CN113754775A - 一种抗pd-l1和her2的双特异性抗体 - Google Patents

一种抗pd-l1和her2的双特异性抗体 Download PDF

Info

Publication number
CN113754775A
CN113754775A CN202010487636.9A CN202010487636A CN113754775A CN 113754775 A CN113754775 A CN 113754775A CN 202010487636 A CN202010487636 A CN 202010487636A CN 113754775 A CN113754775 A CN 113754775A
Authority
CN
China
Prior art keywords
ser
gly
val
thr
leu
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202010487636.9A
Other languages
English (en)
Inventor
朱祯平
黄浩旻
张学赛
李晴柔
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Sunshine Guojian Pharmaceutical Shanghai Co Ltd
Original Assignee
Sunshine Guojian Pharmaceutical Shanghai Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Sunshine Guojian Pharmaceutical Shanghai Co Ltd filed Critical Sunshine Guojian Pharmaceutical Shanghai Co Ltd
Priority to CN202010487636.9A priority Critical patent/CN113754775A/zh
Priority to CN202180032220.0A priority patent/CN115605513B/zh
Priority to PCT/CN2021/095189 priority patent/WO2021244328A1/zh
Priority to TW110119871A priority patent/TW202146457A/zh
Publication of CN113754775A publication Critical patent/CN113754775A/zh
Pending legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL

Abstract

本发明提供了一种抗PD‑L1和HER2的双特异性抗体。本发明的双特异性抗体能够同时特异性结合PD‑L1和HER2两个靶点，具有与单抗相似甚至更优的生物学活性。

Description

一种抗PD-L1和HER2的双特异性抗体
技术领域
本发明涉及抗体领域，更具体地，本发明公开了一种抗PD-L1和HER2的双特异性抗体。
背景技术
人程序性细胞死亡受体-1(PD-1)是一种有288个氨基酸的I型膜蛋白，是已知的主要免疫检查点(Immune Checkpoint)之一(Blank et al,2005,Cancer Immunotherapy,54:307-314)。PD-1表达在已经激活的T淋巴细胞，它与配体PD-L1(程序性细胞死亡受体-配体1，programmed cell death-Ligand 1)和PD-L2(程序性细胞死亡受体-配体2，programmedcell death-Ligand 2)结合可以抑制T淋巴细胞的活性及相关的体内细胞免疫反应。PD-L2主要表达在巨噬细胞和树突状细胞，而PD-L1则广泛表达于B、T淋巴细胞及外周细胞如微血管上皮细胞，肺、肝、心等组织细胞中。大量研究表明，PD-1和PD-L1的相互作用不但是维持体内免疫***平衡所必须，也是导致PD-L1表达阳性肿瘤细胞规避免疫监视的主要机制和原因。通过阻断癌细胞对PD-1/PD-L1信号通路的负调控，激活免疫***，能够促进T细胞相关的肿瘤特异性细胞免疫反应，从而打开了一扇新的肿瘤治疗方法的大门--肿瘤免疫疗法。
PD-1(由基因Pdcd1编码)为与CD28和CTLA-4有关的免疫球蛋白超家族成员。研究成果显示，当PD-1与其配体(PD-L1和/或PD-L2)结合时会负调节抗原受体信号转导。目前已弄清鼠PD-1结构以及小鼠PD-1与人PD-L1的共结晶结构(Zhang，X.等，Immunity 20：337-347(2004)；Lin等，Proc.Natl.Acad.Sci.USA105：3011-6(2008))。PD-1及类似的家族成员为I型跨膜糖蛋白，其含有负责配体结合的Ig可变型(V-型)结构域和负责结合信号转导分子的胞质尾区。PD-1胞质尾区含有两个基于酪氨酸的信号转导模体ITIM(免疫受体酪氨酸抑制作用模体)和ITSM(免疫受体酪氨酸转换作用模体)。
PD-1在肿瘤的免疫逃避机制中起到了重要的作用。肿瘤免疫疗法，即利用人体自身的免疫***抵御癌症，是一种突破性的肿瘤治疗方法，但是肿瘤微环境可保护肿瘤细胞免受有效的免疫破坏，因此如何打破肿瘤微环境成为抗肿瘤研究的重点。现有研究成果已确定了PD-1在肿瘤微环境中的作用：PD-L1在许多小鼠和人肿瘤中表达(并在大多数PD-L1阴性肿瘤细胞系中可由IFN-γ诱导)，并被推定为介导肿瘤免疫逃避的重要靶点(Iwai Y.等，Proc.Natl.Acad.Sci.U.S.A.99：12293-12297(2002)；Strome S.E.等，Cancer Res.，63：6501-6505(2003))。通过免疫组织化学评估活组织检查，已经在人的很多原发性肿瘤中发现PD-1(在肿瘤浸润淋巴细胞上)和/或PD-L1在肿瘤细胞上的表达。这样的组织包括肺癌、肝癌、卵巢癌、***、皮肤癌、结肠癌、神经胶质瘤、膀胱癌、乳腺癌、肾癌、食道癌、胃癌、口腔鳞状细胞癌、尿道上皮细胞癌和胰腺癌以及头颈肿瘤等。由此可见，阻断PD-1/PD-L1的相互作用可以提高肿瘤特异性T细胞的免疫活性，有助于免疫***清除肿瘤细胞，因此PD-L1成为开发肿瘤免疫治疗药物的热门靶点。
HER2/neu(人表皮生长因子受体2)，又称erbB2，具有酪氨酸蛋白激酶活性，是人表皮生长因子受体家族成员之一，只在成年人的少数正常组织中呈低水平表达。但研究表明，HER2在多种肿瘤中过表达，如在约30％的乳腺癌患者和16％的胃癌患者中均存在过度表达情况，HER2在肿瘤中的过表达可以显著促进肿瘤血管的新生、肿瘤的生长，并增强肿瘤的侵袭和转移能力，是这类患者预后较差的重要指征。因此，早在1998年，第一个靶向于HER2的单克隆抗体药物Herceptin(赫赛汀，Trastuzumab/曲妥珠单抗，Genentech/Roche)被FDA批准上市，并用于HER2过表达的乳腺癌和胃癌的治疗。
双特异性抗体是指能同时特异性结合两种抗原或两种表位的抗体分子。根据对称性，双特异性抗体可以分为结构对称的和不对称的分子。根据结合位点的多少，双特异性抗体可以分为二价、三价、四价和多价分子。双特异性抗体正在逐步成为一类新的治疗性抗体，可以用于治疗各种炎性疾病、癌症和其它疾病。
发明内容
本发明提供了一种抗PD-L1和HER2的双特异性抗体。
因此，本发明的第一个目的在于提供一种抗PD-L1和HER2的双特异性抗体。
本发明的第二个目的在于提供一种编码所述的双特异性抗体的分离的核苷酸。
本发明的第三个目的在于提供一种包含所述的核苷酸的表达载体。
本发明的第四个目的在于提供一种包含所述的表达载体的宿主细胞。
本发明的第五个目的在于提供所述的双特异性抗体的制备方法。
本发明的第六个目的在于提供包含所述的双特异性抗体的药物组合物。
本发明的第七个目的在于提供所述的双特异性抗体或所述的药物组合物在制备治疗癌症的药物中的用途。
本发明的第八个目的在于提供所述的双特异性抗体或所述的药物组合物用于治疗癌症的方法。
为了达到上述目的，本发明提供了以下技术方案：
本发明的第一个方面提供了一种抗PD-L1和HER2的双特异性抗体，包含两条多肽链和两条轻链，其中：
(a)所述的多肽链从N末端至C末端包含VH-PDL1-CH1-CH2-CH3-L1-VH-HER2-L2-VL-HER2或VH-HER2-L2-VL-HER2-L1-VH-PDL1-CH1-CH2-CH3，所述的轻链从N末端至C末端包含VL-PDL1-CL；或
(b)所述的多肽链从N末端至C末端包含VH-HER2-CH1-CH2-CH3-L1-VH-PDL1-L2-VL-PDL1或VH-PDL1-L2-VL-PDL1-L1-VH-HER2-CH1-CH2-CH3，所述的轻链从N末端至C末端包含VL-HER2-CL；
其中，所述的VH-PDL1为结合PD-L1的重链可变区，所述的VL-PDL1为结合PD-L1的轻链可变区，所述的VH-HER2为结合HER2的重链可变区，所述的VL-HER2为结合HER2的轻链可变区，所述的L1和L2为(G4S)x，x为3、4、5或6，所述的CH1-CH2-CH3为重链恒定区，所述的CL为轻链恒定区，所述的VH-PDL1与所述的VL-PDL1形成特异性结合PD-L1的抗原结合位点，所述的VH-HER2与所述的VL-HER2形成特异性结合HER2的抗原结合位点。
根据本发明的优选实施例，所述的L1为(G4S)3，所述的L2为(G4S)4。
根据本发明的优选实施例，所述的VH-PDL1包含氨基酸序列如SEQ ID NO：1-3所示的重链CDR，所述的VL-PDL1包含氨基酸序列如SEQ ID NO：4-6所示的轻链CDR，所述的VH-HER2包含氨基酸序列如SEQ ID NO：7-9或SEQ ID NO：13-15所示的重链CDR，所述的VL-HER2包含氨基酸序列如SEQ ID NO：10-12或SEQ ID NO：16-18所示的轻链CDR。
根据本发明的优选实施例，所述的VH-PDL1具有如SEQ ID NO：19所示的氨基酸序列，所述的VL-PDL1具有如SEQ ID NO：20所示的氨基酸序列，所述的VH-HER2具有如SEQ IDNO：21或SEQ ID NO：23所示的氨基酸序列，所述的VL-HER2具有如SEQ ID NO：22或SEQ IDNO：24所示的氨基酸序列。
根据本发明的优选实施例，所述的多肽链具有如SEQ ID NO：31或SEQ ID NO：33所示的氨基酸序列，所述的轻链具有如SEQ ID NO：26所示的氨基酸序列；或所述的多肽链具有如SEQ ID NO：32或SEQ ID NO：34所示的氨基酸序列，所述的轻链具有如SEQ ID NO：28所示的氨基酸序列；或所述的多肽链具有如SEQ ID NO：35或SEQ ID NO：36所示的氨基酸序列，所述的轻链具有如SEQ ID NO：30所示的氨基酸序列。
根据本发明，所述的重链恒定区包括IgG1、IgG2、IgG3或IgG4重链恒定区，所述的轻链恒定区包括κ或λ轻链恒定区。
本发明的第二个方面提供了一种分离的核苷酸，所述的核苷酸编码所述的双特异性抗体。
本发明的第三个方面提供了一种表达载体，所述的表达载体含有如上所述的核苷酸。
本发明的第四个方面提供了一种宿主细胞，所述的宿主细胞含有如上所述的表达载体。
本发明的第五个方面提供了所述的双特异性抗体的制备方法，所述方法包含以下步骤：
(a)在表达条件下，培养如上所述的宿主细胞，从而表达所述的双特异性抗体；
(b)分离并纯化(a)所述的双特异性抗体。
本发明的第六个方面提供了一种药物组合物，所述药物组合物含有如上所述的双特异性抗体和药学上可接受的载体。
本发明的第七个方面提供了如上所述的双特异性抗体或如上所述的药物组合物在制备治疗癌症的药物中的用途。
根据本发明，所述癌症选自由以下组成的组：黑素瘤、肾癌、***癌、胰腺癌、乳腺癌、结肠癌、肺癌、食道癌、头颈鳞状细胞癌、肝癌、卵巢癌、***、甲状腺癌、成胶质细胞瘤、神经胶质瘤、白血病、淋巴瘤及其它赘生性恶性疾病等。
本发明的第八个方面提供了一种治疗癌症的方法，包括向有需要的受试者施用如上所述的双特异性抗体或如上所述的药物组合物。
根据本发明，所述癌症选自由以下组成的组：黑素瘤、肾癌、***癌、胰腺癌、乳腺癌、结肠癌、肺癌、食道癌、头颈鳞状细胞癌、肝癌、卵巢癌、***、甲状腺癌、成胶质细胞瘤、神经胶质瘤、白血病、淋巴瘤及其它赘生性恶性疾病等。
有益效果：本发明提供了一种抗PD-L1和HER2的双特异性抗体，体外和体内实验结果显示其能够同时特异性结合PD-L1和HER2两个靶点，具有与单抗相似甚至更优的生物学活性。
附图说明
图1为ELISA测定双特异性抗体对PDL1-ECD-his的亲和力结果。
图2为ELISA测定双特异性抗体对HER2-ECD-his的亲和力结果，其中，图2A为M8与607组合的双特异性抗体，图2B为M8和612组合的双特异性抗体。
图3为双特异性抗体竞争性抑制PD-1与PD-L1的结合结果。
图4为双特异性抗体抑制PD-1和PD-L1高表达细胞株的结合活性测定结果。
图5为双特异性抗体抑制BT474细胞增殖的活性测定结果。
图6为双特异性抗体抑制N87细胞增殖的活性测定结果。
图7为双特异性抗体抑制BT474细胞增殖的活性测定结果。
图8为双特异性抗体抑制N87细胞增殖的活性测定结果。
具体实施方式
本发明中，术语“抗体(Antibody，缩写Ab)”和“免疫球蛋白G(Immunoglobulin G，缩写IgG)”是有相同结构特征的约150000道尔顿的异四聚糖蛋白，其由两条相同的轻链(L)和两条相同的重链(H)组成。每条轻链通过一个共价二硫键与重链相连，而不同免疫球蛋白同种型(isotype)的重链间的二硫键数目不同。每条重链和轻链也有规则间隔的链内二硫键。每条重链的一端有可变区(VH)，其后是恒定区，重链恒定区由三个结构域CH1、CH2、以及CH3构成。每条轻链的一端有可变区(VL)，另一端有恒定区，轻链恒定区包括一个结构域CL；轻链的恒定区与重链恒定区的CH1结构域配对，轻链的可变区与重链的可变区配对。恒定区不直接参与抗体与抗原的结合，但是它们表现出不同的效应功能，例如参与抗体依赖的细胞介导的细胞毒性作用(ADCC，antibody-dependent cell-mediated cytotoxicity)等。重链恒定区包括IgG1、IgG2、IgG3、IgG4亚型；轻链恒定区包括κ(Kappa)或λ(Lambda)。抗体的重链和轻链通过重链的CH1结构域和轻链的CL结构域之间的二硫键共价连接在一起，抗体的两条重链通过铰链区之间形成的多肽间二硫键共价连接在一起。
本发明中，术语“双特异性抗体(双抗)”是指能同时特异性结合两种抗原(靶点)或两种表位的抗体分子。
本发明中，术语“单克隆抗体(单抗)”指从一类基本均一的群体获得的抗体，即该群体中包含的单个抗体是相同的，除少数可能存在的天然发生的突变外。单克隆抗体高特异性地针对单个抗原位点。而且，与常规多克隆抗体制剂(通常是具有针对不同抗原决定簇的不同抗体的混合物)不同，各单克隆抗体是针对抗原上的单个决定簇。除了它们的特异性外，单克隆抗体的好处还在于它们可以通过杂交瘤培养来合成，不会被其它免疫球蛋白污染。修饰语“单克隆”表示了抗体的特性，是从基本均一的抗体群中获得的，这不应被解释成需要用任何特殊方法来生产抗体。
本发明中，术语“Fab”和“Fc”是指木瓜蛋白酶可将抗体裂解为两个完全相同的Fab段和一个Fc段。Fab段由抗体的重链的VH和CH1以及轻链的VL和CL结构域组成。Fc段即可结晶片段(fragment crystallizable，Fc)，由抗体的CH2和CH3结构域组成。Fc段无抗原结合活性，是抗体与效应分子或细胞相互作用的部位。
本发明中，术语“scFv”为单链抗体(single chain antibody fragment，scFv)，由抗体重链可变区和轻链可变区通过15～25个氨基酸的短肽(linker)连接而成。
本发明中，术语“可变”表示抗体中可变区的某些部分在序列上有所不同，它形成各种特定抗体对其特定抗原的结合和特异性。然而，可变性并不均匀地分布在整个抗体可变区中。它集中于重链可变区和轻链可变区中称为互补决定区(complementarity-determining region，CDR)或超变区中的三个片段中。可变区中较保守的部分称为框架区(frame region，FR)。天然重链和轻链的可变区中各自包含四个FR区，它们大致上呈β-折叠构型，由形成连接环的三个CDR相连，在某些情况下可形成部分β折叠结构。每条链中的CDR通过FR区紧密地靠在一起并与另一链的CDR一起形成了抗体的抗原结合部位(参见Kabat等，NIH Publ.No.91-3242，卷I，647-669页(1991))。
本发明中，术语“抗”、“结合”、“特异性结合”是指两分子间的非随机的结合反应，如抗体和其所针对的抗原之间的反应。通常，抗体以小于大约10-7M，例如小于大约10-8M、10-9M、10-10M、10-11M或更小的平衡解离常数(KD)结合该抗原。本发明中，术语“KD”是指特定抗体-抗原相互作用的平衡解离常数，其用于描述抗体与抗原之间的结合亲和力。平衡解离常数越小，抗体-抗原结合越紧密，抗体与抗原之间的亲和力越高。例如，使用表面等离子体共振术(Surface Plasmon Resonance，缩写SPR)在BIACORE仪中测定抗体与抗原的结合亲和力或使用ELISA测定抗体与抗原结合的相对亲和力。
本发明中，术语“表位”是指与抗体特异性结合的多肽决定簇。本发明的表位是抗原中被抗体结合的区域。
本发明中，术语“表达载体”可以为pTT5，pSECtag系列，pCGS3系列，pcDNA系列载体等，以及其它用于哺乳动物表达***的载体等，表达载体中包括连接有合适的转录和翻译调节序列的融合DNA序列。
本发明中，术语“宿主细胞”是指适用于表达上述表达载体的细胞，可以是真核细胞，如哺乳动物或昆虫宿主细胞培养***均可用于本发明的融合蛋白的表达，CHO(中国仓鼠卵巢，Chinese Hamster Ovary)，HEK293，COS，BHK以及上述细胞的衍生细胞均可适用于本发明。
本发明中，术语“药物组合物”是指本发明的双特异性抗体可以和药学上可以接受的载体一起组成药物制剂组合物从而更稳定地发挥疗效，这些制剂可以保证本发明公开的双特异性抗体的氨基酸核心序列的构象完整性，同时还保护蛋白质的多官能团防止其降解(包括但不限于凝聚、脱氨或氧化)。
以下实施例、实验例是对本发明进行进一步的说明，不应理解为对本发明的限制。实施例不包括对传统方法的详细描述，如那些用于构建载体和质粒的方法，将编码蛋白的基因***到这样的载体和质粒的方法或将质粒引入宿主细胞的方法。这样的方法对于本领域中具有普通技术的人员是众所周知的，并且在许多出版物中都有所描述，包括Sambrook,J.,Fritsch,E.F.and Maniais，T.(1989)Molecular Cloning：A Laboratory Manual，2ndedition，Cold spring Harbor Laboratory Press。
实施例1抗PD-L1和HER2双特异性抗体的构建
本发明的双特异性抗体的结构通式如下：
1)Ab-L1-scFv；
2)scFv-L1-Ab；
其中Ab为抗体，包含两条重链和两条轻链；
scFv为单链抗体，是由另一个抗体的重链可变区和轻链可变区通过连接序列L2连接而成，并通过连接序列L1分别连接在Ab的两条重链的N末端或C末端；
L1和L2为连接序列(G4S)x，x可以为3、4、5或6。
本发明使用抗人PD-L1抗体M8(序列来源于PCT/CN2020/090442)、抗人HER2抗体607(根据曲妥珠单抗序列制备，序列来源于US5821337)和抗人HER2抗体612(序列来源于WO2020/025013A1)的组合构建双特异性抗体，具体结构如表1所示。
表1、本发明的双特异性抗体的结构
Figure BDA0002519816030000071
Figure BDA0002519816030000081
其中，PDL1-HC代表M8的重链，PDL1-LC代表M8的轻链，607-VH代表607的重链可变区，607-VL代表607的轻链可变区，其余类似。
本发明构建的双特异性抗体相关的序列信息如表2所示，其中，CDR根据Kabat规则编码。
表2、本发明的抗体的序列信息
Figure BDA0002519816030000082
Figure BDA0002519816030000091
通过基因合成及常规的分子克隆方法，通过EcoRI和HindIII两个酶切位点构建到pTT5表达载体(购自NRC biotechnology Research Institute)，获得各双特异性抗体及其对应的单克隆抗体的重链和轻链的表达载体，将上述各重链序列表达载体与其对应的轻链表达载体共转染HEK293F细胞(购自Thermo Fisher，货号A14527)，表达并纯化获得各双特异性抗体PDL1H-607scFv、607H-PDL1scFv、607scFv-PDL1H、PDL1scFv-607H、PDL1scFv-612H和612H-PDL1scFv，以及对应的单抗M8、607、612。
