH-huIGH-G11-huIGH-H1-<G4S>-<(EAAAK)2>-<G4S>-抗IL13-VH-huIGHG1(杵)
<400> 34
Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr
20 25 30
Trp Ile His Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Met Ile Asp Pro Ser Asp Gly Glu Thr Arg Leu Asn Gln Arg Phe
50 55 60
Gln Gly Arg Ala Thr Leu Thr Val Asp Glu Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Arg Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Thr Arg Leu Lys Glu Tyr Gly Asn Tyr Asp Ser Phe Tyr Phe Asp Val
100 105 110
Trp Gly Ala Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly
115 120 125
Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
130 135 140
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
145 150 155 160
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
165 170 175
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
180 185 190
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
195 200 205
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
210 215 220
Ser Cys Asp Lys Thr His Thr Gly Gly Gly Gly Ser Glu Ala Ala Ala
225 230 235 240
Lys Glu Ala Ala Ala Lys Gly Gly Gly Gly Ser Glu Val Gln Leu Lys
245 250 255
Glu Ser Gly Pro Gly Leu Val Ala Pro Gly Gly Ser Leu Ser Ile Thr
260 265 270
Cys Thr Val Ser Gly Phe Ser Leu Thr Asp Ser Ser Ile Asn Trp Val
275 280 285
Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu Gly Met Ile Trp Gly
290 295 300
Asp Gly Arg Ile Asp Tyr Ala Asp Ala Leu Lys Ser Arg Leu Ser Ile
305 310 315 320
Ser Lys Asp Ser Ser Lys Ser Gln Val Phe Leu Glu Met Thr Ser Leu
325 330 335
Arg Thr Asp Asp Thr Ala Thr Tyr Tyr Cys Ala Arg Asp Gly Tyr Phe
340 345 350
Pro Tyr Ala Met Asp Phe Trp Gly Gln Gly Thr Ser Val Thr Val Ser
355 360 365
Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser
370 375 380
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
385 390 395 400
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
405 410 415
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
420 425 430
Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln
435 440 445
Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
450 455 460
Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro
465 470 475 480
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
485 490 495
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
500 505 510
Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
515 520 525
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
530 535 540
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
545 550 555 560
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
565 570 575
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
580 585 590
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp
595 600 605
Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe
610 615 620
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
625 630 635 640
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
645 650 655
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
660 665 670
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
675 680 685
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
690 695
<210> 35
<211> 686
<212> PRT
<213> 人工序列
<220>
<223> 抗IL4-VL-huIGKC-[PCS1]-<(AP)7A>-[PCS1]-抗IL13-VL-huIGKC-<G4S>-Fc-huIgG1(臼-RF)
<400> 35
Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Val Ser Val Gly
1 5 10 15
Asp Thr Ile Thr Leu Thr Cys His Ala Ser Gln Asn Ile Asp Val Trp
20 25 30
Leu Ser Trp Phe Gln Gln Lys Pro Gly Asn Ile Pro Lys Leu Leu Ile
35 40 45
Tyr Lys Ala Ser Asn Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Gly Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Ala His Ser Tyr Pro Phe
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys Arg Arg Lys Arg Ala Pro Ala Pro Ala Pro
210 215 220
Ala Pro Ala Pro Ala Pro Ala Pro Ala Arg Arg Lys Arg Asp Ile Val
225 230 235 240
Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly Gln Arg Ala
245 250 255
Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Ser Tyr Gly Gln Ser
260 265 270
Tyr Met His Trp Tyr Gln Gln Lys Ala Gly Gln Pro Pro Lys Leu Leu
275 280 285
Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala Arg Phe Ser
290 295 300
Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asp Pro Val Gln
305 310 315 320
Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Asn Ala Glu Asp Ser
325 330 335
Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala
340 345 350
Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser
355 360 365
Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu
370 375 380
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser
385 390 395 400
Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu
405 410 415
Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val
420 425 430
Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys
435 440 445
Ser Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser Asp Lys Thr His
450 455 460
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
465 470 475 480
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
485 490 495
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
500 505 510
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
515 520 525
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
530 535 540
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
545 550 555 560
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
565 570 575
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro
580 585 590
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala
595 600 605
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
610 615 620
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
625 630 635 640
Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg
645 650 655
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
660 665 670
His Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
675 680 685
<210> 36
<211> 685
<212> PRT
<213> 人工序列
<220>
<223> 抗IL4-VL-huIGKC-[PCS1]-<GS>-<(KQGKQ)2>-<GS>-[PCS1]-抗IL13-VL-huIGKC-<G4S>-Fc-huIgG1(臼-RF)
<400> 36
Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Val Ser Val Gly
1 5 10 15
Asp Thr Ile Thr Leu Thr Cys His Ala Ser Gln Asn Ile Asp Val Trp
20 25 30
Leu Ser Trp Phe Gln Gln Lys Pro Gly Asn Ile Pro Lys Leu Leu Ile
35 40 45
Tyr Lys Ala Ser Asn Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Gly Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Ala His Ser Tyr Pro Phe
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys Arg Arg Lys Arg Gly Ser Lys Gln Gly Lys
210 215 220
Gln Lys Gln Gly Lys Gln Gly Ser Arg Arg Lys Arg Asp Ile Val Leu
225 230 235 240
Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly Gln Arg Ala Thr
245 250 255
Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Ser Tyr Gly Gln Ser Tyr
260 265 270
Met His Trp Tyr Gln Gln Lys Ala Gly Gln Pro Pro Lys Leu Leu Ile
275 280 285
Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala Arg Phe Ser Gly
290 295 300
Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asp Pro Val Gln Ala
305 310 315 320
Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Asn Ala Glu Asp Ser Arg
325 330 335
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
340 345 350
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
355 360 365
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
370 375 380
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
385 390 395 400
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
405 410 415
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
420 425 430
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
435 440 445
Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser Asp Lys Thr His Thr
450 455 460
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
465 470 475 480
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
485 490 495
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
500 505 510
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
515 520 525
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
530 535 540
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
545 550 555 560
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
565 570 575
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro
580 585 590
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val
595 600 605
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
610 615 620
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
625 630 635 640
Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
645 650 655
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
660 665 670
Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
675 680 685
<210> 37
<211> 768
<212> PRT
<213> 智人
<400> 37
Gln Lys Val Phe Thr Asn Thr Trp Ala Val Arg Ile Pro Gly Gly Pro
1 5 10 15
Ala Val Ala Asn Ser Val Ala Arg Lys His Gly Phe Leu Asn Leu Gly
20 25 30
Gln Ile Phe Gly Asp Tyr Tyr His Phe Trp His Arg Gly Val Thr Lys
35 40 45
Arg Ser Leu Ser Pro His Arg Pro Arg His Ser Arg Leu Gln Arg Glu
50 55 60
Pro Gln Val Gln Trp Leu Glu Gln Gln Val Ala Lys Arg Arg Thr Lys
65 70 75 80
Arg Asp Val Tyr Gln Glu Pro Thr Asp Pro Lys Phe Pro Gln Gln Trp
85 90 95
Tyr Leu Ser Gly Val Thr Gln Arg Asp Leu Asn Val Lys Ala Ala Trp
100 105 110
Ala Gln Gly Tyr Thr Gly His Gly Ile Val Val Ser Ile Leu Asp Asp
115 120 125
Gly Ile Glu Lys Asn His Pro Asp Leu Ala Gly Asn Tyr Asp Pro Gly
130 135 140
Ala Ser Phe Asp Val Asn Asp Gln Asp Pro Asp Pro Gln Pro Arg Tyr
145 150 155 160