实施例2 ELISA法测定双特异性抗体靶向HER2和PD-L1的亲和力
PDL1-ECD-his(NCBI登记号为NP_054862.1)和HER2-ECD-his蛋白(NCBI登记号为NP_004439.2)制备方法如下：根据NCBI提供的序列分别合成PD-L1和HER2胞外域基因并在其N端加上信号肽序列，C末端加上6×His标签，通过EcoRI和HindIII两个酶切位点分别构建到pTT5表达载体中，转染HEK-293F细胞表达并纯化获得。
将PDL1-ECD-his或HER2-ECD-his蛋白稀释并分别包被96孔ELISA板，0.05μg/孔，4℃包被过夜，PBST(PBS含0.05％Tween20)洗涤3次，用PBS配制2％BSA，200μL/孔，于室温封闭2h，PBST洗涤2次后，用PBST配制的1％BSA将PDL1H-607scFv、607H-PDL1scFv、607scFv-PDL1H、PDL1scFv-607H、PDL1scFv-612H、612H-PDL1scFv和M8稀释至不同浓度并加至ELISA孔，100μL/孔，每个浓度设置3个复孔，室温孵育1h，PBST洗涤3次，用PBST配制的1％BSA按照适当比例稀释二抗(HRP-anti human IgG Fc，购自Millipore公司，货号AP101P)，加入ELISA孔，100μL/孔，室温孵育1h，PBST洗涤3次后添加TMB显色液，100μL/孔，显色至预期颜色，用2M H2SO4终止显色反应，50μL/孔，使各反应液振荡均匀并于酶标仪测定OD450nm，分析数据，计算EC50。
实验结果分别如图1和图2所示，各双特异性抗体对PDL1-ECD-his和HER2-ECD-his的亲和力(EC50)分别如表3和表4所示，可见各双特异性抗体与PDL1-ECD-his的亲和力与M8相当，均在同一个水平，PDL1scFv-607H稍优。而对HER2-ECD-his的亲和力，在607与M8的双特异性抗体组合中，则以607scFv-PDL1H和PDL1H-607scFv更优，与607单抗的亲和力相当，PDL1scFv-607H和607H-PDL1scFv则相对偏弱；在612与M8的双特异性抗体组合中，PDL1scFv-612H和612H-PDL1scFv对HER2-ECD-his的亲和力相当，均稍弱于612单抗。
表3、各双特异抗体对PDL1-ECD-his的EC50
Figure BDA0002519816030000101
表4、各双特异抗体对HER2-ECD-his的EC50
Figure BDA0002519816030000102
实施例3双特异性抗体竞争性抑制PD-1与PD-L1结合活力测定
将PDL1-ECD-Fc蛋白(制备方法同上，只是将C端标签换成人Fc序列)稀释并包被96孔ELISA板，0.2μg/孔，4℃包被过夜，PBST(PBS含0.05％Tween20)洗涤3次，用PBS配制2％BSA，200μL/孔，于室温封闭2h，PBST洗涤2次后，用含1％BSA的PBST配制的500ng/ml生物素标记的PD1-Fc(即PD1-Fc-biotin，NCBI登记号为NP_005009.2，根据NCBI上登记信息获取PD-1的胞外域基因，克隆及制备方法同PDL1-ECD-Fc)溶液将PDL1H-607scFv、607H-PDL1scFv、607scFv-PDL1H、PDL1scFv-607H、PDL1scFv-612H、612H-PDL1scFv和M8稀释至不同浓度并加至ELISA孔，100μL/孔，每个浓度设置3个复孔，室温孵育1h，PBST洗涤3次，用PBST配制的1％BSA按照适当比例稀释二抗(HRP标记的streptavidin，购自Sigma，货号S4672-5MG)，加入ELISA孔，100μL/孔，室温孵育1h，PBST洗涤3次后添加TMB显色液，100μL/孔，显色至预期颜色，用2M H2SO4终止显色反应，50μL/孔，使各反应液振荡均匀并于酶标仪测定OD450nm，分析数据，计算EC50。
实验结果如图3所示，各双特异性抗体竞争性抑制PD-1与PD-L1结合的活力(IC50)如表5所示，可见各双特异性抗体竞争抑制PD-1与PD-L1活性相当。
表5、各双特异抗体竞争性抑制PD-1与PD-L1结合的IC50
Figure BDA0002519816030000111
实施例4双特异性抗体抑制PD-1和PD-L1高表达细胞株的结合活性
PD-1高表达细胞系PD-1Effector Cells和PD-L1高表达细胞系PD-L1 aAPC/CHO-K1 Cells均购自Promega公司(货号：J1252)。
1)将处于对数生长期的PDL1-aAPC/CHO-K1细胞用胰酶消化，离心，计数，用10％FBS Ham/F12培养基(购自Thermo Fisher，货号11765054)重悬，按照100μL/孔，每孔铺40000个细胞，接种至白板透明底96孔板中，于CO2细胞培养箱37℃生长过夜。
2)用含1％FBS的1640培养基(购自Thermo Fisher，货号61870036)分别配制最高浓度为200nM的M8、607H-PDL1scFv、PDL1scFv-607H、PDL1H-607scFv、607scFv-PDL1H、PDL1scFv-612H、612H-PDL1scFv，并按照3倍梯度稀释(工作浓度最高剂量为100nM)，同时以1％FBS 1640培养基稀释PD-1Effector Cells至1.25×106个/mL。
3)弃净上述培养有PDL1-aAPC/CHO-K1细胞的96孔板上清，加入40μL稀释好的PD-1Effector Cell悬液以及40μL稀释好的抗体，轻拍孔板摇匀，于37℃细胞培养箱继续培养6h。
4)按照80μL/孔加入已恢复至室温的Bio-Glo Luciferase Assay Reagent(购自Promega，货号：G7941)，室温孵育10min。
5)于多功能酶标仪中测定其发光强度，通过GraphPad Prism 6分析数据。
实验结果如图4所示，各双特异性抗体抑制PD-1和PD-L1高表达细胞株的结合活性的IC50如表6所示，可见PDL1H-607scFv、607scFv-PDL1H活性相对较优，PDL1scFv-612H和PDL1scFv-607H次之。
表6、各双特异抗体抑制PD-1和PD-L1高表达细胞株的结合的IC50
Figure BDA0002519816030000121
实施例5双特异性抗体对HER2高表达细胞株细胞增殖抑制活性测定
1)将处于对数生长期的BT474和N87细胞用胰酶消化，计数，用完全培养基，即含10％FBS的1640培养基(购自Thermo Fisher，货号61870036)重悬，按照150μL/孔，分别接种至96孔细胞培养板中，乳腺癌细胞BT474(购自中国科学院细胞库，目录号：TCHu143)每孔铺10000个，胃癌细胞N87(购自中国科学院细胞库，目录号：SCSP-534)每孔铺8000个，于CO2细胞培养箱37℃培养过夜。
2)用完全培养基按照3倍梯度稀释待测抗体，各组均连续稀释9梯度，各抗体最高浓度为400nM(最终工作浓度为100nM)，加入50μL上述抗体至96孔细胞培养板，每孔终体积为200μL，设置未给药组作为阴性对照，于37℃细胞培养箱继续培养5d，每个浓度平行做2个复孔。
3)弃净细胞培养上清液，按照100μL/孔加入CCK8反应液(以完全培养基按1：10比例稀释，购自Dojindo，货号CK04)，于37℃孵育。
4)待细胞培养孔颜色显色至预期深浅，于多功能酶标仪中在450nm波长处测定其吸光度，通过GraphPad Prism 6分析数据。
5)按照下述公式计算细胞存活率及生长抑制率：
存活率＝(OD给药-OD空白)/(OD对照-OD空白)×100％。生长抑制率＝1-存活率。
实验结果分别如图5、图6、图7和图8所示，各双特异性抗体对HER2高表达细胞株BT474和N87增殖抑制的IC50分别如表7、表8、表9、表10所示，可见，对于607和M8的双抗组合，PDL1scFv-607H、607H-PDL1scFv对BT474细胞增殖的抑制活性更优；对N87细胞增殖抑制活性，PDL1H-607scFv活性最弱，虽然PDL1scFv-607H、607H-PDL1scFv的IC50相当，但在高浓度下PDL1scFv-607H对N87细胞的最大杀伤效应最强。对于612和M8的双抗组合，则PDL1scFv-612H和612H-PDL1scFv对细胞增殖抑制活性相当。
表7、双特异性抗体抑制BT474细胞增殖的IC50
Figure BDA0002519816030000131
表8、双特异性抗体抑制N87细胞增殖的IC50
Figure BDA0002519816030000132
表9、双特异性抗体抑制BT474细胞增殖的IC50
Figure BDA0002519816030000133
表10、双特异性抗体抑制N87细胞增殖的IC50
Figure BDA0002519816030000134
序列表
<110> 三生国健药业（上海）股份有限公司
<120> 一种抗PD-L1和HER2的双特异性抗体
<160> 36
<170> SIPOSequenceListing 1.0
<210> 1
<211> 5
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Ser Tyr Gly Val His
1 5
<210> 2
<211> 16
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Leu Ile Trp Ser Gly Gly Gly Thr Asp Tyr Asn Pro Ser Leu Lys Ser
1 5 10 15
<210> 3
<211> 9
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Gln Leu Gly Leu Arg Ala Met Asp Tyr
1 5
<210> 4
<211> 11
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Arg Ala Ser Gln Ser Ile Gly Thr Thr Ile His
1 5 10
<210> 5
<211> 7
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Tyr Ala Ser Gln Ser Phe Ser
1 5
<210> 6
<211> 9
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Gln Gln Ser Asn Ser Trp Pro Leu Thr
1 5
<210> 7
<211> 5
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
Asp Thr Tyr Ile His
1 5
<210> 8
<211> 17
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 8
Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val Lys
1 5 10 15
Gly
<210> 9
<211> 11
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 9
Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr
1 5 10
<210> 10
<211> 11
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 10
Arg Ala Ser Gln Asp Val Asn Thr Ala Val Ala
1 5 10
<210> 11
<211> 7
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 11
Ser Ala Ser Phe Leu Tyr Ser
1 5
<210> 12
<211> 9
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 12
Gln Gln His Tyr Thr Thr Pro Pro Thr
1 5
<210> 13
<211> 5
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 13
Asp Tyr Ala Ile His
1 5
<210> 14
<211> 17
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 14
Val Phe Ser Ile Tyr Tyr Glu Asn Ile Asn Tyr Asn Gln Lys Phe Lys
1 5 10 15
Gly
<210> 15
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 15
Arg Asp Gly Gly Thr Ile Asn Tyr
1 5
<210> 16
<211> 16
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 16
Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu His
1 5 10 15
<210> 17
<211> 7
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 17
Lys Val Ser Asn Arg Phe Ser
1 5
<210> 18
<211> 9
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 18
Ser Gln Ser Thr His Ile Pro Trp Thr
1 5
<210> 19
<211> 117
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 19
Gln Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Ser Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr
20 25 30
Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Gly Leu Ile Trp Ser Gly Gly Gly Thr Asp Tyr Asn Pro Ser Leu Lys
50 55 60
Ser Arg Leu Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Phe
65 70 75 80
Lys Ile Ser Ser Leu Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Gln Leu Gly Leu Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser
100 105 110
Val Thr Val Ser Ser
115
<210> 20
<211> 107
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 20
Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Leu Ser Val Thr Pro Lys
1 5 10 15
Glu Lys Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Thr Thr
20 25 30
Ile His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile
35 40 45
Lys Tyr Ala Ser Gln Ser Phe Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Val Glu Ala
65 70 75 80
Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Asn Ser Trp Pro Leu
85 90 95
Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 21
<211> 120
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 21
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 22
<211> 107
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 22
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Arg 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 His Tyr Thr Thr Pro Pro
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 23
<211> 117
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 23
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Ala Ile His Trp Val Arg Gln Ala Pro Gly Gln Ser Leu Glu Trp Ile