Thr Gln Met Asn Asp Asn Arg His Gly Thr Arg Cys Ala Gly Glu Val
165 170 175
Ala Ala Val Ala Asn Asn Gly Val Cys Gly Val Gly Val Ala Tyr Asn
180 185 190
Ala Arg Ile Gly Gly Val Arg Met Leu Asp Gly Glu Val Thr Asp Ala
195 200 205
Val Glu Ala Arg Ser Leu Gly Leu Asn Pro Asn His Ile His Ile Tyr
210 215 220
Ser Ala Ser Trp Gly Pro Glu Asp Asp Gly Lys Thr Val Asp Gly Pro
225 230 235 240
Ala Arg Leu Ala Glu Glu Ala Phe Phe Arg Gly Val Ser Gln Gly Arg
245 250 255
Gly Gly Leu Gly Ser Ile Phe Val Trp Ala Ser Gly Asn Gly Gly Arg
260 265 270
Glu His Asp Ser Cys Asn Cys Asp Gly Tyr Thr Asn Ser Ile Tyr Thr
275 280 285
Leu Ser Ile Ser Ser Ala Thr Gln Phe Gly Asn Val Pro Trp Tyr Ser
290 295 300
Glu Ala Cys Ser Ser Thr Leu Ala Thr Thr Tyr Ser Ser Gly Asn Gln
305 310 315 320
Asn Glu Lys Gln Ile Val Thr Thr Asp Leu Arg Gln Lys Cys Thr Glu
325 330 335
Ser His Thr Gly Thr Ser Ala Ser Ala Pro Leu Ala Ala Gly Ile Ile
340 345 350
Ala Leu Thr Leu Glu Ala Asn Lys Asn Leu Thr Trp Arg Asp Met Gln
355 360 365
His Leu Val Val Gln Thr Ser Lys Pro Ala His Leu Asn Ala Asn Asp
370 375 380
Trp Ala Thr Asn Gly Val Gly Arg Lys Val Ser His Ser Tyr Gly Tyr
385 390 395 400
Gly Leu Leu Asp Ala Gly Ala Met Val Ala Leu Ala Gln Asn Trp Thr
405 410 415
Thr Val Ala Pro Gln Arg Lys Cys Ile Ile Asp Ile Leu Thr Glu Pro
420 425 430
Lys Asp Ile Gly Lys Arg Leu Glu Val Arg Lys Thr Val Thr Ala Cys
435 440 445
Leu Gly Glu Pro Asn His Ile Thr Arg Leu Glu His Ala Gln Ala Arg
450 455 460
Leu Thr Leu Ser Tyr Asn Arg Arg Gly Asp Leu Ala Ile His Leu Val
465 470 475 480
Ser Pro Met Gly Thr Arg Ser Thr Leu Leu Ala Ala Arg Pro His Asp
485 490 495
Tyr Ser Ala Asp Gly Phe Asn Asp Trp Ala Phe Met Thr Thr His Ser
500 505 510
Trp Asp Glu Asp Pro Ser Gly Glu Trp Val Leu Glu Ile Glu Asn Thr
515 520 525
Ser Glu Ala Asn Asn Tyr Gly Thr Leu Thr Lys Phe Thr Leu Val Leu
530 535 540
Tyr Gly Thr Ala Pro Glu Gly Leu Pro Val Pro Pro Glu Ser Ser Gly
545 550 555 560
Cys Lys Thr Leu Thr Ser Ser Gln Ala Cys Val Val Cys Glu Glu Gly
565 570 575
Phe Ser Leu His Gln Lys Ser Cys Val Gln His Cys Pro Pro Gly Phe
580 585 590
Ala Pro Gln Val Leu Asp Thr His Tyr Ser Thr Glu Asn Asp Val Glu
595 600 605
Thr Ile Arg Ala Ser Val Cys Ala Pro Cys His Ala Ser Cys Ala Thr
610 615 620
Cys Gln Gly Pro Ala Leu Thr Asp Cys Leu Ser Cys Pro Ser His Ala
625 630 635 640
Ser Leu Asp Pro Val Glu Gln Thr Cys Ser Arg Gln Ser Gln Ser Ser
645 650 655
Arg Glu Ser Pro Pro Gln Gln Gln Pro Pro Arg Leu Pro Pro Glu Val
660 665 670
Glu Ala Gly Gln Arg Leu Arg Ala Gly Leu Leu Pro Ser His Leu Pro
675 680 685
Glu Val Val Ala Gly Leu Ser Cys Ala Phe Ile Val Leu Val Phe Val
690 695 700
Thr Val Phe Leu Val Leu Gln Leu Arg Ser Gly Phe Ser Phe Arg Gly
705 710 715 720
Val Lys Val Tyr Thr Met Asp Arg Gly Leu Ile Ser Tyr Lys Gly Leu
725 730 735
Pro Pro Glu Ala Trp Gln Glu Glu Cys Pro Ser Asp Ser Glu Glu Asp
740 745 750
Glu Gly Arg Gly Glu Arg Thr Ala Phe Ile Lys Asp Gln Ser Ala Leu
755 760 765
<210> 38
<211> 775
<212> PRT
<213> 人工序列
<220>
<223> 前肽弗林蛋白酶-成熟弗林蛋白酶-<GAA>-KDEL信号肽
<400> 38
Gln Lys Val Phe Thr Asn Thr Trp Ala Val Arg Ile Pro Gly Gly Pro
1 5 10 15
Ala Val Ala Asn Ser Val Ala Arg Lys His Gly Phe Leu Asn Leu Gly
20 25 30
Gln Ile Phe Gly Asp Tyr Tyr His Phe Trp His Arg Gly Val Thr Lys
35 40 45
Arg Ser Leu Ser Pro His Arg Pro Arg His Ser Arg Leu Gln Arg Glu
50 55 60
Pro Gln Val Gln Trp Leu Glu Gln Gln Val Ala Lys Arg Arg Thr Lys
65 70 75 80
Arg Asp Val Tyr Gln Glu Pro Thr Asp Pro Lys Phe Pro Gln Gln Trp
85 90 95
Tyr Leu Ser Gly Val Thr Gln Arg Asp Leu Asn Val Lys Ala Ala Trp
100 105 110
Ala Gln Gly Tyr Thr Gly His Gly Ile Val Val Ser Ile Leu Asp Asp
115 120 125
Gly Ile Glu Lys Asn His Pro Asp Leu Ala Gly Asn Tyr Asp Pro Gly
130 135 140
Ala Ser Phe Asp Val Asn Asp Gln Asp Pro Asp Pro Gln Pro Arg Tyr
145 150 155 160
Thr Gln Met Asn Asp Asn Arg His Gly Thr Arg Cys Ala Gly Glu Val
165 170 175
Ala Ala Val Ala Asn Asn Gly Val Cys Gly Val Gly Val Ala Tyr Asn
180 185 190
Ala Arg Ile Gly Gly Val Arg Met Leu Asp Gly Glu Val Thr Asp Ala
195 200 205
Val Glu Ala Arg Ser Leu Gly Leu Asn Pro Asn His Ile His Ile Tyr
210 215 220
Ser Ala Ser Trp Gly Pro Glu Asp Asp Gly Lys Thr Val Asp Gly Pro
225 230 235 240
Ala Arg Leu Ala Glu Glu Ala Phe Phe Arg Gly Val Ser Gln Gly Arg
245 250 255
Gly Gly Leu Gly Ser Ile Phe Val Trp Ala Ser Gly Asn Gly Gly Arg
260 265 270
Glu His Asp Ser Cys Asn Cys Asp Gly Tyr Thr Asn Ser Ile Tyr Thr
275 280 285
Leu Ser Ile Ser Ser Ala Thr Gln Phe Gly Asn Val Pro Trp Tyr Ser
290 295 300
Glu Ala Cys Ser Ser Thr Leu Ala Thr Thr Tyr Ser Ser Gly Asn Gln
305 310 315 320
Asn Glu Lys Gln Ile Val Thr Thr Asp Leu Arg Gln Lys Cys Thr Glu
325 330 335
Ser His Thr Gly Thr Ser Ala Ser Ala Pro Leu Ala Ala Gly Ile Ile
340 345 350
Ala Leu Thr Leu Glu Ala Asn Lys Asn Leu Thr Trp Arg Asp Met Gln
355 360 365
His Leu Val Val Gln Thr Ser Lys Pro Ala His Leu Asn Ala Asn Asp
370 375 380
Trp Ala Thr Asn Gly Val Gly Arg Lys Val Ser His Ser Tyr Gly Tyr
385 390 395 400
Gly Leu Leu Asp Ala Gly Ala Met Val Ala Leu Ala Gln Asn Trp Thr
405 410 415
Thr Val Ala Pro Gln Arg Lys Cys Ile Ile Asp Ile Leu Thr Glu Pro
420 425 430
Lys Asp Ile Gly Lys Arg Leu Glu Val Arg Lys Thr Val Thr Ala Cys
435 440 445
Leu Gly Glu Pro Asn His Ile Thr Arg Leu Glu His Ala Gln Ala Arg
450 455 460
Leu Thr Leu Ser Tyr Asn Arg Arg Gly Asp Leu Ala Ile His Leu Val
465 470 475 480
Ser Pro Met Gly Thr Arg Ser Thr Leu Leu Ala Ala Arg Pro His Asp
485 490 495
Tyr Ser Ala Asp Gly Phe Asn Asp Trp Ala Phe Met Thr Thr His Ser
500 505 510
Trp Asp Glu Asp Pro Ser Gly Glu Trp Val Leu Glu Ile Glu Asn Thr
515 520 525
Ser Glu Ala Asn Asn Tyr Gly Thr Leu Thr Lys Phe Thr Leu Val Leu
530 535 540
Tyr Gly Thr Ala Pro Glu Gly Leu Pro Val Pro Pro Glu Ser Ser Gly
545 550 555 560
Cys Lys Thr Leu Thr Ser Ser Gln Ala Cys Val Val Cys Glu Glu Gly
565 570 575
Phe Ser Leu His Gln Lys Ser Cys Val Gln His Cys Pro Pro Gly Phe
580 585 590
Ala Pro Gln Val Leu Asp Thr His Tyr Ser Thr Glu Asn Asp Val Glu
595 600 605
Thr Ile Arg Ala Ser Val Cys Ala Pro Cys His Ala Ser Cys Ala Thr
610 615 620
Cys Gln Gly Pro Ala Leu Thr Asp Cys Leu Ser Cys Pro Ser His Ala
625 630 635 640
Ser Leu Asp Pro Val Glu Gln Thr Cys Ser Arg Gln Ser Gln Ser Ser
645 650 655
Arg Glu Ser Pro Pro Gln Gln Gln Pro Pro Arg Leu Pro Pro Glu Val
660 665 670
Glu Ala Gly Gln Arg Leu Arg Ala Gly Leu Leu Pro Ser His Leu Pro
675 680 685
Glu Val Val Ala Gly Leu Ser Cys Ala Phe Ile Val Leu Val Phe Val
690 695 700
Thr Val Phe Leu Val Leu Gln Leu Arg Ser Gly Phe Ser Phe Arg Gly
705 710 715 720
Val Lys Val Tyr Thr Met Asp Arg Gly Leu Ile Ser Tyr Lys Gly Leu
725 730 735
Pro Pro Glu Ala Trp Gln Glu Glu Cys Pro Ser Asp Ser Glu Glu Asp
740 745 750
Glu Gly Arg Gly Glu Arg Thr Ala Phe Ile Lys Asp Gln Ser Ala Leu
755 760 765
Gly Ala Ala Lys Asp Glu Leu
770 775
<210> 39
<211> 1828
<212> PRT
<213> 智人
<400> 39
Arg Val Tyr Thr Asn His Trp Ala Val Lys Ile Ala Gly Gly Phe Pro
1 5 10 15
Glu Ala Asn Arg Ile Ala Ser Lys Tyr Gly Phe Ile Asn Ile Gly Gln
20 25 30
Ile Gly Ala Leu Lys Asp Tyr Tyr His Phe Tyr His Ser Arg Thr Ile
35 40 45
Lys Arg Ser Val Ile Ser Ser Arg Gly Thr His Ser Phe Ile Ser Met
50 55 60
Glu Pro Lys Val Glu Trp Ile Gln Gln Gln Val Val Lys Lys Arg Thr
65 70 75 80
Lys Arg Asp Tyr Asp Phe Ser Arg Ala Gln Ser Thr Tyr Phe Asn Asp
85 90 95
Pro Lys Trp Pro Ser Met Trp Tyr Met His Cys Ser Asp Asn Thr His
100 105 110
Pro Cys Gln Ser Asp Met Asn Ile Glu Gly Ala Trp Lys Arg Gly Tyr
115 120 125
Thr Gly Lys Asn Ile Val Val Thr Ile Leu Asp Asp Gly Ile Glu Arg
130 135 140
Thr His Pro Asp Leu Met Gln Asn Tyr Asp Ala Leu Ala Ser Cys Asp
145 150 155 160
Val Asn Gly Asn Asp Leu Asp Pro Met Pro Arg Tyr Asp Ala Ser Asn
165 170 175
Glu Asn Lys His Gly Thr Arg Cys Ala Gly Glu Val Ala Ala Ala Ala
180 185 190
Asn Asn Ser His Cys Thr Val Gly Ile Ala Phe Asn Ala Lys Ile Gly
195 200 205
Gly Val Arg Met Leu Asp Gly Asp Val Thr Asp Met Val Glu Ala Lys
210 215 220
Ser Val Ser Phe Asn Pro Gln His Val His Ile Tyr Ser Ala Ser Trp
225 230 235 240
Gly Pro Asp Asp Asp Gly Lys Thr Val Asp Gly Pro Ala Pro Leu Thr
245 250 255
Arg Gln Ala Phe Glu Asn Gly Val Arg Met Gly Arg Arg Gly Leu Gly
260 265 270
Ser Val Phe Val Trp Ala Ser Gly Asn Gly Gly Arg Ser Lys Asp His
275 280 285
Cys Ser Cys Asp Gly Tyr Thr Asn Ser Ile Tyr Thr Ile Ser Ile Ser
290 295 300
Ser Thr Ala Glu Ser Gly Lys Lys Pro Trp Tyr Leu Glu Glu Cys Ser
305 310 315 320
Ser Thr Leu Ala Thr Thr Tyr Ser Ser Gly Glu Ser Tyr Asp Lys Lys
325 330 335
Ile Ile Thr Thr Asp Leu Arg Gln Arg Cys Thr Asp Asn His Thr Gly
340 345 350
Thr Ser Ala Ser Ala Pro Met Ala Ala Gly Ile Ile Ala Leu Ala Leu
355 360 365
Glu Ala Asn Pro Phe Leu Thr Trp Arg Asp Val Gln His Val Ile Val
370 375 380
Arg Thr Ser Arg Ala Gly His Leu Asn Ala Asn Asp Trp Lys Thr Asn
385 390 395 400
Ala Ala Gly Phe Lys Val Ser His Leu Tyr Gly Phe Gly Leu Met Asp
405 410 415
Ala Glu Ala Met Val Met Glu Ala Glu Lys Trp Thr Thr Val Pro Arg
420 425 430
Gln His Val Cys Val Glu Ser Thr Asp Arg Gln Ile Lys Thr Ile Arg
435 440 445
Pro Asn Ser Ala Val Arg Ser Ile Tyr Lys Ala Ser Gly Cys Ser Asp
450 455 460
Asn Pro Asn Arg His Val Asn Tyr Leu Glu His Val Val Val Arg Ile
465 470 475 480
Thr Ile Thr His Pro Arg Arg Gly Asp Leu Ala Ile Tyr Leu Thr Ser
485 490 495
Pro Ser Gly Thr Arg Ser Gln Leu Leu Ala Asn Arg Leu Phe Asp His
500 505 510
Ser Met Glu Gly Phe Lys Asn Trp Glu Phe Met Thr Ile His Cys Trp
515 520 525