35 40 45
Gly Val Phe Ser Ile Tyr Tyr Glu Asn Ile Asn Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Arg Ala Thr Met Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Arg Asp Gly Gly Thr Ile Asn Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser
115
<210> 24
<211> 112
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 24
Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly
1 5 10 15
Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser
20 25 30
Asn Gly Asn Thr Tyr Leu His Trp Tyr Gln Gln Arg Pro Gly Gln Ser
35 40 45
Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Phe Cys Ser Gln Ser
85 90 95
Thr His Ile Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105 110
<210> 25
<211> 447
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 25
Gln Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Ser Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr
20 25 30
Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Gly Leu Ile Trp Ser Gly Gly Gly Thr Asp Tyr Asn Pro Ser Leu Lys
50 55 60
Ser Arg Leu Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Phe
65 70 75 80
Lys Ile Ser Ser Leu Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Gln Leu Gly Leu Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser
100 105 110
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
115 120 125
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
130 135 140
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
145 150 155 160
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
165 170 175
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
180 185 190
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
195 200 205
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His
210 215 220
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
225 230 235 240
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
245 250 255
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
260 265 270
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
275 280 285
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
290 295 300
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
305 310 315 320
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
325 330 335
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
340 345 350
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
355 360 365
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
370 375 380
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
385 390 395 400
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
405 410 415
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
420 425 430
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 26
<211> 214
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 26
Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Leu Ser Val Thr Pro Lys
1 5 10 15
Glu Lys Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Thr Thr
20 25 30
Ile His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile
35 40 45
Lys Tyr Ala Ser Gln Ser Phe Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Val Glu Ala
65 70 75 80
Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Asn Ser Trp Pro Leu
85 90 95
Thr Phe Gly Ala 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> 27
<211> 450
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 27
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
145 150 155 160
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
225 230 235 240
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
290 295 300
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
355 360 365
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
385 390 395 400
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445
Gly Lys
450
<210> 28
<211> 214
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 28
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Arg 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 His Tyr Thr Thr 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
210
<210> 29
<211> 447
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 29
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Ala Ile His Trp Val Arg Gln Ala Pro Gly Gln Ser Leu Glu Trp Ile
35 40 45
Gly Val Phe Ser Ile Tyr Tyr Glu Asn Ile Asn Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Arg Ala Thr Met Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Arg Asp Gly Gly Thr Ile Asn Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
115 120 125
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
130 135 140
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
145 150 155 160
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
165 170 175
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
180 185 190
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
195 200 205
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His
210 215 220
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
225 230 235 240
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
245 250 255
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
260 265 270
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
275 280 285
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
290 295 300
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
305 310 315 320
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
325 330 335
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
340 345 350
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
355 360 365
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
370 375 380
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
385 390 395 400
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
405 410 415
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
420 425 430
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 30
<211> 219
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 30
Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly
1 5 10 15
Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser
20 25 30
Asn Gly Asn Thr Tyr Leu His Trp Tyr Gln Gln Arg Pro Gly Gln Ser
35 40 45
Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Phe Cys Ser Gln Ser
85 90 95
Thr His Ile Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105 110
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
115 120 125
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
130 135 140
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
145 150 155 160
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
165 170 175
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
180 185 190
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
195 200 205
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215
<210> 31
<211> 709
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 31
Gln Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Ser Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr
20 25 30
Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Gly Leu Ile Trp Ser Gly Gly Gly Thr Asp Tyr Asn Pro Ser Leu Lys
50 55 60
Ser Arg Leu Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Phe
65 70 75 80
Lys Ile Ser Ser Leu Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Gln Leu Gly Leu Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser
100 105 110
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
115 120 125
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
130 135 140
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
145 150 155 160
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
165 170 175
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
180 185 190
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
195 200 205
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His
210 215 220
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
225 230 235 240
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
245 250 255
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
260 265 270