Gly Glu Arg Ala Ala Gly Asp Trp Val Leu Glu Val Tyr Asp Thr Pro
530 535 540
Ser Gln Leu Arg Asn Phe Lys Thr Pro Gly Lys Leu Lys Glu Trp Ser
545 550 555 560
Leu Val Leu Tyr Gly Thr Ser Val Gln Pro Tyr Ser Pro Thr Asn Glu
565 570 575
Phe Pro Lys Val Glu Arg Phe Arg Tyr Ser Arg Val Glu Asp Pro Thr
580 585 590
Asp Asp Tyr Gly Thr Glu Asp Tyr Ala Gly Pro Cys Asp Pro Glu Cys
595 600 605
Ser Glu Val Gly Cys Asp Gly Pro Gly Pro Asp His Cys Asn Asp Cys
610 615 620
Leu His Tyr Tyr Tyr Lys Leu Lys Asn Asn Thr Arg Ile Cys Val Ser
625 630 635 640
Ser Cys Pro Pro Gly His Tyr His Ala Asp Lys Lys Arg Cys Arg Lys
645 650 655
Cys Ala Pro Asn Cys Glu Ser Cys Phe Gly Ser His Gly Asp Gln Cys
660 665 670
Met Ser Cys Lys Tyr Gly Tyr Phe Leu Asn Glu Glu Thr Asn Ser Cys
675 680 685
Val Thr His Cys Pro Asp Gly Ser Tyr Gln Asp Thr Lys Lys Asn Leu
690 695 700
Cys Arg Lys Cys Ser Glu Asn Cys Lys Thr Cys Thr Glu Phe His Asn
705 710 715 720
Cys Thr Glu Cys Arg Asp Gly Leu Ser Leu Gln Gly Ser Arg Cys Ser
725 730 735
Val Ser Cys Glu Asp Gly Arg Tyr Phe Asn Gly Gln Asp Cys Gln Pro
740 745 750
Cys His Arg Phe Cys Ala Thr Cys Ala Gly Ala Gly Ala Asp Gly Cys
755 760 765
Ile Asn Cys Thr Glu Gly Tyr Phe Met Glu Asp Gly Arg Cys Val Gln
770 775 780
Ser Cys Ser Ile Ser Tyr Tyr Phe Asp His Ser Ser Glu Asn Gly Tyr
785 790 795 800
Lys Ser Cys Lys Lys Cys Asp Ile Ser Cys Leu Thr Cys Asn Gly Pro
805 810 815
Gly Phe Lys Asn Cys Thr Ser Cys Pro Ser Gly Tyr Leu Leu Asp Leu
820 825 830
Gly Met Cys Gln Met Gly Ala Ile Cys Lys Asp Gly Glu Tyr Val Asp
835 840 845
Glu His Gly His Cys Gln Thr Cys Glu Ala Ser Cys Ala Lys Cys Gln
850 855 860
Gly Pro Thr Gln Glu Asp Cys Thr Thr Cys Pro Met Thr Arg Ile Phe
865 870 875 880
Asp Asp Gly Arg Cys Val Ser Asn Cys Pro Ser Trp Lys Phe Glu Phe
885 890 895
Glu Asn Gln Cys His Pro Cys His His Thr Cys Gln Arg Cys Gln Gly
900 905 910
Ser Gly Pro Thr His Cys Thr Ser Cys Gly Ala Asp Asn Tyr Gly Arg
915 920 925
Glu His Phe Leu Tyr Gln Gly Glu Cys Gly Asp Ser Cys Pro Glu Gly
930 935 940
His Tyr Ala Thr Glu Gly Asn Thr Cys Leu Pro Cys Pro Asp Asn Cys
945 950 955 960
Glu Leu Cys His Ser Val His Val Cys Thr Arg Cys Met Lys Gly Tyr
965 970 975
Phe Ile Ala Pro Thr Asn His Thr Cys Gln Lys Leu Glu Cys Gly Gln
980 985 990
Gly Glu Val Gln Asp Pro Asp Tyr Glu Glu Cys Val Pro Cys Glu Glu
995 1000 1005
Gly Cys Leu Gly Cys Ser Leu Asp Asp Pro Gly Thr Cys Thr Ser
1010 1015 1020
Cys Ala Met Gly Tyr Tyr Arg Phe Asp His His Cys Tyr Lys Thr
1025 1030 1035
Cys Pro Glu Lys Thr Tyr Ser Glu Glu Val Glu Cys Lys Ala Cys
1040 1045 1050
Asp Ser Asn Cys Gly Ser Cys Asp Gln Asn Gly Cys Tyr Trp Cys
1055 1060 1065
Glu Glu Gly Phe Phe Leu Leu Gly Gly Ser Cys Val Arg Lys Cys
1070 1075 1080
Gly Pro Gly Phe Tyr Gly Asp Gln Glu Met Gly Glu Cys Glu Ser
1085 1090 1095
Cys His Arg Ala Cys Glu Thr Cys Thr Gly Pro Gly His Asp Glu
1100 1105 1110
Cys Ser Ser Cys Gln Glu Gly Leu Gln Leu Leu Arg Gly Met Cys
1115 1120 1125
Val His Ala Thr Lys Thr Gln Glu Glu Gly Lys Phe Trp Asn Asp
1130 1135 1140
Ile Leu Arg Lys Leu Gln Pro Cys His Ser Ser Cys Lys Thr Cys
1145 1150 1155
Asn Gly Ser Ala Thr Leu Cys Thr Ser Cys Pro Lys Gly Ala Tyr
1160 1165 1170
Leu Leu Ala Gln Ala Cys Val Ser Ser Cys Pro Gln Gly Thr Trp
1175 1180 1185
Pro Ser Val Arg Ser Gly Ser Cys Glu Asn Cys Thr Glu Ala Cys
1190 1195 1200
Ala Ile Cys Ser Gly Ala Asp Leu Cys Lys Lys Cys Gln Met Gln
1205 1210 1215
Pro Gly His Pro Leu Phe Leu His Glu Gly Arg Cys Tyr Ser Lys
1220 1225 1230
Cys Pro Glu Gly Ser Tyr Ala Glu Asp Gly Ile Cys Glu Arg Cys
1235 1240 1245
Ser Ser Pro Cys Arg Thr Cys Glu Gly Asn Ala Thr Asn Cys His
1250 1255 1260
Ser Cys Glu Gly Gly His Val Leu His His Gly Val Cys Gln Glu
1265 1270 1275
Asn Cys Pro Glu Arg His Val Ala Val Lys Gly Val Cys Lys His
1280 1285 1290
Cys Pro Glu Met Cys Gln Asp Cys Ile His Glu Lys Thr Cys Lys
1295 1300 1305
Glu Cys Thr Pro Glu Phe Phe Leu His Asp Asp Met Cys His Gln
1310 1315 1320
Ser Cys Pro Arg Gly Phe Tyr Ala Asp Ser Arg His Cys Val Pro
1325 1330 1335
Cys His Lys Asp Cys Leu Glu Cys Ser Gly Pro Lys Ala Asp Asp
1340 1345 1350
Cys Glu Leu Cys Leu Glu Ser Ser Trp Val Leu Tyr Asp Gly Leu
1355 1360 1365
Cys Leu Glu Glu Cys Pro Ala Gly Thr Tyr Tyr Glu Lys Glu Thr
1370 1375 1380
Lys Glu Cys Arg Asp Cys His Lys Ser Cys Leu Thr Cys Ser Ser
1385 1390 1395
Ser Gly Thr Cys Thr Thr Cys Gln Lys Gly Leu Ile Met Asn Pro
1400 1405 1410
Arg Gly Ser Cys Met Ala Asn Glu Lys Cys Ser Pro Ser Glu Tyr
1415 1420 1425
Trp Asp Glu Asp Ala Pro Gly Cys Lys Pro Cys His Val Lys Cys
1430 1435 1440
Phe His Cys Met Gly Pro Ala Glu Asp Gln Cys Gln Thr Cys Pro
1445 1450 1455
Met Asn Ser Leu Leu Leu Asn Thr Thr Cys Val Lys Asp Cys Pro
1460 1465 1470
Glu Gly Tyr Tyr Ala Asp Glu Asp Ser Asn Arg Cys Ala His Cys
1475 1480 1485
His Ser Ser Cys Arg Thr Cys Glu Gly Arg His Ser Arg Gln Cys
1490 1495 1500
His Ser Cys Arg Pro Gly Trp Phe Gln Leu Gly Lys Glu Cys Leu
1505 1510 1515
Leu Gln Cys Arg Glu Gly Tyr Tyr Ala Asp Asn Ser Thr Gly Arg
1520 1525 1530
Cys Glu Arg Cys Asn Arg Ser Cys Lys Gly Cys Gln Gly Pro Arg
1535 1540 1545
Pro Thr Asp Cys Leu Ser Cys Asp Arg Phe Phe Phe Leu Leu Arg
1550 1555 1560
Ser Lys Gly Glu Cys His Arg Ser Cys Pro Asp His Tyr Tyr Val
1565 1570 1575
Glu Gln Ser Thr Gln Thr Cys Glu Arg Cys His Pro Thr Cys Asp
1580 1585 1590
Gln Cys Lys Gly Lys Gly Ala Leu Asn Cys Leu Ser Cys Val Trp
1595 1600 1605
Ser Tyr His Leu Met Gly Gly Ile Cys Thr Ser Asp Cys Leu Val
1610 1615 1620
Gly Glu Tyr Arg Val Gly Glu Gly Glu Lys Phe Asn Cys Glu Lys
1625 1630 1635
Cys His Glu Ser Cys Met Glu Cys Lys Gly Pro Gly Ala Lys Asn
1640 1645 1650
Cys Thr Leu Cys Pro Ala Asn Leu Val Leu His Met Asp Asp Ser
1655 1660 1665
His Cys Leu His Cys Cys Asn Thr Ser Asp Pro Pro Ser Ala Gln
1670 1675 1680
Glu Cys Cys Asp Cys Gln Asp Thr Thr Asp Glu Cys Ile Leu Arg
1685 1690 1695
Thr Ser Lys Val Arg Pro Ala Thr Glu His Phe Lys Thr Ala Leu
1700 1705 1710
Phe Ile Thr Ser Ser Met Met Leu Val Leu Leu Leu Gly Ala Ala
1715 1720 1725
Val Val Val Trp Lys Lys Ser Arg Gly Arg Val Gln Pro Ala Ala
1730 1735 1740
Lys Ala Gly Tyr Glu Lys Leu Ala Asp Pro Asn Lys Ser Tyr Ser
1745 1750 1755
Ser Tyr Lys Ser Ser Tyr Arg Glu Ser Thr Ser Phe Glu Glu Asp
1760 1765 1770
Gln Val Ile Glu Tyr Arg Asp Arg Asp Tyr Asp Glu Asp Asp Asp
1775 1780 1785
Asp Asp Ile Val Tyr Met Gly Gln Asp Gly Thr Val Tyr Arg Lys
1790 1795 1800
Phe Lys Tyr Gly Leu Leu Asp Asp Asp Asp Ile Asp Glu Leu Glu
1805 1810 1815
Tyr Asp Asp Glu Ser Tyr Ser Tyr Tyr Gln
1820 1825
<210> 40
<211> 748
<212> PRT
<213> 智人
<400> 40
Leu Ala Gly Thr Gly Gly Pro Asp Gly Gln Gly Thr Gly Gly Pro Ser
1 5 10 15
Trp Ala Val His Leu Glu Ser Leu Glu Gly Asp Gly Glu Glu Glu Thr
20 25 30
Leu Glu Gln Gln Ala Asp Ala Leu Ala Gln Ala Ala Gly Leu Val Asn
35 40 45
Ala Gly Arg Ile Gly Glu Leu Gln Gly His Tyr Leu Phe Val Gln Pro
50 55 60
Ala Gly His Arg Pro Ala Leu Glu Val Glu Ala Ile Arg Gln Gln Val
65 70 75 80
Glu Ala Val Leu Ala Gly His Glu Ala Val Arg Trp His Ser Glu Gln
85 90 95
Arg Leu Leu Arg Arg Ala Lys Arg Ser Val His Phe Asn Asp Pro Lys
100 105 110
Tyr Pro Gln Gln Trp His Leu Asn Asn Arg Arg Ser Pro Gly Arg Asp
115 120 125
Ile Asn Val Thr Gly Val Trp Glu Arg Asn Val Thr Gly Arg Gly Val
130 135 140
Thr Val Val Val Val Asp Asp Gly Val Glu His Thr Ile Gln Asp Ile
145 150 155 160
Ala Pro Asn Tyr Ser Pro Glu Gly Ser Tyr Asp Leu Asn Ser Asn Asp
165 170 175
Pro Asp Pro Met Pro His Pro Asp Val Glu Asn Gly Asn His His Gly
180 185 190
Thr Arg Cys Ala Gly Glu Ile Ala Ala Val Pro Asn Asn Ser Phe Cys
195 200 205
Ala Val Gly Val Ala Tyr Gly Ser Arg Ile Ala Gly Ile Arg Val Leu
210 215 220
Asp Gly Pro Leu Thr Asp Ser Met Glu Ala Val Ala Phe Asn Lys His
225 230 235 240
Tyr Gln Ile Asn Asp Ile Tyr Ser Cys Ser Trp Gly Pro Asp Asp Asp
245 250 255
Gly Lys Thr Val Asp Gly Pro His Gln Leu Gly Lys Ala Ala Leu Gln
260 265 270
His Gly Val Ile Ala Gly Arg Gln Gly Phe Gly Ser Ile Phe Val Val
275 280 285
Ala Ser Gly Asn Gly Gly Gln His Asn Asp Asn Cys Asn Tyr Asp Gly
290 295 300
Tyr Ala Asn Ser Ile Tyr Thr Val Thr Ile Gly Ala Val Asp Glu Glu
305 310 315 320
Gly Arg Met Pro Phe Tyr Ala Glu Glu Cys Ala Ser Met Leu Ala Val
325 330 335
Thr Phe Ser Gly Gly Asp Lys Met Leu Arg Ser Ile Val Thr Thr Asp
340 345 350
Trp Asp Leu Gln Lys Gly Thr Gly Cys Thr Glu Gly His Thr Gly Thr
355 360 365
Ser Ala Ala Ala Pro Leu Ala Ala Gly Met Ile Ala Leu Met Leu Gln
370 375 380
Val Arg Pro Cys Leu Thr Trp Arg Asp Val Gln His Ile Ile Val Phe
385 390 395 400
Thr Ala Thr Arg Tyr Glu Asp Arg Arg Ala Glu Trp Val Thr Asn Glu
405 410 415
Ala Gly Phe Ser His Ser His Gln His Gly Phe Gly Leu Leu Asn Ala
420 425 430
Trp Arg Leu Val Asn Ala Ala Lys Ile Trp Thr Ser Val Pro Tyr Leu
435 440 445
Ala Ser Tyr Val Ser Pro Val Leu Lys Glu Asn Lys Ala Ile Pro Gln
450 455 460
Ser Pro Arg Ser Leu Glu Val Leu Trp Asn Val Ser Arg Met Asp Leu
465 470 475 480
Glu Met Ser Gly Leu Lys Thr Leu Glu His Val Ala Val Thr Val Ser
485 490 495
Ile Thr His Pro Arg Arg Gly Ser Leu Glu Leu Lys Leu Phe Cys Pro
500 505 510
Ser Gly Met Met Ser Leu Ile Gly Ala Pro Arg Ser Met Asp Ser Asp
515 520 525
Pro Asn Gly Phe Asn Asp Trp Thr Phe Ser Thr Val Arg Cys Trp Gly
530 535 540
Glu Arg Ala Arg Gly Thr Tyr Arg Leu Val Ile Arg Asp Val Gly Asp
545 550 555 560
Glu Ser Phe Gln Val Gly Ile Leu Arg Gln Trp Gln Leu Thr Leu Tyr
565 570 575
Gly Ser Val Trp Ser Ala Val Asp Ile Arg Asp Arg Gln Arg Leu Leu
580 585 590
Glu Ser Ala Met Ser Gly Lys Tyr Leu His Asp Asp Phe Ala Leu Pro
595 600 605
Cys Pro Pro Gly Leu Lys Ile Pro Glu Glu Asp Gly Tyr Thr Ile Thr
610 615 620
Pro Asn Thr Leu Lys Thr Leu Val Leu Val Gly Cys Phe Thr Val Phe
625 630 635 640
Trp Thr Val Tyr Tyr Met Leu Glu Val Tyr Leu Ser Gln Arg Asn Val
645 650 655
Ala Ser Asn Gln Val Cys Arg Ser Gly Pro Cys His Trp Pro His Arg
660 665 670
Ser Arg Lys Ala Lys Glu Glu Gly Thr Glu Leu Glu Ser Val Pro Leu
675 680 685
Cys Ser Ser Lys Asp Pro Asp Glu Val Glu Thr Glu Ser Arg Gly Pro
690 695 700
Pro Thr Thr Ser Asp Leu Leu Ala Pro Asp Leu Leu Glu Gln Gly Asp
705 710 715 720
Trp Ser Leu Ser Gln Asn Lys Ser Ala Leu Asp Cys Pro His Gln His
725 730 735
Leu Asp Val Pro His Gly Lys Glu Glu Gln Ile Cys
740 745
<210> 41
<211> 906
<212> PRT
<213> 智人
<400> 41
Pro Pro Pro Arg Pro Val Tyr Thr Asn His Trp Ala Val Gln Val Leu
1 5 10 15
Gly Gly Pro Ala Glu Ala Asp Arg Val Ala Ala Ala His Gly Tyr Leu
20 25 30
Asn Leu Gly Gln Ile Gly Asn Leu Glu Asp Tyr Tyr His Phe Tyr His
35 40 45
Ser Lys Thr Phe Lys Arg Ser Thr Leu Ser Ser Arg Gly Pro His Thr
50 55 60
Phe Leu Arg Met Asp Pro Gln Val Lys Trp Leu Gln Gln Gln Glu Val
65 70 75 80
Lys Arg Arg Val Lys Arg Gln Val Arg Ser Asp Pro Gln Ala Leu Tyr
85 90 95
Phe Asn Asp Pro Ile Trp Ser Asn Met Trp Tyr Leu His Cys Gly Asp
100 105 110
Lys Asn Ser Arg Cys Arg Ser Glu Met Asn Val Gln Ala Ala Trp Lys
115 120 125
Arg Gly Tyr Thr Gly Lys Asn Val Val Val Thr Ile Leu Asp Asp Gly
130 135 140
Ile Glu Arg Asn His Pro Asp Leu Ala Pro Asn Tyr Asp Ser Tyr Ala
145 150 155 160
Ser Tyr Asp Val Asn Gly Asn Asp Tyr Asp Pro Ser Pro Arg Tyr Asp
165 170 175
Ala Ser Asn Glu Asn Lys His Gly Thr Arg Cys Ala Gly Glu Val Ala
180 185 190
Ala Ser Ala Asn Asn Ser Tyr Cys Ile Val Gly Ile Ala Tyr Asn Ala
195 200 205
Lys Ile Gly Gly Ile Arg Met Leu Asp Gly Asp Val Thr Asp Val Val
210 215 220
Glu Ala Lys Ser Leu Gly Ile Arg Pro Asn Tyr Ile Asp Ile Tyr Ser
225 230 235 240
Ala Ser Trp Gly Pro Asp Asp Asp Gly Lys Thr Val Asp Gly Pro Gly
245 250 255
Arg Leu Ala Lys Gln Ala Phe Glu Tyr Gly Ile Lys Lys Gly Arg Gln
260 265 270
Gly Leu Gly Ser Ile Phe Val Trp Ala Ser Gly Asn Gly Gly Arg Glu
275 280 285
Gly Asp Tyr Cys Ser Cys Asp Gly Tyr Thr Asn Ser Ile Tyr Thr Ile
290 295 300
Ser Val Ser Ser Ala Thr Glu Asn Gly Tyr Lys Pro Trp Tyr Leu Glu
305 310 315 320
Glu Cys Ala Ser Thr Leu Ala Thr Thr Tyr Ser Ser Gly Ala Phe Tyr
325 330 335
Glu Arg Lys Ile Val Thr Thr Asp Leu Arg Gln Arg Cys Thr Asp Gly
340 345 350
His Thr Gly Thr Ser Val Ser Ala Pro Met Val Ala Gly Ile Ile Ala
355 360 365
Leu Ala Leu Glu Ala Asn Ser Gln Leu Thr Trp Arg Asp Val Gln His
370 375 380
Leu Leu Val Lys Thr Ser Arg Pro Ala His Leu Lys Ala Ser Asp Trp
385 390 395 400
Lys Val Asn Gly Ala Gly His Lys Val Ser His Phe Tyr Gly Phe Gly
405 410 415
Leu Val Asp Ala Glu Ala Leu Val Val Glu Ala Lys Lys Trp Thr Ala
420 425 430
Val Pro Ser Gln His Met Cys Val Ala Ala Ser Asp Lys Arg Pro Arg
435 440 445
Ser Ile Pro Leu Val Gln Val Leu Arg Thr Thr Ala Leu Thr Ser Ala
450 455 460
Cys Ala Glu His Ser Asp Gln Arg Val Val Tyr Leu Glu His Val Val
465 470 475 480
Val Arg Thr Ser Ile Ser His Pro Arg Arg Gly Asp Leu Gln Ile Tyr
485 490 495
Leu Val Ser Pro Ser Gly Thr Lys Ser Gln Leu Leu Ala Lys Arg Leu
500 505 510
Leu Asp Leu Ser Asn Glu Gly Phe Thr Asn Trp Glu Phe Met Thr Val
515 520 525
His Cys Trp Gly Glu Lys Ala Glu Gly Gln Trp Thr Leu Glu Ile Gln
530 535 540
Asp Leu Pro Ser Gln Val Arg Asn Pro Glu Lys Gln Gly Lys Leu Lys
545 550 555 560
Glu Trp Ser Leu Ile Leu Tyr Gly Thr Ala Glu His Pro Tyr His Thr
565 570 575
Phe Ser Ala His Gln Ser Arg Ser Arg Met Leu Glu Leu Ser Ala Pro
580 585 590
Glu Leu Glu Pro Pro Lys Ala Ala Leu Ser Pro Ser Gln Val Glu Val
595 600 605
Pro Glu Asp Glu Glu Asp Tyr Thr Ala Gln Ser Thr Pro Gly Ser Ala
610 615 620
Asn Ile Leu Gln Thr Ser Val Cys His Pro Glu Cys Gly Asp Lys Gly
625 630 635 640
Cys Asp Gly Pro Asn Ala Asp Gln Cys Leu Asn Cys Val His Phe Ser
645 650 655
Leu Gly Ser Val Lys Thr Ser Arg Lys Cys Val Ser Val Cys Pro Leu
660 665 670
Gly Tyr Phe Gly Asp Thr Ala Ala Arg Arg Cys Arg Arg Cys His Lys
675 680 685
Gly Cys Glu Thr Cys Ser Ser Arg Ala Ala Thr Gln Cys Leu Ser Cys
690 695 700
Arg Arg Gly Phe Tyr His His Gln Glu Met Asn Thr Cys Val Thr Leu
705 710 715 720
Cys Pro Ala Gly Phe Tyr Ala Asp Glu Ser Gln Lys Asn Cys Leu Lys
725 730 735
Cys His Pro Ser Cys Lys Lys Cys Val Asp Glu Pro Glu Lys Cys Thr
740 745 750
Val Cys Lys Glu Gly Phe Ser Leu Ala Arg Gly Ser Cys Ile Pro Asp
755 760 765
Cys Glu Pro Gly Thr Tyr Phe Asp Ser Glu Leu Ile Arg Cys Gly Glu
770 775 780
Cys His His Thr Cys Gly Thr Cys Val Gly Pro Gly Arg Glu Glu Cys
785 790 795 800
Ile His Cys Ala Lys Asn Phe His Phe His Asp Trp Lys Cys Val Pro
805 810 815
Ala Cys Gly Glu Gly Phe Tyr Pro Glu Glu Met Pro Gly Leu Pro His
820 825 830
Lys Val Cys Arg Arg Cys Asp Glu Asn Cys Leu Ser Cys Ala Gly Ser
835 840 845
Ser Arg Asn Cys Ser Arg Cys Lys Thr Gly Phe Thr Gln Leu Gly Thr
850 855 860
Ser Cys Ile Thr Asn His Thr Cys Ser Asn Ala Asp Glu Thr Phe Cys
865 870 875 880
Glu Met Val Lys Ser Asn Arg Leu Cys Glu Arg Lys Leu Phe Ile Gln
885 890 895
Phe Cys Cys Arg Thr Cys Leu Leu Ala Gly
900 905
<210> 42
<211> 10
<212> PRT
<213> 人工序列
<220>
<223> PCS 通式
<220>
<221> misc_feature
<222> (4)..(5)
<223> Xaa can be any naturally occurring amino acid
<400> 42
His Arg Arg Xaa Xaa Arg Ser Val Asp Glu
1 5 10
<210> 43
<211> 10
<212> PRT
<213> 人工序列
<220>
<223> PCS2
<400> 43
His Arg Arg Arg Lys Arg Ser Val Asp Glu
1 5 10
<210> 44
<211> 10
<212> PRT
<213> 人工序列
<220>
<223> PCS2a
<400> 44
His Arg Arg Gln Gln Arg Ser Val Asp Glu
1 5 10
<210> 45
<211> 15
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 45
Gly Ser His His His His His His His His Gly Gly Gly Gly Ser
1 5 10 15
<210> 46
<211> 20
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 46
Gly Gly Gly Gly Ser Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Gly
1 5 10 15
Gly Gly Gly Ser
20
<210> 47
<211> 15
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 47
Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala
1 5 10 15
<210> 48
<211> 14
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 48
Gly Ser Lys Gln Gly Lys Gln Lys Gln Gly Lys Gln Gly Ser
1 5 10
<210> 49
<211> 4
<212> PRT
<213> 人工序列
<220>
<223> PCS1
<400> 49
Arg Arg Lys Arg
1
<210> 50
<211> 10
<212> PRT
<213> 人工序列
<220>
<223> PCS3
<400> 50
Pro Met Lys Lys Arg Arg Ala Gly Val Pro
1 5 10
<210> 51
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 接头
<220>
<221> REPEAT
<222> (1)..(5)
<400> 51
Gly Gly Gly Gly Ser
1 5
<210> 52
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 接头
<220>
<221> REPEAT
<222> (1)..(5)
<400> 52
Glu Ala Ala Ala Lys
1 5
<210> 53
<211> 3
<212> PRT
<213> 人工序列
<220>
<223> 接头
<220>
<221> REPEAT
<222> (1)..(2)
<400> 53
Ala Pro Ala
1
<210> 54
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 接头
<220>
<221> REPEAT
<222> (1)..(5)
<400> 54
Lys Gln Gly Lys Gln
1 5
<210> 55
<211> 15
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 55
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10 15
<210> 56
<211> 341
<212> PRT
<213> 人工序列
<220>
<223> 抗OX40-VL-<(G4S)2>-抗PD1-VL-<(G4S)2>-huIGKC
<400> 56
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Tyr Thr Ser Arg Leu Arg Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly His Thr Leu Pro Pro
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser
100 105 110
Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu
115 120 125
Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln
130 135 140
Ser Val Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala
145 150 155 160
Pro Arg Leu Leu Ile Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro
165 170 175
Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
180 185 190
Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser
195 200 205
Ser Asn Trp Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
210 215 220
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg Thr Val Ala Ala Pro
225 230 235 240
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr
245 250 255
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys
260 265 270
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu
275 280 285
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser
290 295 300
Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala
305 310 315 320
Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe
325 330 335
Asn Arg Gly Glu Cys
340
<210> 57
<211> 559
<212> PRT
<213> 人工序列
<220>
<223> 抗PD1-VH-抗OX40-VH-huIGHG1(LALA-杵)
<400> 57
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser
20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
100 105 110
Ser Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly
115 120 125
Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp
130 135 140
Ser Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp
145 150 155 160
Ile Gly Asp Met Tyr Pro Asp Asn Gly Asp Ser Ser Tyr Asn Gln Lys
165 170 175
Phe Arg Glu Arg Val Thr Ile Thr Arg Asp Thr Ser Thr Ser Thr Ala
180 185 190
Tyr Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr
195 200 205
Cys Val Leu Ala Pro Arg Trp Tyr Phe Ser Val Trp Gly Gln Gly Thr
210 215 220
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
225 230 235 240
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
245 250 255
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
260 265 270
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
275 280 285