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
275 280 285
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
290 295 300
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
305 310 315 320
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
325 330 335
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
340 345 350
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
355 360 365
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
370 375 380
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
385 390 395 400
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
405 410 415
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
420 425 430
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ala Gly
435 440 445
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val
450 455 460
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu
465 470 475 480
Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile
485 490 495
His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg
500 505 510
Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val Lys Gly
515 520 525
Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln
530 535 540
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ser Arg
545 550 555 560
Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr
565 570 575
Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
580 585 590
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln
595 600 605
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr
610 615 620
Cys Arg Ala Ser Gln Asp Val Asn Thr Ala Val Ala Trp Tyr Gln Gln
625 630 635 640
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu
645 650 655
Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp
660 665 670
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr
675 680 685
Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr
690 695 700
Lys Val Glu Ile Lys
705
<210> 32
<211> 709
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 32
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
145 150 155 160
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
225 230 235 240
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
290 295 300
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
355 360 365
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
385 390 395 400
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445
Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
450 455 460
Ser Gln Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Val Lys Pro Ser
465 470 475 480
Gln Ser Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser
485 490 495
Tyr Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp
500 505 510
Ile Gly Leu Ile Trp Ser Gly Gly Gly Thr Asp Tyr Asn Pro Ser Leu
515 520 525
Lys Ser Arg Leu Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser
530 535 540
Phe Lys Ile Ser Ser Leu Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys
545 550 555 560
Ala Arg Gln Leu Gly Leu Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr
565 570 575
Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
580 585 590
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln
595 600 605
Ser Pro Asp Phe Leu Ser Val Thr Pro Lys Glu Lys Val Thr Ile Thr
610 615 620
Cys Arg Ala Ser Gln Ser Ile Gly Thr Thr Ile His Trp Tyr Gln Gln
625 630 635 640
Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile Lys Tyr Ala Ser Gln Ser
645 650 655
Phe Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
660 665 670
Phe Thr Leu Thr Ile Asn Ser Val Glu Ala Glu Asp Ala Ala Thr Tyr
675 680 685
Tyr Cys Gln Gln Ser Asn Ser Trp Pro Leu Thr Phe Gly Ala Gly Thr
690 695 700
Lys Leu Glu Ile Lys
705
<210> 33
<211> 709
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 33
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly
115 120 125
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met
130 135 140
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr
145 150 155 160
Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala Val Ala Trp Tyr
165 170 175
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser
180 185 190
Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly
195 200 205
Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala
210 215 220
Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro Thr Phe Gly Gln
225 230 235 240
Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly
245 250 255
Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Ser Gly Gly Gly
260 265 270
Leu Val Lys Pro Ser Gln Ser Leu Ser Leu Thr Cys Thr Val Ser Gly
275 280 285
Phe Ser Leu Thr Ser Tyr Gly Val His Trp Val Arg Gln Pro Pro Gly
290 295 300
Lys Gly Leu Glu Trp Ile Gly Leu Ile Trp Ser Gly Gly Gly Thr Asp
305 310 315 320
Tyr Asn Pro Ser Leu Lys Ser Arg Leu Thr Ile Ser Arg Asp Thr Ser
325 330 335
Lys Asn Gln Val Ser Phe Lys Ile Ser Ser Leu Thr Ala Ala Asp Thr
340 345 350
Ala Val Tyr Tyr Cys Ala Arg Gln Leu Gly Leu Arg Ala Met Asp Tyr
355 360 365
Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly
370 375 380
Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
385 390 395 400
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
405 410 415
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
420 425 430
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
435 440 445
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
450 455 460
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
465 470 475 480
Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
485 490 495
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
500 505 510
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
515 520 525
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
530 535 540
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
545 550 555 560
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
565 570 575
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
580 585 590
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
595 600 605
Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln
610 615 620
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
625 630 635 640
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
645 650 655
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
660 665 670
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
675 680 685
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
690 695 700
Leu Ser Pro Gly Lys
705
<210> 34
<211> 709
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 34
Gln Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Ser Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr
20 25 30
Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Gly Leu Ile Trp Ser Gly Gly Gly Thr Asp Tyr Asn Pro Ser Leu Lys
50 55 60
Ser Arg Leu Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Phe
65 70 75 80
Lys Ile Ser Ser Leu Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Gln Leu Gly Leu Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser
100 105 110
Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
115 120 125
Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser
130 135 140
Pro Asp Phe Leu Ser Val Thr Pro Lys Glu Lys Val Thr Ile Thr Cys
145 150 155 160
Arg Ala Ser Gln Ser Ile Gly Thr Thr Ile His Trp Tyr Gln Gln Lys
165 170 175
Pro Asp Gln Ser Pro Lys Leu Leu Ile Lys Tyr Ala Ser Gln Ser Phe
180 185 190
Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
195 200 205
Thr Leu Thr Ile Asn Ser Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr
210 215 220
Cys Gln Gln Ser Asn Ser Trp Pro Leu Thr Phe Gly Ala Gly Thr Lys
225 230 235 240
Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
245 250 255
Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
260 265 270
Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile
275 280 285
Lys Asp Thr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
290 295 300
Glu Trp Val Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala
305 310 315 320
Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn
325 330 335
Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
340 345 350
Tyr Tyr Cys Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr
355 360 365
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly
370 375 380
Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
385 390 395 400
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
405 410 415
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
420 425 430
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
435 440 445
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
450 455 460
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
465 470 475 480
Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
485 490 495
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
500 505 510
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
515 520 525
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
530 535 540
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
545 550 555 560
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
565 570 575
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
580 585 590
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
595 600 605
Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln
610 615 620
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
625 630 635 640
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
645 650 655
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
660 665 670
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
675 680 685
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
690 695 700
Leu Ser Pro Gly Lys
705
<210> 35
<211> 706
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 35
Gln Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Ser Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr
20 25 30
Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Gly Leu Ile Trp Ser Gly Gly Gly Thr Asp Tyr Asn Pro Ser Leu Lys
50 55 60
Ser Arg Leu Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Phe
65 70 75 80
Lys Ile Ser Ser Leu Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Gln Leu Gly Leu Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser
100 105 110
Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
115 120 125
Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser
130 135 140
Pro Asp Phe Leu Ser Val Thr Pro Lys Glu Lys Val Thr Ile Thr Cys
145 150 155 160
Arg Ala Ser Gln Ser Ile Gly Thr Thr Ile His Trp Tyr Gln Gln Lys
165 170 175
Pro Asp Gln Ser Pro Lys Leu Leu Ile Lys Tyr Ala Ser Gln Ser Phe
180 185 190
Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
195 200 205
Thr Leu Thr Ile Asn Ser Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr
210 215 220
Cys Gln Gln Ser Asn Ser Trp Pro Leu Thr Phe Gly Ala Gly Thr Lys
225 230 235 240
Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
245 250 255
Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys
260 265 270
Pro Gly Ala Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe
275 280 285
Thr Asp Tyr Ala Ile His Trp Val Arg Gln Ala Pro Gly Gln Ser Leu
290 295 300
Glu Trp Ile Gly Val Phe Ser Ile Tyr Tyr Glu Asn Ile Asn Tyr Asn
305 310 315 320
Gln Lys Phe Lys Gly Arg Ala Thr Met Thr Val Asp Lys Ser Thr Ser
325 330 335
Thr Ala Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val
340 345 350
Tyr Tyr Cys Ala Arg Arg Asp Gly Gly Thr Ile Asn Tyr Trp Gly Gln
355 360 365
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
370 375 380
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
385 390 395 400
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
405 410 415
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
420 425 430
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
435 440 445
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
450 455 460
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
465 470 475 480
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
485 490 495
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
500 505 510
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
515 520 525
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
530 535 540
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
545 550 555 560
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
565 570 575
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
580 585 590
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
595 600 605
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
610 615 620
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
625 630 635 640
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
645 650 655
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
660 665 670
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
675 680 685
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
690 695 700
Gly Lys
705
<210> 36
<211> 706
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 36
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Ala Ile His Trp Val Arg Gln Ala Pro Gly Gln Ser Leu Glu Trp Ile
35 40 45
Gly Val Phe Ser Ile Tyr Tyr Glu Asn Ile Asn Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Arg Ala Thr Met Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Arg Asp Gly Gly Thr Ile Asn Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
115 120 125
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
130 135 140
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
145 150 155 160
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
165 170 175
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
180 185 190
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
195 200 205
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His
210 215 220
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
225 230 235 240
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
245 250 255
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
260 265 270
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
275 280 285
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
290 295 300
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
305 310 315 320
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
325 330 335
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
340 345 350
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
355 360 365
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
370 375 380
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
385 390 395 400
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
405 410 415
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
420 425 430
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ala Gly
435 440 445
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val
450 455 460
Gln Leu Gln Gln Ser Gly Gly Gly Leu Val Lys Pro Ser Gln Ser Leu
465 470 475 480
Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr Gly Val
485 490 495
His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly Leu
500 505 510
Ile Trp Ser Gly Gly Gly Thr Asp Tyr Asn Pro Ser Leu Lys Ser Arg
515 520 525
Leu Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Phe Lys Ile
530 535 540
Ser Ser Leu Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gln
545 550 555 560
Leu Gly Leu Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr
565 570 575
Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
580 585 590
Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Asp
595 600 605
Phe Leu Ser Val Thr Pro Lys Glu Lys Val Thr Ile Thr Cys Arg Ala
610 615 620
Ser Gln Ser Ile Gly Thr Thr Ile His Trp Tyr Gln Gln Lys Pro Asp
625 630 635 640
Gln Ser Pro Lys Leu Leu Ile Lys Tyr Ala Ser Gln Ser Phe Ser Gly
645 650 655
Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
660 665 670
Thr Ile Asn Ser Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln
675 680 685
Gln Ser Asn Ser Trp Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu
690 695 700
Ile Lys
705