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
290 295 300
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
305 310 315 320
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
325 330 335
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser
340 345 350
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
355 360 365
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
370 375 380
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
385 390 395 400
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
405 410 415
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
420 425 430
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
435 440 445
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
450 455 460
Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys
465 470 475 480
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
485 490 495
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
500 505 510
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
515 520 525
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
530 535 540
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
545 550 555
<210> 58
<211> 214
<212> PRT
<213> 人工序列
<220>
<223> 抗GITR-VL-huIGKC
<400> 58
Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Ala
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 59
<211> 453
<212> PRT
<213> 人工序列
<220>
<223> 抗GITR-VH-huIGHG1(LALA-臼-RF)
<400> 59
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Trp Tyr Glu Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Gly Ser Met Val Arg Gly Asp Tyr Tyr Tyr Gly Met Asp
100 105 110
Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys
115 120 125
Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly
130 135 140
Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
145 150 155 160
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
165 170 175
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
180 185 190
Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
195 200 205
Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val 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 Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
355 360 365
Gln Val Ser Leu Ser Cys Ala 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
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val 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 Arg Phe Thr Gln Lys Ser Leu
435 440 445
Ser Leu Ser Pro Gly
450
<210> 60
<211> 717
<212> PRT
<213> 人工序列
<220>
<223> 抗GITR-VL-huIGKC-[PCS2]-<(G4S)6>-[PCS2]-抗GITR-VH-huIGHG1(LALA-臼-RF)
<400> 60
Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Ala
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys His Arg Arg Arg Lys Arg Ser Val Asp Glu
210 215 220
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
225 230 235 240
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser His Arg
245 250 255
Arg Arg Lys Arg Ser Val Asp Glu Gln Val Gln Leu Val Glu Ser Gly
260 265 270
Gly Gly Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala
275 280 285
Ser Gly Phe Thr Phe Ser Ser Tyr Gly Met His Trp Val Arg Gln Ala
290 295 300
Pro Gly Lys Gly Leu Glu Trp Val Ala Val Ile Trp Tyr Glu Gly Ser
305 310 315 320
Asn Lys Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg
325 330 335
Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala
340 345 350
Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Gly Ser Met Val Arg
355 360 365
Gly Asp Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val
370 375 380
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
385 390 395 400
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
405 410 415
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
420 425 430
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
435 440 445
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
450 455 460
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
465 470 475 480
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
485 490 495
Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe
500 505 510
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
515 520 525
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
530 535 540
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
545 550 555 560
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
565 570 575
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
580 585 590
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
595 600 605
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro
610 615 620
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val
625 630 635 640
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
645 650 655
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
660 665 670
Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
675 680 685
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
690 695 700
Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
705 710 715
<210> 61
<211> 717
<212> PRT
<213> 人工序列
<220>
<223> 抗GITR-VL-huIGKC-[PCS2a]-<(G4S)6>-[PCS2a]-抗GITR-VH-huIGHG1(LALA-臼-RF)
<400> 61
Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Ala
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys His Arg Arg Gln Gln Arg Ser Val Asp Glu
210 215 220
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
225 230 235 240
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser His Arg
245 250 255
Arg Gln Gln Arg Ser Val Asp Glu Gln Val Gln Leu Val Glu Ser Gly
260 265 270
Gly Gly Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala
275 280 285
Ser Gly Phe Thr Phe Ser Ser Tyr Gly Met His Trp Val Arg Gln Ala
290 295 300
Pro Gly Lys Gly Leu Glu Trp Val Ala Val Ile Trp Tyr Glu Gly Ser
305 310 315 320
Asn Lys Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg
325 330 335
Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala
340 345 350
Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Gly Ser Met Val Arg
355 360 365
Gly Asp Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val
370 375 380
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
385 390 395 400
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
405 410 415
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
420 425 430
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
435 440 445
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
450 455 460
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
465 470 475 480
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
485 490 495
Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe
500 505 510
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
515 520 525
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
530 535 540
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
545 550 555 560
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
565 570 575
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
580 585 590
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
595 600 605
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro
610 615 620
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val
625 630 635 640
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
645 650 655
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
660 665 670
Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
675 680 685
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
690 695 700
Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
705 710 715
<210> 62
<211> 341
<212> PRT
<213> 人工序列
<220>
<223> 抗PD1-VL-<(G4S)2>-抗GITR-VL-<(G4S)2>-huIGKC
<400> 62
Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser Asn Trp Pro Arg
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser
100 105 110
Gly Gly Gly Gly Ser Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu
115 120 125
Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
130 135 140
Gly Ile Ser Ser Ala Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala
145 150 155 160
Pro Lys Leu Leu Ile Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro
165 170 175
Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
180 185 190
Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe
195 200 205
Asn Ser Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
210 215 220
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg Thr Val Ala Ala Pro
225 230 235 240
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr
245 250 255
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys
260 265 270
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu
275 280 285
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser
290 295 300
Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala
305 310 315 320
Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe
325 330 335
Asn Arg Gly Glu Cys
340
<210> 63
<211> 566
<212> PRT
<213> 人工序列
<220>
<223> 抗GITR-VH-抗PD1-VH-huIGHG1(LALA-杵)
<400> 63
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Trp Tyr Glu Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Gly Ser Met Val Arg Gly Asp Tyr Tyr Tyr Gly Met Asp
100 105 110
Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gln Val Gln Leu
115 120 125
Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu Arg Leu
130 135 140
Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser Gly Met His Trp
145 150 155 160
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Val Ile Trp
165 170 175
Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe
180 185 190
Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe Leu Gln Met Asn
195 200 205
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Thr Asn Asp
210 215 220
Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr
225 230 235 240
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
245 250 255
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
260 265 270
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
275 280 285
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
290 295 300
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
305 310 315 320
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
325 330 335
Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
340 345 350
Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
355 360 365
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
370 375 380
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
385 390 395 400
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
405 410 415
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
420 425 430
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
435 440 445
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
450 455 460
Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys
465 470 475 480
Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
485 490 495
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
500 505 510
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
515 520 525
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
530 535 540
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
545 550 555 560
Leu Ser Leu Ser Pro Gly
565
<210> 64
<211> 710
<212> PRT
<213> 人工序列
<220>
<223> 抗OX40-VL-huIGKC-[PCS2]-<(G4S)6>-[PCS2]-抗OX40-VH-huIGHG1(LALA-臼-RF)
<400> 64
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Tyr Thr Ser Arg Leu Arg Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly His Thr Leu Pro Pro
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys His Arg Arg Arg Lys Arg Ser Val Asp Glu
210 215 220
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
225 230 235 240
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser His Arg
245 250 255
Arg Arg Lys Arg Ser Val Asp Glu Glu Val Gln Leu Val Gln Ser Gly
260 265 270
Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala
275 280 285
Ser Gly Tyr Thr Phe Thr Asp Ser Tyr Met Ser Trp Val Arg Gln Ala
290 295 300
Pro Gly Gln Gly Leu Glu Trp Ile Gly Asp Met Tyr Pro Asp Asn Gly
305 310 315 320
Asp Ser Ser Tyr Asn Gln Lys Phe Arg Glu Arg Val Thr Ile Thr Arg
325 330 335
Asp Thr Ser Thr Ser Thr Ala Tyr Leu Glu Leu Ser Ser Leu Arg Ser
340 345 350
Glu Asp Thr Ala Val Tyr Tyr Cys Val Leu Ala Pro Arg Trp Tyr Phe
355 360 365
Ser Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr
370 375 380
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
385 390 395 400
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
405 410 415
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
420 425 430
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
435 440 445
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
450 455 460
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
465 470 475 480
Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
485 490 495
Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
500 505 510
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
515 520 525
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
530 535 540
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
545 550 555 560
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
565 570 575
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
580 585 590
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
595 600 605
Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
610 615 620
Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp
625 630 635 640
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
645 650 655
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser
660 665 670
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
675 680 685
Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln Lys Ser
690 695 700
Leu Ser Leu Ser Pro Gly
705 710
<210> 65
<211> 710
<212> PRT
<213> 人工序列
<220>
<223> 抗OX40-VL-huIGKC-[PCS2a]-<(G4S)6>-[PCS2a]-抗OX40-VH-huIGHG1(LALA-臼-RF)
<400> 65
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Tyr Thr Ser Arg Leu Arg Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly His Thr Leu Pro Pro
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys His Arg Arg Gln Gln Arg Ser Val Asp Glu
210 215 220
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
225 230 235 240
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser His Arg
245 250 255
Arg Gln Gln Arg Ser Val Asp Glu Glu Val Gln Leu Val Gln Ser Gly
260 265 270
Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala
275 280 285
Ser Gly Tyr Thr Phe Thr Asp Ser Tyr Met Ser Trp Val Arg Gln Ala
290 295 300
Pro Gly Gln Gly Leu Glu Trp Ile Gly Asp Met Tyr Pro Asp Asn Gly
305 310 315 320
Asp Ser Ser Tyr Asn Gln Lys Phe Arg Glu Arg Val Thr Ile Thr Arg
325 330 335
Asp Thr Ser Thr Ser Thr Ala Tyr Leu Glu Leu Ser Ser Leu Arg Ser
340 345 350
Glu Asp Thr Ala Val Tyr Tyr Cys Val Leu Ala Pro Arg Trp Tyr Phe
355 360 365
Ser Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr
370 375 380
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
385 390 395 400
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
405 410 415
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
420 425 430
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
435 440 445
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
450 455 460
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
465 470 475 480
Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
485 490 495
Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
500 505 510
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
515 520 525
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
530 535 540
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
545 550 555 560
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
565 570 575
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
580 585 590
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
595 600 605
Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
610 615 620
Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp
625 630 635 640
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
645 650 655
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser
660 665 670
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
675 680 685
Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln Lys Ser
690 695 700
Leu Ser Leu Ser Pro Gly
705 710
<210> 66
<211> 341
<212> PRT
<213> 人工序列
<220>
<223> 抗GITR-VL-<(G4S)2>-抗PD1-VL-<(G4S)2>-huIGKC
<400> 66
Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Ala
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser
100 105 110
Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu
115 120 125
Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln
130 135 140
Ser Val Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala
145 150 155 160
Pro Arg Leu Leu Ile Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro
165 170 175
Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
180 185 190
Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser
195 200 205
Ser Asn Trp Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
210 215 220
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg Thr Val Ala Ala Pro
225 230 235 240
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr
245 250 255
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys
260 265 270
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu
275 280 285
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser
290 295 300
Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala
305 310 315 320
Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe
325 330 335
Asn Arg Gly Glu Cys
340
<210> 67
<211> 566
<212> PRT
<213> 人工序列
<220>
<223> 抗PD1-VH-抗GITR-VH-huIGHG1(LALA-杵)
<400> 67
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser
20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
100 105 110
Ser Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly
115 120 125
Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser
130 135 140
Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
145 150 155 160
Val Ala Val Ile Trp Tyr Glu Gly Ser Asn Lys Tyr Tyr Ala Asp Ser
165 170 175
Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
180 185 190
Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
195 200 205
Cys Ala Arg Gly Gly Ser Met Val Arg Gly Asp Tyr Tyr Tyr Gly Met
210 215 220
Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr
225 230 235 240
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
245 250 255
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
260 265 270
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
275 280 285
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
290 295 300
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
305 310 315 320
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
325 330 335
Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
340 345 350
Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
355 360 365
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
370 375 380
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
385 390 395 400
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
405 410 415
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
420 425 430
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
435 440 445
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
450 455 460
Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys
465 470 475 480
Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
485 490 495
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
500 505 510
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
515 520 525
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
530 535 540
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
545 550 555 560
Leu Ser Leu Ser Pro Gly
565
<210> 68
<211> 710
<212> PRT
<213> 人工序列
<220>
<223> 抗OX40-VL-huIGKC-[PCS2]-<(G4S)6>-[PCS2]-抗OX40-VH-huIGHG1(LALA-臼-RF)
<400> 68
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Tyr Thr Ser Arg Leu Arg Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly His Thr Leu Pro Pro
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys His Arg Arg Arg Lys Arg Ser Val Asp Glu
210 215 220
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
225 230 235 240
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser His Arg
245 250 255
Arg Arg Lys Arg Ser Val Asp Glu Glu Val Gln Leu Val Gln Ser Gly
260 265 270
Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala
275 280 285
Ser Gly Tyr Thr Phe Thr Asp Ser Tyr Met Ser Trp Val Arg Gln Ala
290 295 300
Pro Gly Gln Gly Leu Glu Trp Ile Gly Asp Met Tyr Pro Asp Asn Gly
305 310 315 320
Asp Ser Ser Tyr Asn Gln Lys Phe Arg Glu Arg Val Thr Ile Thr Arg
325 330 335
Asp Thr Ser Thr Ser Thr Ala Tyr Leu Glu Leu Ser Ser Leu Arg Ser
340 345 350
Glu Asp Thr Ala Val Tyr Tyr Cys Val Leu Ala Pro Arg Trp Tyr Phe
355 360 365
Ser Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr
370 375 380
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
385 390 395 400
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
405 410 415
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
420 425 430
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
435 440 445
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
450 455 460
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
465 470 475 480
Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
485 490 495
Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
500 505 510
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
515 520 525
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
530 535 540
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
545 550 555 560
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
565 570 575
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
580 585 590
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
595 600 605
Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
610 615 620
Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp
625 630 635 640
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
645 650 655
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser
660 665 670
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
675 680 685
Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln Lys Ser
690 695 700
Leu Ser Leu Ser Pro Gly
705 710
<210> 69
<211> 710
<212> PRT
<213> 人工序列
<220>
<223> 抗OX40-VL-huIGKC-[PCS2a]-<(G4S)6>-[PCS2a]-抗OX40-VH-huIGHG1(LALA-臼-RF)
<400> 69
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Tyr Thr Ser Arg Leu Arg Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly His Thr Leu Pro Pro
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys His Arg Arg Gln Gln Arg Ser Val Asp Glu
210 215 220
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
225 230 235 240
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser His Arg
245 250 255
Arg Gln Gln Arg Ser Val Asp Glu Glu Val Gln Leu Val Gln Ser Gly
260 265 270
Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala
275 280 285
Ser Gly Tyr Thr Phe Thr Asp Ser Tyr Met Ser Trp Val Arg Gln Ala
290 295 300
Pro Gly Gln Gly Leu Glu Trp Ile Gly Asp Met Tyr Pro Asp Asn Gly
305 310 315 320
Asp Ser Ser Tyr Asn Gln Lys Phe Arg Glu Arg Val Thr Ile Thr Arg
325 330 335
Asp Thr Ser Thr Ser Thr Ala Tyr Leu Glu Leu Ser Ser Leu Arg Ser
340 345 350
Glu Asp Thr Ala Val Tyr Tyr Cys Val Leu Ala Pro Arg Trp Tyr Phe
355 360 365
Ser Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr
370 375 380
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
385 390 395 400
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
405 410 415
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
420 425 430
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
435 440 445
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
450 455 460
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
465 470 475 480
Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
485 490 495
Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
500 505 510
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
515 520 525
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
530 535 540
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
545 550 555 560
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
565 570 575
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
580 585 590
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
595 600 605
Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
610 615 620
Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp
625 630 635 640
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
645 650 655
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser
660 665 670
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
675 680 685
Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln Lys Ser
690 695 700
Leu Ser Leu Ser Pro Gly
705 710
<210> 70
<211> 465
<212> PRT
<213> 人工序列
<220>
<223> 抗PD1-VL-huIGKC-[PCS2]-<(G4S)3>-[PCS2]-抗4.1BB-VL-huIGKC
<400> 70
Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser Asn Trp Pro Arg
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys His Arg Arg Arg Lys Arg Ser Val Asp Glu
210 215 220
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser His
225 230 235 240
Arg Arg Arg Lys Arg Ser Val Asp Glu Glu Ile Val Leu Thr Gln Ser
245 250 255
Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys
260 265 270
Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys
275 280 285
Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Asp Ala Ser Asn Arg Ala
290 295 300
Thr Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
305 310 315 320
Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr
325 330 335
Cys Gln Gln Arg Ser Asn Trp Pro Pro Ala Leu Thr Phe Gly Gly Gly
340 345 350
Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile
355 360 365
Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val
370 375 380
Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys
385 390 395 400
Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu
405 410 415
Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu
420 425 430
Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr
435 440 445
His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu
450 455 460
Cys
465
<210> 71
<211> 686
<212> PRT
<213> 人工序列
<220>
<223> 抗PD1-VH-huIGH-G11-huIGH-H1-<(G4S)3>-抗4.1BB-VH-huIGHG1-(LALA)
<400> 71
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser
20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
100 105 110
Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser
115 120 125
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
130 135 140
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
145 150 155 160
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
165 170 175
Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln
180 185 190
Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
195 200 205
Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Gly Gly Gly
210 215 220
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu
225 230 235 240
Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu Thr Leu Ser Leu
245 250 255
Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr Tyr Trp Ser Trp
260 265 270
Ile Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Ile Gly Glu Ile Asn
275 280 285
His Gly Gly Tyr Val Thr Tyr Asn Pro Ser Leu Glu Ser Arg Val Thr
290 295 300
Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser
305 310 315 320
Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp Tyr Gly
325 330 335
Pro Gly Asn Tyr Asp Trp Tyr Phe Asp Leu Trp Gly Arg Gly Thr Leu
340 345 350
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
355 360 365
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
370 375 380
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
385 390 395 400
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
405 410 415
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
420 425 430
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
435 440 445
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His
450 455 460
Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val
465 470 475 480
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
485 490 495
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
500 505 510
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
515 520 525
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
530 535 540
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
545 550 555 560
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
565 570 575
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
580 585 590
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
595 600 605
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
610 615 620
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
625 630 635 640
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
645 650 655
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
660 665 670
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
675 680 685
<210> 72
<211> 465
<212> PRT
<213> 人工序列
<220>
<223> 抗PD1-VL-huIGKC-[PCS2a]-<(G4S)3>-[PCS2a]-抗4.1BB-VL-huIGKC
<400> 72
Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser Asn Trp Pro Arg
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys His Arg Arg Gln Gln Arg Ser Val Asp Glu
210 215 220
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser His
225 230 235 240
Arg Arg Gln Gln Arg Ser Val Asp Glu Glu Ile Val Leu Thr Gln Ser
245 250 255
Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys
260 265 270
Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys
275 280 285
Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Asp Ala Ser Asn Arg Ala
290 295 300
Thr Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
305 310 315 320
Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr
325 330 335
Cys Gln Gln Arg Ser Asn Trp Pro Pro Ala Leu Thr Phe Gly Gly Gly
340 345 350
Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile
355 360 365
Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val
370 375 380
Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys
385 390 395 400
Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu
405 410 415
Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu
420 425 430
Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr
435 440 445
His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu
450 455 460
Cys
465
<210> 73
<211> 463
<212> PRT
<213> 人工序列
<220>
<223> 抗PD1-VL-huIGKC-[PCS2]-<(G4S)3>-[PCS2]-抗OX40-VL-huIGKC
<400> 73
Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser Asn Trp Pro Arg
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys His Arg Arg Arg Lys Arg Ser Val Asp Glu
210 215 220
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser His
225 230 235 240
Arg Arg Arg Lys Arg Ser Val Asp Glu Asp Ile Gln Met Thr Gln Ser
245 250 255
Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys
260 265 270
Arg Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn Trp Tyr Gln Gln Lys
275 280 285
Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu Arg
290 295 300
Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
305 310 315 320
Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr
325 330 335
Cys Gln Gln Gly His Thr Leu Pro Pro Thr Phe Gly Gln Gly Thr Lys
340 345 350
Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro
355 360 365
Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu
370 375 380
Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp
385 390 395 400
Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp
405 410 415
Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys
420 425 430
Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln
435 440 445
Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
450 455 460
<210> 74
<211> 682
<212> PRT
<213> 人工序列
<220>
<223> 抗PD1-VH-huIGH-G11-huIGH-H1-<(G4S)3>-抗OX40-VH-huIGHG1-(LALA)
<400> 74
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser
20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
100 105 110
Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser
115 120 125
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
130 135 140
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
145 150 155 160
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
165 170 175
Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln
180 185 190
Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
195 200 205
Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Gly Gly Gly
210 215 220
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu
225 230 235 240
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val
245 250 255
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Ser Tyr Met Ser Trp
260 265 270
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile Gly Asp Met Tyr
275 280 285
Pro Asp Asn Gly Asp Ser Ser Tyr Asn Gln Lys Phe Arg Glu Arg Val
290 295 300
Thr Ile Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr Leu Glu Leu Ser
305 310 315 320
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Val Leu Ala Pro
325 330 335
Arg Trp Tyr Phe Ser Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser
340 345 350
Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser
355 360 365
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
370 375 380
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
385 390 395 400
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
405 410 415
Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln
420 425 430
Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
435 440 445
Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro
450 455 460
Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro
465 470 475 480
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
485 490 495
Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
500 505 510
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
515 520 525
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
530 535 540
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
545 550 555 560
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
565 570 575
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
580 585 590
Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
595 600 605
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
610 615 620
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
625 630 635 640
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
645 650 655
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
660 665 670
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
675 680
<210> 75
<211> 463
<212> PRT
<213> 人工序列
<220>
<223> 抗PD1-VL-huIGKC-[PCS2a]-<(G4S)3>-[PCS2a]-抗OX40-VL-huIGKC
<400> 75
Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser Asn Trp Pro Arg
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys His Arg Arg Gln Gln Arg Ser Val Asp Glu
210 215 220
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser His
225 230 235 240
Arg Arg Gln Gln Arg Ser Val Asp Glu Asp Ile Gln Met Thr Gln Ser
245 250 255
Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys
260 265 270
Arg Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn Trp Tyr Gln Gln Lys
275 280 285
Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu Arg
290 295 300
Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
305 310 315 320
Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr
325 330 335
Cys Gln Gln Gly His Thr Leu Pro Pro Thr Phe Gly Gln Gly Thr Lys
340 345 350
Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro
355 360 365
Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu
370 375 380
Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp
385 390 395 400
Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp
405 410 415
Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys
420 425 430
Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln
435 440 445
Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
450 455 460

Claims (15)

1.一种蛋白质，其包含至少一个具有下式的多肽链
PP1-PCL-PP2，
其中
PP1是第一多肽，
PP2是第二多肽，并且
PCL是蛋白酶可切割接头，所述蛋白酶可切割接头包含至少一个蛋白酶切割位点，所述至少一个蛋白酶切割位点包含氨基酸序列HRRX1X2RSVDE(SEQ ID NO:42)，其中X1和X2独立地选自氨基酸，优选天然存在的氨基酸。
2.根据权利要求1所述的蛋白质，其中所述至少一个蛋白酶切割位点包含氨基酸序列HRRRKRSVDE(SEQ ID NO:43)或氨基酸序列HRRQQRSVDE(SEQ ID NO:44)。
3.根据权利要求1或2所述的蛋白质，其中所述接头的蛋白酶切割导致所述蛋白质的活性的改变，其中优选地，所述活性的改变是与至少一种抗原的结合或增加的结合。
4.根据权利要求1至3中任一项所述的蛋白质，其中所述蛋白酶可切割的接头包含两个蛋白酶切割位点，其中这两个蛋白酶切割位点分别位于所述蛋白酶可切割接头的N末端和C末端，其中这两个蛋白酶切割位点可以相同或不同。
5.根据权利要求1至4中任一项所述的蛋白质，其中PP1包含至少一个免疫球蛋白恒定区和/或至少一个免疫球蛋白可变区，和/或其中PP2包含至少一个免疫球蛋白恒定区和/或至少一个免疫球蛋白可变区，其中优选地，PP1包含免疫球蛋白恒定区并且PP2包含免疫球蛋白可变区，
PP1包含免疫球蛋白可变区并且PP2包含免疫球蛋白恒定区，
PP1包含免疫球蛋白恒定区并且PP2包含免疫球蛋白恒定区，或
PP1包含免疫球蛋白可变区并且PP2包含免疫球蛋白可变区。
6.根据权利要求1至5中任一项所述的蛋白质，其中所述蛋白质是抗体或抗体衍生物，其中优选地，所述蛋白质是单链抗体，优选多特异性单链抗体。
7.根据权利要求1至6中任一项所述的蛋白质，其包含具有选自以下的式的多肽链：
(a)VL-CL-PCL-VH-CH1-铰链-CH2-CH3，
其中
VL是免疫球蛋白轻链可变区；
CL是免疫球蛋白轻链恒定区；
VH是免疫球蛋白重链可变区；
CH1是免疫球蛋白CH1重链恒定区；
铰链是免疫球蛋白铰链区；
CH2是免疫球蛋白CH2重链恒定区；
CH3是免疫球蛋白CH3重链恒定区；并且
PCL是所述蛋白酶可切割接头；
(b)VLa-CLa-PCL-VLb-CLb，
其中
VLa是第一免疫球蛋白轻链可变区；
VLb是第二免疫球蛋白轻链可变区；
CLa是第一免疫球蛋白轻链恒定区；
CLb是第二免疫球蛋白轻链恒定区；并且
PCL是所述蛋白酶可切割接头；以及
(c)VLa-CLa-PCL-VLb-CLb-L-铰链-CH2-CH3
其中
VLa是第一免疫球蛋白轻链可变区；
VLb是第二免疫球蛋白轻链可变区；
CLa是第一免疫球蛋白轻链恒定区；
CLb是第二免疫球蛋白轻链恒定区；
L是任选的接头；
铰链是免疫球蛋白铰链区；
CH2是免疫球蛋白CH2重链恒定区；
CH3是免疫球蛋白CH3重链恒定区；并且
PCL是所述蛋白酶可切割接头。
8.一种核酸或核酸组，其编码根据权利要求1至7中任一项所述的蛋白质。
9.一种载体或载体组，其包含根据权利要求8所述的核酸或核酸组。
10.一种宿主细胞，其包含根据权利要求1至7中任一项所述的蛋白质、根据权利要求8所述的核酸或核酸组、或根据权利要求9所述的载体或载体组，其中优选地，所述宿主细胞是哺乳动物细胞。
11.一种生产蛋白质的方法，其包括以下步骤：
i)培养根据权利要求10所述的宿主细胞；以及
ii)将所述蛋白质与所述宿主细胞分离。
12.一种通过根据权利要求11所述的方法可获得的蛋白质。
13.一种通过弗林蛋白酶和/或弗林蛋白酶样蛋白酶切割根据权利要求1至7中任一项所述的蛋白质可获得的蛋白质。
14.根据权利要求1至7、12或13中任一项所述的蛋白质，其中所述蛋白质是治疗活性蛋白质。
15.根据权利要求14所述的蛋白质，其用于疗法。
CN202180018397.5A 2020-03-05 2021-03-05 蛋白酶加工的分子 Pending CN115210265A (zh)

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