Claims (15)

1.抗PD-L1和HER2的双特异性抗体，其特征在于，包含两条多肽链和两条轻链，其中：
(a)所述的多肽链从N末端至C末端包含VH-PDL1-CH1-CH2-CH3-L1-VH-HER2-L2-VL-HER2或VH-HER2-L2-VL-HER2-L1-VH-PDL1-CH1-CH2-CH3，所述的轻链从N末端至C末端包含VL-PDL1-CL；或
(b)所述的多肽链从N末端至C末端包含VH-HER2-CH1-CH2-CH3-L1-VH-PDL1-L2-VL-PDL1或VH-PDL1-L2-VL-PDL1-L1-VH-HER2-CH1-CH2-CH3，所述的轻链从N末端至C末端包含VL-HER2-CL；
其中，所述的VH-PDL1为结合PD-L1的重链可变区，所述的VL-PDL1为结合PD-L1的轻链可变区，所述的VH-HER2为结合HER2的重链可变区，所述的VL-HER2为结合HER2的轻链可变区，所述的L1和L2为(G4S)x，x为3、4、5或6，所述的CH1-CH2-CH3为重链恒定区，所述的CL为轻链恒定区，所述的VH-PDL1与所述的VL-PDL1形成特异性结合PD-L1的抗原结合位点，所述的VH-HER2与所述的VL-HER2形成特异性结合HER2的抗原结合位点。
2.如权利要求1所述的双特异性抗体，其特征在于，所述的L1为(G4S)3，所述的L2为(G4S)4。
3.如权利要求1所述的双特异性抗体，其特征在于，所述的VH-PDL1包含氨基酸序列如SEQ ID NO：1-3所示的重链CDR，所述的VL-PDL1包含氨基酸序列如SEQ ID NO：4-6所示的轻链CDR，所述的VH-HER2包含氨基酸序列如SEQ ID NO：7-9或SEQ ID NO：13-15所示的重链CDR，所述的VL-HER2包含氨基酸序列如SEQ ID NO：10-12或SEQ ID NO：16-18所示的轻链CDR。
4.如权利要求3所述的双特异性抗体，其特征在于，所述的VH-PDL1具有如SEQ ID NO：19所示的氨基酸序列，所述的VL-PDL1具有如SEQ ID NO：20所示的氨基酸序列，所述的VH-HER2具有如SEQ ID NO：21或SEQ ID NO：23所示的氨基酸序列，所述的VL-HER2具有如SEQID NO：22或SEQ ID NO：24所示的氨基酸序列。
5.如权利要求4所述的双特异性抗体，其特征在于，所述的多肽链具有如SEQ ID NO：31或SEQ ID NO：33所示的氨基酸序列，所述的轻链具有如SEQ ID NO：26所示的氨基酸序列；或所述的多肽链具有如SEQ ID NO：32或SEQ ID NO：34所示的氨基酸序列，所述的轻链具有如SEQ ID NO：28所示的氨基酸序列；或所述的多肽链具有如SEQ ID NO：35或SEQ ID NO：36所示的氨基酸序列，所述的轻链具有如SEQ ID NO：30所示的氨基酸序列。
6.如权利要求1所述的双特异性抗体，其特征在于，所述的重链恒定区包括IgG1、IgG2、IgG3或IgG4重链恒定区，所述的轻链恒定区包括κ或λ轻链恒定区。
7.一种分离的核苷酸，其特征在于，所述的核苷酸编码如权利要求1-6中任一项所述的双特异性抗体。
8.一种表达载体，其特征在于，所述的表达载体含有如权利要求7所述的核苷酸。
9.一种宿主细胞，其特征在于，所述的宿主细胞含有如权利要求8所述的表达载体。
10.如权利要求1-6中任一项所述的双特异性抗体的制备方法，其特征在于，所述方法包含以下步骤：
(a)在表达条件下，培养如权利要求9所述的宿主细胞，从而表达所述的双特异性抗体；
(b)分离并纯化(a)所述的双特异性抗体。
11.一种药物组合物，其特征在于，所述药物组合物含有如权利要求1-6中任一项所述的双特异性抗体和药学上可接受的载体。
12.如权利要求1-6中任一项所述的双特异性抗体或如权利要求11所述的药物组合物在制备治疗癌症的药物中的用途。
13.如权利要求12所述的用途，其特征在于，所述癌症选自由以下组成的组：黑素瘤、肾癌、***癌、胰腺癌、乳腺癌、结肠癌、肺癌、食道癌、头颈鳞状细胞癌、肝癌、卵巢癌、***、甲状腺癌、成胶质细胞瘤、神经胶质瘤、白血病、淋巴瘤及其它赘生性恶性疾病。
14.一种治疗癌症的方法，其特征在于，包括向有需要的受试者施用如权利要求1-6中任一项所述的双特异性抗体或如权利要求11所述的药物组合物。
15.如权利要求14所述的方法，其特征在于，所述癌症选自由以下组成的组：黑素瘤、肾癌、***癌、胰腺癌、乳腺癌、结肠癌、肺癌、食道癌、头颈鳞状细胞癌、肝癌、卵巢癌、***、甲状腺癌、成胶质细胞瘤、神经胶质瘤、白血病、淋巴瘤及其它赘生性恶性疾病。
CN202010487636.9A 2020-06-02 2020-06-02 一种抗pd-l1和her2的双特异性抗体 Pending CN113754775A (zh)

Priority Applications (4)

Application Number Priority Date Filing Date Title
CN202010487636.9A CN113754775A (zh) 2020-06-02 2020-06-02 一种抗pd-l1和her2的双特异性抗体
CN202180032220.0A CN115605513B (zh) 2020-06-02 2021-05-21 一种抗pd-l1和her2的双特异性抗体
PCT/CN2021/095189 WO2021244328A1 (zh) 2020-06-02 2021-05-21 一种抗pd-l1和her2的双特异性抗体
TW110119871A TW202146457A (zh) 2020-06-02 2021-06-01 一種抗pd-l1和her2的雙特異性抗體

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202010487636.9A CN113754775A (zh) 2020-06-02 2020-06-02 一种抗pd-l1和her2的双特异性抗体

Publications (1)

Publication Number Publication Date
CN113754775A true CN113754775A (zh) 2021-12-07

Family

ID=78782246

Family Applications (2)

Application Number Title Priority Date Filing Date
CN202010487636.9A Pending CN113754775A (zh) 2020-06-02 2020-06-02 一种抗pd-l1和her2的双特异性抗体
CN202180032220.0A Active CN115605513B (zh) 2020-06-02 2021-05-21 一种抗pd-l1和her2的双特异性抗体

Family Applications After (1)

Application Number Title Priority Date Filing Date
CN202180032220.0A Active CN115605513B (zh) 2020-06-02 2021-05-21 一种抗pd-l1和her2的双特异性抗体

Country Status (3)

Country Link
CN (2) CN113754775A (zh)
TW (1) TW202146457A (zh)
WO (1) WO2021244328A1 (zh)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021226984A1 (zh) * 2020-05-15 2021-11-18 三生国健药业（上海）股份有限公司 一种抗pd-1和pd-l1的四价双特异性抗体
CN113943371B (zh) * 2021-11-01 2023-05-05 达石药业(广东)有限公司 一种抗her2/抗pd-l1双功能抗体及其应用
CN114409797B (zh) * 2021-12-24 2024-02-20 合肥天港免疫药物有限公司 重组抗体及其应用
CN117247457A (zh) * 2022-06-10 2023-12-19 三优生物医药(上海)有限公司 靶向her2和pd-l1的双特异性抗体及其制备方法和应用

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ATE255131T1 (de) * 1991-06-14 2003-12-15 Genentech Inc Humanisierter heregulin antikörper
CA3021669A1 (en) * 2016-05-09 2017-11-16 Igm Biosciences, Inc. Anti-pd-l1 antibodies
GB201611530D0 (en) * 2016-07-01 2016-08-17 Alligator Bioscience Ab Novel polypeptides
CN110790840A (zh) * 2018-08-01 2020-02-14 三生国健药业（上海）股份有限公司 结合人her2的抗体、其制备方法和用途
CN111196856A (zh) * 2018-11-19 2020-05-26 三生国健药业（上海）股份有限公司 抗her2/pd1双特异性抗体

Also Published As

Publication number Publication date
TW202146457A (zh) 2021-12-16
CN115605513B (zh) 2024-03-19
CN115605513A (zh) 2023-01-13
WO2021244328A1 (zh) 2021-12-09

Similar Documents

Publication Publication Date Title
JP7386796B2 (ja) 抗－ｈｅｒ２抗体又はその抗原結合フラグメント、及びこれを含むキメラ抗原受容体
JP6359372B2 (ja) ＤＡＲＰｉｎを含む二重特異キメラ蛋白質
AU2012319299B2 (en) Anti c-Met antibody and uses thereof
US9808507B2 (en) Anti-c-Met/anti-Ang2 bispecific antibody
CN113754775A (zh) 一种抗pd-l1和her2的双特异性抗体
WO2020156555A1 (zh) 抗her2的双特异性抗体及其应用
US10246507B2 (en) Polypeptide, anti-VEGF antibody, and anti-c-Met/anti-VEGF bispecific antibodies comprising the same
TWI797609B (zh) 抗pd-1和pd-l1的四價雙特異性抗體
US10000569B2 (en) Anti-cMet/anti-EGFR/anti-HER3 multispecific antibodies and use thereof
WO2022194201A1 (zh) 一种靶向cldn18.2的抗体或其抗原结合片段及其应用
EP4269442A1 (en) Mesothelin binding molecule and application thereof
WO2021244554A1 (zh) 抗pdl1×egfr的双特异性抗体
WO2021244392A1 (zh) 一种抗pd1×pdl1的双特异性抗体
CN113754774A (zh) 一种抗pd-l1和egfr的四价双特异性抗体
WO2021244552A1 (zh) 抗pdl1×kdr的双特异性抗体
WO2023109888A1 (zh) 抗ang2-vegf双特异性抗体及其用途
WO2023236844A1 (zh) 靶向her2和pd-l1的双特异性抗体及其制备方法和应用
CN114805590A (zh) 结合her2的双特异性抗体
CN116284427A (zh) 抗muc17/cd3双特异性抗体、其制备方法及用途
CN116284408A (zh) 结合人muc17的抗体、其制备方法及用途
CN116554324A (zh) 一种特异性识别4-1bb的抗体、其制备方法及其用途
CN113754777A (zh) 一种抗PD-L1/TGF-β融合蛋白

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication