CN116925234A

CN116925234A - 一种编码抗vegf-a和ang-2双特异性抗体的aav载体

Info

Publication number: CN116925234A
Application number: CN202210356939.6A
Authority: CN
Inventors: 才源; 马珍; 周佩佩; 张明亮
Original assignee: Hefei Xingmou Biotechnology Co ltd
Current assignee: Hefei Xingmou Biotechnology Co ltd
Priority date: 2022-04-02
Filing date: 2022-04-02
Publication date: 2023-10-24
Anticipated expiration: 2042-04-02
Also published as: WO2023186131A1; CN116925234B; CN118574856A

Abstract

本公开提供了用于治疗受试者的湿性年龄相关性黄斑变性或糖尿病性黄斑水肿的药物组合物和方法。提供了一种双特异性抗体、核酸、以及AAV病毒载体和病毒颗粒。双特异性抗体表达盒包括编码启动子、可操作地连接至双特异性抗体的可选择的信号肽，其中所述双特异性抗体包含特异于VEGF‑A和ANG‑2的结合结构域。

Description

一种编码抗VEGF-A和ANG-2双特异性抗体的AAV载体

发明领域

本发明涉及免疫学和基因递送领域。更具体地，本申请涉及用于生产感兴趣的蛋白诸如抗体的组合物、系统和方法。

背景技术

年龄相关性黄斑变性(age-related macular degeneration,AMD)是由各种因素诱导并与年龄相关的一组黄斑疾病，其共同特点是黄斑部视网膜及视网膜色素上皮和脉络膜发生病变，并导致患者视功能障碍和中心视力进行性下降。我国乃至世界AMD的患病率不断上升且随着年龄增加而升高，是50岁以上人群不可逆视力损伤的主要原因之一。随着人口的老龄化，预计到2040年AMD患者将达2.88亿人。AMD临床分型没有统一标准，国内外目前均倾向于先分期再分型，晚期分成2种类型，分别为干性型(萎缩型)和新生血管性型(渗出型、湿性型)，其中，新生血管性年龄相关性黄斑变性(neovascular age-related maculardegeneration,nAMD)也称湿性年龄相关性黄斑变性(wet age-related maculardegeneration,wAMD)，是导致视力丧失的主要临床类型，其主要特点是脉络膜新生血管(choroidal neovascularization,CNV)出现在黄斑区，从而导致黄斑区发生出血、渗出。糖尿病性视网膜病变(diabetic macular edema,DME)目前是西方发达国家重要致盲的原因之一，随着我国人民生活水平的提高，人口老龄化，DME的发病率已逐渐增高，严重影响患者的视功能和生存质量。

VEGF是wAMD和DME的发病机制中最重要的因素之一，它能特异地作用于血管内皮细胞，促使血管内皮细胞的增殖，诱导新生血管的生成以及血管渗漏的增加。目前研究发现，阻断VEGF的表达可诱导血管重塑的发生，同时促使尚未成熟的新生血管回退，因此VEGF是一个有前景的wAMD和DME的治疗靶点。自2006年以雷珠单抗(Ranibizumab)为代表的抗VEGF药物应用以来，抗VEGF药物已被广泛应用于治疗wAMD和DME。在近10年间，由于同时应用多种抗VEGF治疗方法，因wAMD和DME导致的不可逆盲已有大幅减少。

尽管靶向VEGF的药物在临床上取得了长足的进步和突破，它可以显著降低血管渗漏和水肿的程度，改善视力，并且未发现严重的并发症。但是，这些注射入体内的蛋白质药物会随着代谢而迅速清除，因此需要多次眼内注射以维持治疗效果。长期治疗增加了患者的经济负担，重复注射会增加患者的痛苦和发生不良反应的可能性，在从常规给药转变为低频给药时会出现一定程度的视力下降，一些患者在治疗后容易复发。针对wAMD和DME现有疗法的局限性，在本领域上出现了对更加经济且持续时间更长、效果更优的治疗策略的需求。

本发明旨在提供一种抗VEGF-A和抗ANG-2基因疗法。

发明内容

本发明提供了一种双特异性抗体，其包括抗VEGF-A和ANG-2的结合结构域，其优选为单链抗体串联分子(tandem scFv，ta-scFv)，所述双特异性抗体重链可变区(VH)和轻链可变区(VL)从N-末端到C-末端按照如下顺序排列：

1)VH_抗VEGF-A-VL_抗VEGF-A-VH_抗ANG-2-VL_抗ANG-2；

2)VL_抗ANG-2-VH_抗VEGF-A-VL_抗VEGF-A-VH_抗ANG-2；或

3)_VL抗VEGF-A-VH_抗ANG-2-VL_抗ANG-2-VH_抗VEGF-A；

其中，所述VH_抗VEGF-A包括SEQ ID NO:1所示的CDR1，SEQ ID NO:2所示的CDR2；SEQID NO:3所示的CDR3；

所述VL_抗VEGF-A包括SEQ ID NO:4所示的CDR1，SEQ ID NO:5所示的CDR2；SEQ ID NO:6或SEQ ID NO:47所示的CDR3；

所述VH_抗ANG-2包括SEQ ID NO:7所示的CDR1，SEQ ID NO:8所示的CDR2；SEQ ID NO:9所示的CDR3；

所述VL_抗ANG-2包括SEQ ID NO:10所示的CDR1，SEQ ID NO:11所示的CDR2；SEQ IDNO:12所示的CDR3。

在一个实施例中，所述VH_抗VEGF-A包括SEQ ID NO:13所示的序列或与SEQ ID NO:13序列同一性为70％、80％、90％、95％、或99％的序列；

所述VL_抗VEGF-A包括SEQ ID NO:14所示的序列或与SEQ ID NO:14或SEQ ID NO:48序列同一性为70％、80％、90％、95％、或99％的序列；

所述VH_抗ANG-2包括SEQ ID NO:15所示的序列或与SEQ ID NO:15序列同一性为70％、80％、90％、95％、或99％的序列；

所述VL_抗ANG-2包括SEQ ID NO:16所示的序列或与SEQ ID NO:16序列同一性为70％、80％、90％、95％、或99％的序列。

在一个实施例中，所述抗体包括SEQ ID NO:17、18、19、49、51或53所示的序列或与SEQ ID NO:17、18、19、49、51或53序列同一性为70％、80％、90％、95％、或99％的序列。

在一个实施例中，所述双特异性抗体构建体的N-末端包含信号肽序列或标签序列，优选所述信号肽序列为CD5-sp信号肽，其包含如SEQ ID NO：23所示的序列。

在一个实施例中，所述抗体包括SEQ ID NO:20、21、22、50、52或54所示的序列或与SEQ ID NO:20、21、22、50、52或54序列同一性为70％、80％、90％、95％、或99％的序列。

在一个实施例中，重链可变区(VH)和轻链可变区(VL)之间可操作性地由(G₄S)_n链接，其中n为大于1的整数，优选为1至4间任意整数，例如G₄S、(G₄S)₂、(G₄S)₃或(G₄S)₄，例如，

1)VH_抗VEGF-A-(G₄S)_m+X-VL_抗VEGF-A-(G₄S)_m-VH_抗ANG-2-(G₄S)_m+X-VL_抗ANG-2；其中m≥1，X≥2，优选为VH_抗VEGF-A-(G₄S)₃-VL_抗VEGF-A-G₄S-VH_抗ANG-2-(G₄S)_3-VL_抗ANG-2；

2)VL_抗ANG-2-(G₄S)_m-VH_抗VEGF-A-(G₄S)_m+X-VL_抗VEGF-A-(G₄S)_m-VH_抗ANG-2，其中m≥1，X≥2，优选为VL_抗ANG-2-G₄S-VH_抗VEGF-A-(G₄S)₃-VL_抗VEGF-A-G₄S-VH_抗ANG-2；或

3)VL_抗VEGF-A-(G₄S)_m-VH_抗ANG-2-(G₄S)_m+X-VL_抗ANG-2-(G₄S)_m-VH_抗VEGF-A，其中m≥1，X≥2，优选为VL_抗VEGF-A-G₄S-VH_抗ANG-2-(G₄S)₃-VL_抗ANG-2-G₄S-VH_抗VEGF-A。

进一步，本发明提供了一种核酸序列，其编码前述双特异性抗体。

本发明还提供了一种载体，包括编码前述双特异性抗体的核酸序列。

在一个实施例中，所述载体优选为AAV病毒。

在一个实施例中，AAV病毒载体进一步包括：5’ITR和3’ITR、启动子和polyA序列等。

又一方面，本发明提供了一种AAV病毒颗粒，包括如前任意的AAV病毒载体和衣壳蛋白，优选其中所述衣壳蛋白的血清型为AAV1、AAV2、AAV4、AAV5、AAV6、AAV7、AAV8、或AAV9等。

本发明提供了一种药物组合物，包括如前所述的双特异性抗体、核酸序列、载体诸如AAV病毒载体和如前所述的AAV病毒颗粒中的至少一种，以及药学上可接受的载体。

本发明提供如前所述双特异性抗体、核酸序列、载体诸如AAV病毒载体、如前所述的AAV病毒颗粒、或如前所述的药物组合物在制备用于治疗或预防癌症、眼内新生血管综合征、类风湿性关节炎、银屑病、增殖性视网膜病变、年龄相关性黄斑变性或糖尿病性黄斑水肿的药物中的用途。

在一个实施例中，所述年龄相关性黄斑变性为湿性年龄相关性黄斑变性。

在一个具体实施例中，所述药物通过玻璃体腔或视网膜下腔注射施用。

附图说明

图1示出了XMVA01、XMVA04和XMVA09的结构示意图。

图2示出了XMVA09的载体信息。

图3示出了XMVA01、XMVA04和XMVA09载体转染细胞后表达VEGF165中和蛋白的含量。

图4示出了在细胞中表达XMVA01、XMVA04和XMVA09对人视网膜微血管内皮细胞(HRMEC)在ECGS刺激下增殖的影响。

图5示出了在细胞中表达XMVA01、XMVA04和XMVA09对HRMECs管腔形成的影响的定量图。

图6示出了在细胞中表达XMVA01、XMVA04和XMVA09对HRMECs管腔形成的影响的细胞图。

图7示出了XMVA10的结构示意图。

图8示出了XMVA11的结构示意图。

图9示出了XMVA13的结构示意图。

图10示出了XMVA14的结构示意图。

图11示出了XMVA15的结构示意图。

发明详述

以下根据实施例，并且结合附图，详细描述本发明。从下文的详细描述中，本发明的上述方面和本发明的其他方面将是明显的。本发明的范围不局限于下列实施例。

本发明中的抗体是多特异性的、可以为人源化的、单链的、嵌合的、合成的、重组的、杂合的、突变的、以及嫁接的抗体；本发明中的抗体形式为抗体轻链可变区(VL)和抗体重链可变区(VH)拼接而成的scFv，该抗体可以结合两种不同的抗原，例如VEGF-A和ANG-2。

抗体轻链可变区(VL)和抗体重链可变区(VH)，VH和VL区可以进一步细分成：称为互补决定区(CDR)的高变区，以及穿插分布的称为框架区(FWR)的更保守区域。本发明公开的抗体和抗原结合片段的CDR由Kabat编号所定义或识别。在一个实施方案中，每个VH和VL一般包括从氨基端到羧基端按以下顺序排列的3个CDR和4个FWR：FWR1、CDR1、FWR2、CDR2、FWR3、CDR3、FWR4。本发明公开的抗体和抗原结合片段的CDR由Kabat编号所定义或识别。

本文使用的“载体”是指包括重组多核苷酸的载体，所述重组多核苷酸包括可操作地连接至待表达的核苷酸序列的表达控制序列。

下述实施例中的实验方法，如无特殊说明，均为常规方法。

实施例

实例1：质粒载体的构建

表达anti-VEGF-A/ANG-2基因的AAV载体质粒的构建

抗VEGF的VH氨基酸序列和VL氨基酸序列、抗ANG-2的VH氨基酸序列和VL氨基酸序列，在N端加上分泌信号肽CD5-sp核苷酸序列,分别用(G₄S)₃、G₄S、(G₄S)₃肽接头连接，组成了结构为CD5-sp-VH_抗VEGF-A-(G₄S)₃-VL_抗VEGF-A-G₄S-VH_抗ANG-2-(G₄S)_3-VL_抗ANG-2的开放阅读框，根据人密码子偏好设计核苷酸序列，并在5’端引入BamH I酶切位点，在3’端引入EcoR V酶切位点，根据表1、表2中各自的序列进行构建，分别命名为XMVA01、XMVA04和XMVA09(由南京金斯瑞生物科技公司合成全基因)；

用BamH I/EcoR V双酶切上述构建体和pAAV9neo-CAG质粒，通过连接、转化及克隆筛选鉴定等常规分子生物学操作分别构建了XMVA01、XMVA04和XMVA09载体，其结构示意图见图1。用无内毒素的质粒提取试剂盒(MN)获得高质量的质粒DNA备用。

表1 XMVA09的序列信息

表2. XMVA01和XMVA04序列信息

实例2：XMVA01、XMVA04、XMVA09在细胞中的表达测定

将XMVA01、XMVA04、XMVA09质粒分别转染HEK293T细胞，收集上清，用ELISA检测上清中蛋白的表达。具体操作如下：用含10％胎牛血清的DMEM的完全培养基重悬293T细胞后，接种至培养皿中，37℃、5％CO₂培养箱培养至细胞的汇合度达到70％-90％时，采用Lipofectamine2000(Invitrogen)促转试剂，按照其说明书转染质粒。继续培养72小时后，收集上清液，-80℃备用。

ELISA具体检测方法如下：以0.1μg/mL VEGF165(proteintech)蛋白包被酶标板(Thermo)，每孔100μL，4℃过夜孵育，用含有0.05％Tween(Sangon)的PBS洗涤酶标板3次，每次3分钟。用含有2％BSA(麦客生物)的PBS 200μL/孔室温封闭1小时，再次洗涤3次。在酶标板中分别加入康柏西普(10mg/mL)标准品和XMVA01、XMVA04、XMVA09质粒转染HEK293T细胞后收集的细胞上清，标准品从第一孔以312.5ng/mL浓度开始连续8个稀释度进行倍比稀释，待测上清以1：10或1：50进行倍比稀释，数据取平均值，以未转染质粒的细胞上清液作为对照组(Control组)，37℃孵育1小时，洗涤3次。每孔加入1：5000稀释的辣根过氧化氢酶标记的羊抗人IgG抗体(JacksonImmunoResearch)，37℃孵育1小时，再洗涤3次。每孔加入TMB显色液(Beyotime)100μL/孔，室温避光反应6分钟，向上述孔中加入100μL/孔终止液(Beyotime)终止反应。用酶标仪在450nm处测定OD值，计算待测样品的含量，结果见图3。XMVA01、XMVA04和XMVA09载体转染细胞后能有效表达VEGF165中和蛋白，和对照组相比，具有显著差异。

实例3：在细胞中表达XMVA01、XMVA04、XMVA09对人视网膜微血管内皮细胞(HRMEC) 增殖的影响

具体实验如下：用含1％ECGS的ECM的完全培养基(Science Cell)调整HRMECs的细胞密度为4×10⁴/mL，100μL/孔接种于平底96孔板中，将XMVA01、XMVA04、XMVA09质粒分别转染HEK293T细胞后收集的细胞上清以100μL/孔加入孔内，以未转染质粒的细胞上清液作为对照组(Control组)。37℃、5％CO₂的培养箱中培养72小时，按照Cell Counting Kit-8(CCK-8，Biosharp)说明书分别在实验组和对照组中加入20μL/孔的CCK-8的溶液，孵育4小时后，用酶标仪测定在450nm处的吸光值，结果见图4，XMVA09载体所表达的蛋白能有效抑制ECGS刺激下的HRMECs的增殖,且抑制效果显著优于XMVA01和XMVA04。

实例4：在细胞中表达XMVA01、XMVA04、XMVA09对HRMECs管腔形成的影响

将解冻后的Matrigel(Corning)基质胶均一地铺在平底96孔板中，50μL/孔，37℃培养箱孵育1小时；以XMVA01、XMVA04、XMVA09质粒分别转染HEK293T细胞后收集的细胞上清处理HRMECs，以未转染质粒的细胞上清液作为对照组(Control组)，培养48小时后用不含生长因子和血清的ECM的基础培养基重悬，并调整细胞密度为2×10⁵/mL，100μL/孔接种在含基质胶的96孔板内；于37℃，5％CO₂中培养箱中培养，每间隔2小时观察一次，4-8小时后，在显微镜下观察、拍照，记录经待测上清处理后对HRMECs管腔的形成能力的影响，结果见图5。XMVA09载体所表达的蛋白能有效抑制HRMECs管腔的形成，且抑制效果显著优于XMVA01和XMVA04。

实例5：重组AAV病毒的制备和鉴定

使用三质粒包装系统和纯化重组AAV病毒，辅助质粒(phelper)、AAV的Cap和Rep蛋白表达质粒(AAV2/5)、表达载体目的质粒(XMVA01、XMVA04或XMVA09)按照2:1:1的质量比，和PEI促转剂形成转染复合物，转染HEK293T细胞，进行含目的基因的AAV病毒的包装。分别于转染后第3天和第7天，进行两次上清的收集，即获得含有目的基因的AAV病毒颗粒。采用不同梯度的碘克沙醇(15％、25％、40％和60％)进行密度梯度离心(Beckman的超速离心机)，获得纯化的AAV病毒。对获得的纯化的AAV病毒，通过透射电子显微电镜进行AAV质量的鉴定和qPCR进行AAV病毒滴度的定量。

统计分析

采用双尾分析，统计学水平设在5％或p≤0.05，计算各项分析指标的平均数和标准差(Mean±SD)。上述数据按下列过程分析：

用Levene's Test检验方差齐性；如果没有统计学意义(p>0.05)，用单因素方差分析(ANOVA)进行统计分析。如果ANOVA有统计学意义(p≤0.05)，则进行LSD多重比较。

如果方差不齐(p≤0.05)，则进行Kruskal-Wallis非参数检验。如果Kruskal-Wallis非参数检验结果显著(p≤0.05)，则进一步采用Mann-Whitney U检验进行两两比较。

实例6：其他质粒载体的构建

方案1

抗VEGF的VH氨基酸序列和VL氨基酸序列、抗ANG-2的VH氨基酸序列和和VL氨基酸序列，在N端加上分泌信号肽CD5-sp核苷酸序列,分别用G₄S、(G₄S)₃、G₄S肽接头连接，组成了结构为CD5-sp-VL_抗ANG-2-G₄S-VH_抗VEGF-A-(G₄S)₃-VL_抗VEGF-A-G₄S-VH_抗ANG-2的开放阅读框，根据人密码子偏好设计核苷酸序列，并在5’端引入BamH I酶切位点，在3’端引入EcoR V酶切位点，命名为XMVA10(见图7)。其中抗VEGF的VH氨基酸序列和VL氨基酸序列、抗ANG-2的VH氨基酸序列和VL氨基酸序列与XMVA09相同。

方案2

抗VEGF的VH氨基酸序列和VL氨基酸序列、抗ANG-2的VH氨基酸序列和VL氨基酸序列，在N端加上分泌信号肽CD5-sp核苷酸序列,分别用G₄S、(G₄S)₃、G₄S肽接头连接，组成了结构为CD5-sp-VL_抗VEGF-A-G₄S-VH_抗ANG-2-(G₄S)₃-VL_抗ANG-2-G₄S-VH_抗VEGF-A的开放阅读框，根据人密码子偏好设计核苷酸序列，并在5’端引入BamH I酶切位点，在3’端引入EcoR V酶切位点，命名为XMVA11(见图8)。其中抗VEGF的VH氨基酸序列和VL氨基酸序列、抗ANG-2的VH氨基酸序列和VL氨基酸序列与XMVA09相同。

方案3

抗VEGF的VH氨基酸序列和VL氨基酸序列、抗ANG-2的VH氨基酸序列和和VL氨基酸序列，在N端加上分泌信号肽CD5-sp核苷酸序列,分别用(G₄S)₃、G₄S、(G₄S)₃肽接头连接，组成了结构为CD5-sp-VH_抗VEGF-A-(G₄S)₃-VL_抗VEGF-A-G₄S-VH_抗ANG-2-(G₄S)_3-VL_抗ANG-2的开放阅读框，根据人密码子偏好设计核苷酸序列，并在5’端引入BamH I酶切位点，在3’端引入EcoR V酶切位点，命名为XMVA13(见图9)。

方案4

抗VEGF的VH氨基酸序列和VL氨基酸序列、抗ANG-2的VH氨基酸序列和和VL氨基酸序列，在N端加上分泌信号肽CD5-sp核苷酸序列,分别用G₄S、(G₄S)₃、G₄S肽接头连接，组成了结构为CD5-sp-VL_抗ANG-2-G₄S-VH_抗VEGF-A-(G₄S)₃-VL_抗VEGF-A-G₄S-VH_抗ANG-2的开放阅读框，根据人密码子偏好设计核苷酸序列，并在5’端引入BamH I酶切位点，在3’端引入EcoR V酶切位点，命名为XMVA14(见图10)。其中抗VEGF的VH氨基酸序列和VL氨基酸序列、抗ANG-2的VH氨基酸序列和VL氨基酸序列与XMVA13相同。

方案5

抗VEGF的VH氨基酸序列和VL氨基酸序列、抗ANG-2的VH氨基酸序列和VL氨基酸序列，在N端加上分泌信号肽CD5-sp核苷酸序列,分别用G₄S、(G₄S)₃、G₄S肽接头连接，组成了结构为CD5-sp-VL_抗VEGF-A-G₄S-VH_抗ANG-2-(G₄S)₃-VL_抗ANG-2-G₄S-VH_抗VEGF-A的开放阅读框，根据人密码子偏好设计核苷酸序列，并在5’端引入BamH I酶切位点，在3’端引入EcoR V酶切位点，命名为XMVA15(见图11)。其中抗VEGF的VH氨基酸序列和VL氨基酸序列、抗ANG-2的VH氨基酸序列和VL氨基酸序列与XMVA13相同。

通过连接、转化及克隆筛选鉴定等常规分子生物学操作构建了XMVA10、XMVA11、XMVA13、XMVA14和XMVA15载体，用无内毒素的质粒提取试剂盒(MN)获得高质量的质粒DNA备用。

按照实施例2中所述方法，将质粒分别转染HEK293T细胞，收集上清，用ELISA方法检测上清中蛋白的表达。按照实施例3中所述的方法，在细胞中表达上述载体对人视网膜微血管内皮细胞(HRMEC)增殖的影响。按照实施例4中所述的方法，在细胞中表达上述质粒对HRMECs管腔形成的影响。按照实施例5中所述的方法，制备重组AAV病毒并鉴定。

表3. XMVA10至XMVA15的序列信息

SEQUENCE LISTING

<110> 合肥星眸生物科技有限公司

<120> 一种编码抗VEGF-A和ANG-2双特异性抗体的AAV载体

<130> AJ8339PI2201

<160> 62

<170> PatentIn version 3.3

<210> 1

<211> 5

<212> PRT

<213> Artificial

<220>

<223> VH抗VEGF-A（G6-23）-CDR1

<400> 1

Asp Tyr Trp Ile His

1 5

<210> 2

<211> 17

<212> PRT

<213> Artificial

<220>

<223> VH抗VEGF-A（G6-23）-CDR2

<400> 2

Gly Ile Thr Pro Ala Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 3

<211> 11

<212> PRT

<213> Artificial

<220>

<223> VH抗VEGF-A（G6-23）-CDR3

<400> 3

Phe Val Phe Phe Leu Pro Tyr Ala Met Asp Tyr

1 5 10

<210> 4

<211> 11

<212> PRT

<213> Artificial

<220>

<223> VL抗VEGF-A（G6-23）-CDR1

<400> 4

Arg Ala Ser Gln Asp Val Ser Thr Ala Val Ala

1 5 10

<210> 5

<211> 7

<212> PRT

<213> Artificial

<220>

<223> VL抗VEGF-A（G6-23）-CDR2

<400> 5

Ser Ala Ser Phe Leu Tyr Ser

1 5

<210> 6

<211> 9

<212> PRT

<213> Artificial

<220>

<223> VL抗VEGF-A（G6-23）-CDR3

<400> 6

Lys Gln Gly Tyr Ala Asn Pro Trp Thr

1 5

<210> 7

<211> 5

<212> PRT

<213> Artificial

<220>

<223> VH抗ANG-2（3.19.3）-CDR1

<400> 7

Asn Tyr Gly Met His

1 5

<210> 8

<211> 17

<212> PRT

<213> Artificial

<220>

<223> VH抗ANG-2（3.19.3）-CDR2

<400> 8

Val Ile Ser His Asp Gly Asn Asn Lys Tyr Tyr Val Asp Ser Val Lys

1 5 10 15

Gly

<210> 9

<211> 14

<212> PRT

<213> Artificial

<220>

<223> VH抗ANG-2（3.19.3）-CDR3

<400> 9

Glu Gly Ile Asp Phe Trp Ser Gly Leu Asn Trp Phe Asp Pro

1 5 10

<210> 10

<211> 12

<212> PRT

<213> Artificial

<220>

<223> VL抗ANG-2（3.19.3）-CDR1

<400> 10

Arg Ala Ser Gln Ser Ile Thr Gly Ser Tyr Leu Ala

1 5 10

<210> 11

<211> 7

<212> PRT

<213> Artificial

<220>

<223> VL抗ANG-2（3.19.3）-CDR2

<400> 11

Gly Ala Ser Ser Trp Ala Thr

1 5

<210> 12

<211> 9

<212> PRT

<213> Artificial

<220>

<223> VL抗ANG-2（3.19.3）-CDR3

<400> 12

Gln Gln Tyr Ser Ser Ser Pro Ile Thr

1 5

<210> 13

<211> 120

<212> PRT

<213> Artificial

<220>

<223> VH抗VEGF-A（G6-23）

<400> 13

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Ser Asp Tyr

20 25 30

Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Gly Ile Thr Pro Ala Gly Gly Tyr Thr Tyr 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

Ala Arg Phe Val Phe Phe Leu Pro Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 14

<211> 108

<212> PRT

<213> Artificial

<220>

<223> VL抗VEGF-A（G6-23）

<400> 14

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 Ser 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 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 Lys Gln Gly Tyr Ala Asn Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

100 105

<210> 15

<211> 124

<212> PRT

<213> Artificial

<220>

<223> VH抗ANG-2（3.19.3）

<400> 15

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 Thr Asn Tyr

20 25 30

Gly Met His Trp Gly Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Ser His Asp Gly Asn Asn Lys Tyr Tyr Val 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 Glu Gly Ile Asp Phe Trp Ser Gly Leu Asn Trp Phe Asp Pro

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala

115 120

<210> 16

<211> 109

<212> PRT

<213> Artificial

<220>

<223> VL抗ANG-2（3.19.3）

<400> 16

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Thr Gly Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Cys Gly Ala Ser Ser Trp Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Ser Ser Ser Pro

85 90 95

Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg

100 105

<210> 17

<211> 496

<212> PRT

<213> Artificial

<220>

<223> VH抗VEGF-A- (G4S)3-VL抗VEGF-A- G4S-VH抗ANG-2- (G4S)3-VL抗ANG-2

<400> 17

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Ser Asp Tyr

20 25 30

Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Gly Ile Thr Pro Ala Gly Gly Tyr Thr Tyr 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

Ala Arg Phe Val Phe Phe Leu Pro 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 Asp Ile Gln Met Thr Gln Ser Pro Ser

130 135 140

Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala

145 150 155 160

Ser Gln Asp Val Ser Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly

165 170 175

Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly

180 185 190

Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu

195 200 205

Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Lys

210 215 220

Gln Gly Tyr Ala Asn Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu

225 230 235 240

Ile Lys Arg Gly Gly Gly Gly Ser Gln Val Gln Leu Val Glu Ser Gly

245 250 255

Gly Gly Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala

260 265 270

Ser Gly Phe Thr Phe Thr Asn Tyr Gly Met His Trp Gly Arg Gln Ala

275 280 285

Pro Gly Lys Gly Leu Glu Trp Val Ala Val Ile Ser His Asp Gly Asn

290 295 300

Asn Lys Tyr Tyr Val Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg

305 310 315 320

Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala

325 330 335

Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Glu Gly Ile Asp Phe Trp

340 345 350

Ser Gly Leu Asn Trp Phe Asp Pro Trp Gly Gln Gly Thr Leu Val Thr

355 360 365

Val Ser Ser Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

370 375 380

Gly Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu

385 390 395 400

Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile

405 410 415

Thr Gly Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

420 425 430

Arg Leu Leu Ile Cys Gly Ala Ser Ser Trp Ala Thr Gly Ile Pro Asp

435 440 445

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

450 455 460

Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Ser

465 470 475 480

Ser Ser Pro Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg

485 490 495

<210> 18

<211> 486

<212> PRT

<213> Artificial

<220>

<223> VL抗ANG-2-G4S-VH抗VEGF-A-(G4S)3-VL抗VEGF-A-G4S-VH抗ANG-2

<400> 18

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Thr Gly Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Cys Gly Ala Ser Ser Trp Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Ser Ser Ser Pro

85 90 95

Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg Gly Gly Gly

100 105 110

Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro

115 120 125

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Ser

130 135 140

Asp Tyr Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu

145 150 155 160

Trp Val Ala Gly Ile Thr Pro Ala Gly Gly Tyr Thr Tyr Tyr Ala Asp

165 170 175

Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr

180 185 190

Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

195 200 205

Tyr Cys Ala Arg Phe Val Phe Phe Leu Pro Tyr Ala Met Asp Tyr Trp

210 215 220

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly

225 230 235 240

Gly Gly Gly Ser Gly Gly Gly Gly Ser 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 Val Ser Thr Ala Val Ala Trp Tyr Gln Gln Lys

275 280 285

Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr

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 Lys Gln Gly Tyr Ala Asn Pro Trp Thr Phe Gly Gln Gly Thr Lys

340 345 350

Val Glu Ile Lys Arg Gly Gly Gly Gly Ser Gln Val Gln Leu Val Glu

355 360 365

Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys

370 375 380

Ala Ala Ser Gly Phe Thr Phe Thr Asn Tyr Gly Met His Trp Gly Arg

385 390 395 400

Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Val Ile Ser His Asp

405 410 415

Gly Asn Asn Lys Tyr Tyr Val Asp Ser Val Lys Gly Arg Phe Thr Ile

420 425 430

Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu

435 440 445

Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Glu Gly Ile Asp

450 455 460

Phe Trp Ser Gly Leu Asn Trp Phe Asp Pro Trp Gly Gln Gly Thr Leu

465 470 475 480

Val Thr Val Ser Ser Ala

485

<210> 19

<211> 486

<212> PRT

<213> Artificial

<220>

<223> VL抗VEGF-A-G4S -VH抗ANG-2-(G4S)3-VL抗ANG-2-G4S -VH抗VEGF-A

<400> 19

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 Ser 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 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 Lys Gln Gly Tyr Ala Asn Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly

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 Thr Asn

130 135 140

Tyr Gly Met His Trp Gly Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp

145 150 155 160

Val Ala Val Ile Ser His Asp Gly Asn Asn Lys Tyr Tyr Val 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 Glu Gly Ile Asp Phe Trp Ser Gly Leu Asn Trp Phe Asp

210 215 220

Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Gly Gly Gly

225 230 235 240

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Leu

245 250 255

Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr

260 265 270

Leu Ser Cys Arg Ala Ser Gln Ser Ile Thr Gly Ser Tyr Leu Ala Trp

275 280 285

Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Cys Gly Ala

290 295 300

Ser Ser Trp Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser

305 310 315 320

Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe

325 330 335

Ala Val Tyr Tyr Cys Gln Gln Tyr Ser Ser Ser Pro Ile Thr Phe Gly

340 345 350

Gln Gly Thr Arg Leu Glu Ile Lys Arg Gly Gly Gly Gly Ser Glu Val

355 360 365

Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu

370 375 380

Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Ser Asp Tyr Trp Ile

385 390 395 400

His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Gly

405 410 415

Ile Thr Pro Ala Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val Lys Gly

420 425 430

Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln

435 440 445

Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg

450 455 460

Phe Val Phe Phe Leu Pro Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

465 470 475 480

Leu Val Thr Val Ser Ser

485

<210> 20

<211> 520

<212> PRT

<213> Artificial

<220>

<223> CD5-sp-VH抗VEGF-A- (G4S)3-VL抗VEGF-A- G4S-VH抗ANG-2-

(G4S)3-VL抗ANG-2

<400> 20

Met Pro Met Gly Ser Leu Gln Pro Leu Ala Thr Leu Tyr Leu Leu Gly

1 5 10 15

Met Leu Val Ala Ser Cys Leu Gly Glu Val Gln Leu Val Glu Ser Gly

20 25 30

Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala

35 40 45

Ser Gly Phe Thr Ile Ser Asp Tyr Trp Ile His Trp Val Arg Gln Ala

50 55 60

Pro Gly Lys Gly Leu Glu Trp Val Ala Gly Ile Thr Pro Ala Gly Gly

65 70 75 80

Tyr Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala

85 90 95

Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala

100 105 110

Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Phe Val Phe Phe Leu Pro

115 120 125

Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

130 135 140

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp

145 150 155 160

Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp

165 170 175

Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala Val

180 185 190

Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

195 200 205

Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

210 215 220

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

225 230 235 240

Asp Phe Ala Thr Tyr Tyr Cys Lys Gln Gly Tyr Ala Asn Pro Trp Thr

245 250 255

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly Ser

260 265 270

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

275 280 285

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asn Tyr

290 295 300

Gly Met His Trp Gly Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

305 310 315 320

Ala Val Ile Ser His Asp Gly Asn Asn Lys Tyr Tyr Val Asp Ser Val

325 330 335

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

340 345 350

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

355 360 365

Ala Arg Glu Gly Ile Asp Phe Trp Ser Gly Leu Asn Trp Phe Asp Pro

370 375 380

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Gly Gly Gly Gly

385 390 395 400

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Leu Thr

405 410 415

Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu

420 425 430

Ser Cys Arg Ala Ser Gln Ser Ile Thr Gly Ser Tyr Leu Ala Trp Tyr

435 440 445

Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Cys Gly Ala Ser

450 455 460

Ser Trp Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly

465 470 475 480

Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala

485 490 495

Val Tyr Tyr Cys Gln Gln Tyr Ser Ser Ser Pro Ile Thr Phe Gly Gln

500 505 510

Gly Thr Arg Leu Glu Ile Lys Arg

515 520

<210> 21

<211> 510

<212> PRT

<213> Artificial

<220>

<223> CD5-sp-VL抗ANG-2-G4S-VH抗VEGF-A-(G4S)3-VL抗VEGF-A-G4S-VH抗ANG-2

<400> 21

Met Pro Met Gly Ser Leu Gln Pro Leu Ala Thr Leu Tyr Leu Leu Gly

1 5 10 15

Met Leu Val Ala Ser Cys Leu Gly Glu Ile Val Leu Thr Gln Ser Pro

20 25 30

Gly Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg

35 40 45

Ala Ser Gln Ser Ile Thr Gly Ser Tyr Leu Ala Trp Tyr Gln Gln Lys

50 55 60

Pro Gly Gln Ala Pro Arg Leu Leu Ile Cys Gly Ala Ser Ser Trp Ala

65 70 75 80

Thr Gly Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe

85 90 95

Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr

100 105 110

Cys Gln Gln Tyr Ser Ser Ser Pro Ile Thr Phe Gly Gln Gly Thr Arg

115 120 125

Leu Glu Ile Lys Arg Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu

130 135 140

Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys

145 150 155 160

Ala Ala Ser Gly Phe Thr Ile Ser Asp Tyr Trp Ile His Trp Val Arg

165 170 175

Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Gly Ile Thr Pro Ala

180 185 190

Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile

195 200 205

Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu

210 215 220

Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Phe Val Phe Phe

225 230 235 240

Leu Pro Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

245 250 255

Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

260 265 270

Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val

275 280 285

Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr

290 295 300

Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu

305 310 315 320

Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser

325 330 335

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln

340 345 350

Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Lys Gln Gly Tyr Ala Asn Pro

355 360 365

Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly

370 375 380

Gly Ser Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro

385 390 395 400

Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr

405 410 415

Asn Tyr Gly Met His Trp Gly Arg Gln Ala Pro Gly Lys Gly Leu Glu

420 425 430

Trp Val Ala Val Ile Ser His Asp Gly Asn Asn Lys Tyr Tyr Val Asp

435 440 445

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr

450 455 460

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

465 470 475 480

Tyr Cys Ala Arg Glu Gly Ile Asp Phe Trp Ser Gly Leu Asn Trp Phe

485 490 495

Asp Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala

500 505 510

<210> 22

<211> 510

<212> PRT

<213> Artificial

<220>

<223> CD5-sp-VL抗VEGF-A-G4S -VH抗ANG-2-(G4S)3-VL抗ANG-2-G4S -VH抗VEGF-A

<400> 22

Met Pro Met Gly Ser Leu Gln Pro Leu Ala Thr Leu Tyr Leu Leu Gly

1 5 10 15

Met Leu Val Ala Ser Cys Leu Gly Asp Ile Gln Met Thr Gln Ser Pro

20 25 30

Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg

35 40 45

Ala Ser Gln Asp Val Ser Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro

50 55 60

Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser

65 70 75 80

Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

85 90 95

Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

100 105 110

Lys Gln Gly Tyr Ala Asn Pro Trp Thr Phe Gly Gln Gly Thr Lys Val

115 120 125

Glu Ile Lys Arg Gly Gly Gly Gly Ser Gln Val Gln Leu Val Glu Ser

130 135 140

Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala

145 150 155 160

Ala Ser Gly Phe Thr Phe Thr Asn Tyr Gly Met His Trp Gly Arg Gln

165 170 175

Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Val Ile Ser His Asp Gly

180 185 190

Asn Asn Lys Tyr Tyr Val Asp Ser Val Lys Gly Arg Phe Thr Ile Ser

195 200 205

Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg

210 215 220

Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Glu Gly Ile Asp Phe

225 230 235 240

Trp Ser Gly Leu Asn Trp Phe Asp Pro Trp Gly Gln Gly Thr Leu Val

245 250 255

Thr Val Ser Ser Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

260 265 270

Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser

275 280 285

Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser

290 295 300

Ile Thr Gly Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala

305 310 315 320

Pro Arg Leu Leu Ile Cys Gly Ala Ser Ser Trp Ala Thr Gly Ile Pro

325 330 335

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

340 345 350

Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr

355 360 365

Ser Ser Ser Pro Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

370 375 380

Arg Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly

385 390 395 400

Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly

405 410 415

Phe Thr Ile Ser Asp Tyr Trp Ile His Trp Val Arg Gln Ala Pro Gly

420 425 430

Lys Gly Leu Glu Trp Val Ala Gly Ile Thr Pro Ala Gly Gly Tyr Thr

435 440 445

Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr

450 455 460

Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp

465 470 475 480

Thr Ala Val Tyr Tyr Cys Ala Arg Phe Val Phe Phe Leu Pro Tyr Ala

485 490 495

Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

500 505 510

<210> 23

<211> 24

<212> PRT

<213> Artificial

<220>

<223> CD5-sp信号肽

<400> 23

Met Pro Met Gly Ser Leu Gln Pro Leu Ala Thr Leu Tyr Leu Leu Gly

1 5 10 15

Met Leu Val Ala Ser Cys Leu Gly

20

<210> 24

<211> 15

<212> DNA

<213> Artificial

<220>

<223> VH抗VEGF-A（G6-23）-CDR1

<400> 24

gactactgga ttcac 15

<210> 25

<211> 51

<212> DNA

<213> Artificial

<220>

<223> VH抗VEGF-A（G6-23）-CDR2

<400> 25

ggaatcaccc cagcaggagg ctacacatac tatgccgaca gcgtgaaggg c 51

<210> 26

<211> 33

<212> DNA

<213> Artificial

<220>

<223> VH抗VEGF-A（G6-23）-CDR3

<400> 26

ttcgtgttct tcctgccata cgccatggat tat 33

<210> 27

<211> 360

<212> DNA

<213> Artificial

<220>

<223> VH抗VEGF-A（G6-23）

<400> 27

gaggtgcagc tggtggagag cggaggagga ctggtgcagc caggaggctc cctgcggctg 60

tcttgcgccg ccagcggctt taccatctcc gactactgga ttcactgggt gagacaggca 120

cctggcaagg gactggagtg ggtggcagga atcaccccag caggaggcta cacatactat 180

gccgacagcg tgaagggccg gttcaccatc tccgccgata cctctaagaa cacagcctat 240

ctgcagatga actccctgcg ggccgaggac acagccgtgt actattgcgc cagattcgtg 300

ttcttcctgc catacgccat ggattattgg ggccagggca ccctggtgac agtgagctcc 360

<210> 28

<211> 33

<212> DNA

<213> Artificial

<220>

<223> VL抗VEGF-A（G6-23）-CDR1

<400> 28

agagcaagcc aggacgtgag caccgcagtg gca 33

<210> 29

<211> 21

<212> DNA

<213> Artificial

<220>

<223> VL抗VEGF-A（G6-23）-CDR2

<400> 29

tccgcctctt tcctgtattc t 21

<210> 30

<211> 27

<212> DNA

<213> Artificial

<220>

<223> VL抗VEGF-A（G6-23）-CDR3

<400> 30

aagcagggct atgccaatcc atggacc 27

<210> 31

<211> 324

<212> DNA

<213> Artificial

<220>

<223> VL抗VEGF-A（G6-23）

<400> 31

gatattcaga tgacacagtc cccatctagc ctgtctgcca gcgtgggcga cagggtgacc 60

atcacatgta gagcaagcca ggacgtgagc accgcagtgg catggtacca gcagaagcct 120

ggcaaggccc caaagctgct gatctactcc gcctctttcc tgtattctgg cgtgccaagc 180

aggtttagcg ggtccggatc tggaaccgac ttcaccctga caatctcctc tctgcagcct 240

gaggattttg ccacatacta ttgcaagcag ggctatgcca atccatggac cttcggccag 300

ggcacaaagg tggagatcaa gagg 324

<210> 32

<211> 15

<212> DNA

<213> Artificial

<220>

<223> VH抗ANG-2（3.19.3）-CDR1

<400> 32

aactatggaa tgcac 15

<210> 33

<211> 51

<212> DNA

<213> Artificial

<220>

<223> VH抗ANG-2（3.19.3）-CDR2

<400> 33

gtgatcagcc acgacggcaa caacaagtac tacgtggaca gcgtgaaggg c 51

<210> 34

<211> 42

<212> DNA

<213> Artificial

<220>

<223> VH抗ANG-2（3.19.3）-CDR3

<400> 34

gagggcatcg acttttggag cggcctgaat tggttcgacc ct 42

<210> 35

<211> 372

<212> DNA

<213> Artificial

<220>

<223> VH抗ANG-2（3.19.3）

<400> 35

caggtgcagc tggttgaatc tggtggcgga gttgtgcagc ctggcagaag cctgagactg 60

tcttgtgccg ccagcggctt caccttcacc aactatggaa tgcactgggg cagacaggcc 120

cctggcaaag gacttgaatg ggtcgccgtg atcagccacg acggcaacaa caagtactac 180

gtggacagcg tgaagggcag attcaccatc agccgggaca acagcaagaa caccctgtac 240

ctgcagatga acagcctgag agccgaggac accgccgtgt actattgtgc cagagagggc 300

atcgactttt ggagcggcct gaattggttc gacccttggg gacagggcac cctggttaca 360

gtttcttctg cc 372

<210> 36

<211> 36

<212> DNA

<213> Artificial

<220>

<223> VL抗ANG-2（3.19.3）-CDR1

<400> 36

agagccagcc agagcatcac cggctcttac ctggct 36

<210> 37

<211> 21

<212> DNA

<213> Artificial

<220>

<223> VL抗ANG-2（3.19.3）-CDR2

<400> 37

ggcgcctctt cttgggccac c 21

<210> 38

<211> 27

<212> DNA

<213> Artificial

<220>

<223> VL抗ANG-2（3.19.3）-CDR3

<400> 38

cagcagtaca gcagcagccc catcacc 27

<210> 39

<211> 327

<212> DNA

<213> Artificial

<220>

<223> VL抗ANG-2（3.19.3）

<400> 39

gagatcgtgc tgacacagag ccctggcaca ctgtcactgt ctccaggcga aagagccaca 60

ctgagctgta gagccagcca gagcatcacc ggctcttacc tggcttggta tcagcagaag 120

cctggacagg cccctagact gctgatttgt ggcgcctctt cttgggccac cggcattccc 180

gatagatttt ctggcagcgg ctccggcacc gacttcaccc tgacaatcag cagactggaa 240

cccgaggact tcgccgtgta ctactgccag cagtacagca gcagccccat cacctttggc 300

cagggcacaa gactggaaat caagcgg 327

<210> 40

<211> 1488

<212> DNA

<213> Artificial

<220>

<223> VH抗VEGF-A- (G4S)3-VL抗VEGF-A- G4S-VH抗ANG-2- (G4S)3-VL抗ANG-2

<400> 40

gaggtgcagc tggtggagag cggaggagga ctggtgcagc caggaggctc cctgcggctg 60

tcttgcgccg ccagcggctt taccatctcc gactactgga ttcactgggt gagacaggca 120

cctggcaagg gactggagtg ggtggcagga atcaccccag caggaggcta cacatactat 180

gccgacagcg tgaagggccg gttcaccatc tccgccgata cctctaagaa cacagcctat 240

ctgcagatga actccctgcg ggccgaggac acagccgtgt actattgcgc cagattcgtg 300

ttcttcctgc catacgccat ggattattgg ggccagggca ccctggtgac agtgagctcc 360

ggtggaggtg gctccggtgg cggtggcagc ggcggtggcg gctctgatat tcagatgaca 420

cagtccccat ctagcctgtc tgccagcgtg ggcgacaggg tgaccatcac atgtagagca 480

agccaggacg tgagcaccgc agtggcatgg taccagcaga agcctggcaa ggccccaaag 540

ctgctgatct actccgcctc tttcctgtat tctggcgtgc caagcaggtt tagcgggtcc 600

ggatctggaa ccgacttcac cctgacaatc tcctctctgc agcctgagga ttttgccaca 660

tactattgca agcagggcta tgccaatcca tggaccttcg gccagggcac aaaggtggag 720

atcaagaggg gcggaggggg ctctcaggtg cagctggttg aatctggtgg cggagttgtg 780

cagcctggca gaagcctgag actgtcttgt gccgccagcg gcttcacctt caccaactat 840

ggaatgcact ggggcagaca ggcccctggc aaaggacttg aatgggtcgc cgtgatcagc 900

cacgacggca acaacaagta ctacgtggac agcgtgaagg gcagattcac catcagccgg 960

gacaacagca agaacaccct gtacctgcag atgaacagcc tgagagccga ggacaccgcc 1020

gtgtactatt gtgccagaga gggcatcgac ttttggagcg gcctgaattg gttcgaccct 1080

tggggacagg gcaccctggt tacagtttct tctgccggtg gaggtggctc cggtggcggt 1140

ggcagcggcg gtggcggctc tgagatcgtg ctgacacaga gccctggcac actgtcactg 1200

tctccaggcg aaagagccac actgagctgt agagccagcc agagcatcac cggctcttac 1260

ctggcttggt atcagcagaa gcctggacag gcccctagac tgctgatttg tggcgcctct 1320

tcttgggcca ccggcattcc cgatagattt tctggcagcg gctccggcac cgacttcacc 1380

ctgacaatca gcagactgga acccgaggac ttcgccgtgt actactgcca gcagtacagc 1440

agcagcccca tcacctttgg ccagggcaca agactggaaa tcaagcgg 1488

<210> 41

<211> 1560

<212> DNA

<213> Artificial

<220>

<223> CD5-sp-VH抗VEGF-A- (G4S)3-VL抗VEGF-A- G4S-VH抗ANG-2-

(G4S)3-VL抗ANG-2

<400> 41

atgccaatgg ggtccctaca acccctggca acactatatc tgctaggtat gctcgtggcc 60

tcctgtttag gagaggtgca gctggtggag agcggaggag gactggtgca gccaggaggc 120

tccctgcggc tgtcttgcgc cgccagcggc tttaccatct ccgactactg gattcactgg 180

gtgagacagg cacctggcaa gggactggag tgggtggcag gaatcacccc agcaggaggc 240

tacacatact atgccgacag cgtgaagggc cggttcacca tctccgccga tacctctaag 300

aacacagcct atctgcagat gaactccctg cgggccgagg acacagccgt gtactattgc 360

gccagattcg tgttcttcct gccatacgcc atggattatt ggggccaggg caccctggtg 420

acagtgagct ccggtggagg tggctccggt ggcggtggca gcggcggtgg cggctctgat 480

attcagatga cacagtcccc atctagcctg tctgccagcg tgggcgacag ggtgaccatc 540

acatgtagag caagccagga cgtgagcacc gcagtggcat ggtaccagca gaagcctggc 600

aaggccccaa agctgctgat ctactccgcc tctttcctgt attctggcgt gccaagcagg 660

tttagcgggt ccggatctgg aaccgacttc accctgacaa tctcctctct gcagcctgag 720

gattttgcca catactattg caagcagggc tatgccaatc catggacctt cggccagggc 780

acaaaggtgg agatcaagag gggcggaggg ggctctcagg tgcagctggt tgaatctggt 840

ggcggagttg tgcagcctgg cagaagcctg agactgtctt gtgccgccag cggcttcacc 900

ttcaccaact atggaatgca ctggggcaga caggcccctg gcaaaggact tgaatgggtc 960

gccgtgatca gccacgacgg caacaacaag tactacgtgg acagcgtgaa gggcagattc 1020

accatcagcc gggacaacag caagaacacc ctgtacctgc agatgaacag cctgagagcc 1080

gaggacaccg ccgtgtacta ttgtgccaga gagggcatcg acttttggag cggcctgaat 1140

tggttcgacc cttggggaca gggcaccctg gttacagttt cttctgccgg tggaggtggc 1200

tccggtggcg gtggcagcgg cggtggcggc tctgagatcg tgctgacaca gagccctggc 1260

acactgtcac tgtctccagg cgaaagagcc acactgagct gtagagccag ccagagcatc 1320

accggctctt acctggcttg gtatcagcag aagcctggac aggcccctag actgctgatt 1380

tgtggcgcct cttcttgggc caccggcatt cccgatagat tttctggcag cggctccggc 1440

accgacttca ccctgacaat cagcagactg gaacccgagg acttcgccgt gtactactgc 1500

cagcagtaca gcagcagccc catcaccttt ggccagggca caagactgga aatcaagcgg 1560

<210> 42

<211> 72

<212> DNA

<213> Artificial

<220>

<223> CD5-sp信号肽

<400> 42

atgccaatgg ggtccctaca acccctggca acactatatc tgctaggtat gctcgtggcc 60

tcctgtttag ga 72

<210> 43

<211> 1458

<212> DNA

<213> Artificial

<220>

<223> VL抗ANG-2-G4S-VH抗VEGF-A-(G4S)3-VL抗VEGF-A-G4S-VH抗ANG-2

<400> 43

gagatcgtgc tgacacagag ccctggcaca ctgtcactgt ctccaggcga aagagccaca 60

ctgagctgta gagccagcca gagcatcacc ggctcttacc tggcttggta tcagcagaag 120

cctggacagg cccctagact gctgatttgt ggcgcctctt cttgggccac cggcattccc 180

gatagatttt ctggcagcgg ctccggcacc gacttcaccc tgacaatcag cagactggaa 240

cccgaggact tcgccgtgta ctactgccag cagtacagca gcagccccat cacctttggc 300

cagggcacaa gactggaaat caagcggggc ggagggggct ctgaggtgca gctggtggag 360

agcggaggag gactggtgca gccaggaggc tccctgcggc tgtcttgcgc cgccagcggc 420

tttaccatct ccgactactg gattcactgg gtgagacagg cacctggcaa gggactggag 480

tgggtggcag gaatcacccc agcaggaggc tacacatact atgccgacag cgtgaagggc 540

cggttcacca tctccgccga tacctctaag aacacagcct atctgcagat gaactccctg 600

cgggccgagg acacagccgt gtactattgc gccagattcg tgttcttcct gccatacgcc 660

atggattatt ggggccaggg caccctggtg acagtgagct ccggtggagg tggctccggt 720

ggcggtggca gcggcggtgg cggctctgat attcagatga cacagtcccc atctagcctg 780

tctgccagcg tgggcgacag ggtgaccatc acatgtagag caagccagga cgtgagcacc 840

gcagtggcat ggtaccagca gaagcctggc aaggccccaa agctgctgat ctactccgcc 900

tctttcctgt attctggcgt gccaagcagg tttagcgggt ccggatctgg aaccgacttc 960

accctgacaa tctcctctct gcagcctgag gattttgcca catactattg caagcagggc 1020

tatgccaatc catggacctt cggccagggc acaaaggtgg agatcaagag gggcggaggg 1080

ggctctcagg tgcagctggt tgaatctggt ggcggagttg tgcagcctgg cagaagcctg 1140

agactgtctt gtgccgccag cggcttcacc ttcaccaact atggaatgca ctggggcaga 1200

caggcccctg gcaaaggact tgaatgggtc gccgtgatca gccacgacgg caacaacaag 1260

tactacgtgg acagcgtgaa gggcagattc accatcagcc gggacaacag caagaacacc 1320

ctgtacctgc agatgaacag cctgagagcc gaggacaccg ccgtgtacta ttgtgccaga 1380

gagggcatcg acttttggag cggcctgaat tggttcgacc cttggggaca gggcaccctg 1440

gttacagttt cttctgcc 1458

<210> 44

<211> 1530

<212> DNA

<213> Artificial

<220>

<223> CD5-sp-VL抗ANG-2-G4S-VH抗VEGF-A-(G4S)3-VL抗VEGF-A-G4S-VH抗ANG-2

<400> 44

atgccaatgg ggtccctaca acccctggca acactatatc tgctaggtat gctcgtggcc 60

tcctgtttag gagagatcgt gctgacacag agccctggca cactgtcact gtctccaggc 120

gaaagagcca cactgagctg tagagccagc cagagcatca ccggctctta cctggcttgg 180

tatcagcaga agcctggaca ggcccctaga ctgctgattt gtggcgcctc ttcttgggcc 240

accggcattc ccgatagatt ttctggcagc ggctccggca ccgacttcac cctgacaatc 300

agcagactgg aacccgagga cttcgccgtg tactactgcc agcagtacag cagcagcccc 360

atcacctttg gccagggcac aagactggaa atcaagcggg gcggaggggg ctctgaggtg 420

cagctggtgg agagcggagg aggactggtg cagccaggag gctccctgcg gctgtcttgc 480

gccgccagcg gctttaccat ctccgactac tggattcact gggtgagaca ggcacctggc 540

aagggactgg agtgggtggc aggaatcacc ccagcaggag gctacacata ctatgccgac 600

agcgtgaagg gccggttcac catctccgcc gatacctcta agaacacagc ctatctgcag 660

atgaactccc tgcgggccga ggacacagcc gtgtactatt gcgccagatt cgtgttcttc 720

ctgccatacg ccatggatta ttggggccag ggcaccctgg tgacagtgag ctccggtgga 780

ggtggctccg gtggcggtgg cagcggcggt ggcggctctg atattcagat gacacagtcc 840

ccatctagcc tgtctgccag cgtgggcgac agggtgacca tcacatgtag agcaagccag 900

gacgtgagca ccgcagtggc atggtaccag cagaagcctg gcaaggcccc aaagctgctg 960

atctactccg cctctttcct gtattctggc gtgccaagca ggtttagcgg gtccggatct 1020

ggaaccgact tcaccctgac aatctcctct ctgcagcctg aggattttgc cacatactat 1080

tgcaagcagg gctatgccaa tccatggacc ttcggccagg gcacaaaggt ggagatcaag 1140

aggggcggag ggggctctca ggtgcagctg gttgaatctg gtggcggagt tgtgcagcct 1200

ggcagaagcc tgagactgtc ttgtgccgcc agcggcttca ccttcaccaa ctatggaatg 1260

cactggggca gacaggcccc tggcaaagga cttgaatggg tcgccgtgat cagccacgac 1320

ggcaacaaca agtactacgt ggacagcgtg aagggcagat tcaccatcag ccgggacaac 1380

agcaagaaca ccctgtacct gcagatgaac agcctgagag ccgaggacac cgccgtgtac 1440

tattgtgcca gagagggcat cgacttttgg agcggcctga attggttcga cccttgggga 1500

cagggcaccc tggttacagt ttcttctgcc 1530

<210> 45

<211> 1458

<212> DNA

<213> Artificial

<220>

<223> VL抗VEGF-A-G4S -VH抗ANG-2-(G4S)3-VL抗ANG-2-G4S -VH抗VEGF-A

<400> 45

gatattcaga tgacacagtc cccatctagc ctgtctgcca gcgtgggcga cagggtgacc 60

atcacatgta gagcaagcca ggacgtgagc accgcagtgg catggtacca gcagaagcct 120

ggcaaggccc caaagctgct gatctactcc gcctctttcc tgtattctgg cgtgccaagc 180

aggtttagcg ggtccggatc tggaaccgac ttcaccctga caatctcctc tctgcagcct 240

gaggattttg ccacatacta ttgcaagcag ggctatgcca atccatggac cttcggccag 300

ggcacaaagg tggagatcaa gaggggcgga gggggctctc aggtgcagct ggttgaatct 360

ggtggcggag ttgtgcagcc tggcagaagc ctgagactgt cttgtgccgc cagcggcttc 420

accttcacca actatggaat gcactggggc agacaggccc ctggcaaagg acttgaatgg 480

gtcgccgtga tcagccacga cggcaacaac aagtactacg tggacagcgt gaagggcaga 540

ttcaccatca gccgggacaa cagcaagaac accctgtacc tgcagatgaa cagcctgaga 600

gccgaggaca ccgccgtgta ctattgtgcc agagagggca tcgacttttg gagcggcctg 660

aattggttcg acccttgggg acagggcacc ctggttacag tttcttctgc cggtggaggt 720

ggctccggtg gcggtggcag cggcggtggc ggctctgaga tcgtgctgac acagagccct 780

ggcacactgt cactgtctcc aggcgaaaga gccacactga gctgtagagc cagccagagc 840

atcaccggct cttacctggc ttggtatcag cagaagcctg gacaggcccc tagactgctg 900

atttgtggcg cctcttcttg ggccaccggc attcccgata gattttctgg cagcggctcc 960

ggcaccgact tcaccctgac aatcagcaga ctggaacccg aggacttcgc cgtgtactac 1020

tgccagcagt acagcagcag ccccatcacc tttggccagg gcacaagact ggaaatcaag 1080

cggggcggag ggggctctga ggtgcagctg gtggagagcg gaggaggact ggtgcagcca 1140

ggaggctccc tgcggctgtc ttgcgccgcc agcggcttta ccatctccga ctactggatt 1200

cactgggtga gacaggcacc tggcaaggga ctggagtggg tggcaggaat caccccagca 1260

ggaggctaca catactatgc cgacagcgtg aagggccggt tcaccatctc cgccgatacc 1320

tctaagaaca cagcctatct gcagatgaac tccctgcggg ccgaggacac agccgtgtac 1380

tattgcgcca gattcgtgtt cttcctgcca tacgccatgg attattgggg ccagggcacc 1440

ctggtgacag tgagctcc 1458

<210> 46

<211> 1530

<212> DNA

<213> Artificial

<220>

<223> CD5-sp-VL抗VEGF-A-G4S -VH抗ANG-2-(G4S)3-VL抗ANG-2-G4S -VH抗VEGF-A

<400> 46

atgccaatgg ggtccctaca acccctggca acactatatc tgctaggtat gctcgtggcc 60

tcctgtttag gagatattca gatgacacag tccccatcta gcctgtctgc cagcgtgggc 120

gacagggtga ccatcacatg tagagcaagc caggacgtga gcaccgcagt ggcatggtac 180

cagcagaagc ctggcaaggc cccaaagctg ctgatctact ccgcctcttt cctgtattct 240

ggcgtgccaa gcaggtttag cgggtccgga tctggaaccg acttcaccct gacaatctcc 300

tctctgcagc ctgaggattt tgccacatac tattgcaagc agggctatgc caatccatgg 360

accttcggcc agggcacaaa ggtggagatc aagaggggcg gagggggctc tcaggtgcag 420

ctggttgaat ctggtggcgg agttgtgcag cctggcagaa gcctgagact gtcttgtgcc 480

gccagcggct tcaccttcac caactatgga atgcactggg gcagacaggc ccctggcaaa 540

ggacttgaat gggtcgccgt gatcagccac gacggcaaca acaagtacta cgtggacagc 600

gtgaagggca gattcaccat cagccgggac aacagcaaga acaccctgta cctgcagatg 660

aacagcctga gagccgagga caccgccgtg tactattgtg ccagagaggg catcgacttt 720

tggagcggcc tgaattggtt cgacccttgg ggacagggca ccctggttac agtttcttct 780

gccggtggag gtggctccgg tggcggtggc agcggcggtg gcggctctga gatcgtgctg 840

acacagagcc ctggcacact gtcactgtct ccaggcgaaa gagccacact gagctgtaga 900

gccagccaga gcatcaccgg ctcttacctg gcttggtatc agcagaagcc tggacaggcc 960

cctagactgc tgatttgtgg cgcctcttct tgggccaccg gcattcccga tagattttct 1020

ggcagcggct ccggcaccga cttcaccctg acaatcagca gactggaacc cgaggacttc 1080

gccgtgtact actgccagca gtacagcagc agccccatca cctttggcca gggcacaaga 1140

ctggaaatca agcggggcgg agggggctct gaggtgcagc tggtggagag cggaggagga 1200

ctggtgcagc caggaggctc cctgcggctg tcttgcgccg ccagcggctt taccatctcc 1260

gactactgga ttcactgggt gagacaggca cctggcaagg gactggagtg ggtggcagga 1320

atcaccccag caggaggcta cacatactat gccgacagcg tgaagggccg gttcaccatc 1380

tccgccgata cctctaagaa cacagcctat ctgcagatga actccctgcg ggccgaggac 1440

acagccgtgt actattgcgc cagattcgtg ttcttcctgc catacgccat ggattattgg 1500

ggccagggca ccctggtgac agtgagctcc 1530

<210> 47

<211> 9

<212> PRT

<213> Artificial

<220>

<223> VL抗VEGF-A（G6-31）-CDR3

<400> 47

Gln Gln Gly Tyr Gly Asn Pro Phe Thr

1 5

<210> 48

<211> 108

<212> PRT

<213> Artificial

<220>

<223> VL抗VEGF-A（G6-31）

<400> 48

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 Ser 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 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 Tyr Gly Asn Pro Phe

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

100 105

<210> 49

<211> 496

<212> PRT

<213> Artificial

<220>

<223> VH抗VEGF-A- (G4S)3-VL抗VEGF-A- G4S-VH抗ANG-2- (G4S)3-VL抗ANG-2

<400> 49

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Ser Asp Tyr

20 25 30

Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Gly Ile Thr Pro Ala Gly Gly Tyr Thr Tyr 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

Ala Arg Phe Val Phe Phe Leu Pro 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 Asp Ile Gln Met Thr Gln Ser Pro Ser

130 135 140

Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala

145 150 155 160

Ser Gln Asp Val Ser Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly

165 170 175

Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly

180 185 190

Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu

195 200 205

Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln

210 215 220

Gln Gly Tyr Gly Asn Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu

225 230 235 240

Ile Lys Arg Gly Gly Gly Gly Ser Gln Val Gln Leu Val Glu Ser Gly

245 250 255

Gly Gly Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala

260 265 270

Ser Gly Phe Thr Phe Thr Asn Tyr Gly Met His Trp Gly Arg Gln Ala

275 280 285

Pro Gly Lys Gly Leu Glu Trp Val Ala Val Ile Ser His Asp Gly Asn

290 295 300

Asn Lys Tyr Tyr Val Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg

305 310 315 320

Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala

325 330 335

Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Glu Gly Ile Asp Phe Trp

340 345 350

Ser Gly Leu Asn Trp Phe Asp Pro Trp Gly Gln Gly Thr Leu Val Thr

355 360 365

Val Ser Ser Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

370 375 380

Gly Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu

385 390 395 400

Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile

405 410 415

Thr Gly Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

420 425 430

Arg Leu Leu Ile Cys Gly Ala Ser Ser Trp Ala Thr Gly Ile Pro Asp

435 440 445

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

450 455 460

Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Ser

465 470 475 480

Ser Ser Pro Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg

485 490 495

<210> 50

<211> 520

<212> PRT

<213> Artificial

<220>

<223> CD5-sp-VH抗VEGF-A- (G4S)3-VL抗VEGF-A- G4S-VH抗ANG-2-

(G4S)3-VL抗ANG-2

<400> 50

Met Pro Met Gly Ser Leu Gln Pro Leu Ala Thr Leu Tyr Leu Leu Gly

1 5 10 15

Met Leu Val Ala Ser Cys Leu Gly Glu Val Gln Leu Val Glu Ser Gly

20 25 30

Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala

35 40 45

Ser Gly Phe Thr Ile Ser Asp Tyr Trp Ile His Trp Val Arg Gln Ala

50 55 60

Pro Gly Lys Gly Leu Glu Trp Val Ala Gly Ile Thr Pro Ala Gly Gly

65 70 75 80

Tyr Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala

85 90 95

Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala

100 105 110

Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Phe Val Phe Phe Leu Pro

115 120 125

Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

130 135 140

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp

145 150 155 160

Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp

165 170 175

Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala Val

180 185 190

Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

195 200 205

Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

210 215 220

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

225 230 235 240

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Gly Asn Pro Phe Thr

245 250 255

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly Ser

260 265 270

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

275 280 285

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asn Tyr

290 295 300

Gly Met His Trp Gly Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

305 310 315 320

Ala Val Ile Ser His Asp Gly Asn Asn Lys Tyr Tyr Val Asp Ser Val

325 330 335

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

340 345 350

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

355 360 365

Ala Arg Glu Gly Ile Asp Phe Trp Ser Gly Leu Asn Trp Phe Asp Pro

370 375 380

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Gly Gly Gly Gly

385 390 395 400

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Leu Thr

405 410 415

Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu

420 425 430

Ser Cys Arg Ala Ser Gln Ser Ile Thr Gly Ser Tyr Leu Ala Trp Tyr

435 440 445

Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Cys Gly Ala Ser

450 455 460

Ser Trp Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly

465 470 475 480

Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala

485 490 495

Val Tyr Tyr Cys Gln Gln Tyr Ser Ser Ser Pro Ile Thr Phe Gly Gln

500 505 510

Gly Thr Arg Leu Glu Ile Lys Arg

515 520

<210> 51

<211> 486

<212> PRT

<213> Artificial

<220>

<223> VL抗ANG-2-G4S-VH抗VEGF-A-(G4S)3-VL抗VEGF-A-G4S-VH抗ANG-2

<400> 51

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Thr Gly Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Cys Gly Ala Ser Ser Trp Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Ser Ser Ser Pro

85 90 95

Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg Gly Gly Gly

100 105 110

Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro

115 120 125

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Ser

130 135 140

Asp Tyr Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu

145 150 155 160

Trp Val Ala Gly Ile Thr Pro Ala Gly Gly Tyr Thr Tyr Tyr Ala Asp

165 170 175

Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr

180 185 190

Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

195 200 205

Tyr Cys Ala Arg Phe Val Phe Phe Leu Pro Tyr Ala Met Asp Tyr Trp

210 215 220

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly

225 230 235 240

Gly Gly Gly Ser Gly Gly Gly Gly Ser 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 Val Ser Thr Ala Val Ala Trp Tyr Gln Gln Lys

275 280 285

Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr

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 Tyr Gly Asn Pro Phe Thr Phe Gly Gln Gly Thr Lys

340 345 350

Val Glu Ile Lys Arg Gly Gly Gly Gly Ser Gln Val Gln Leu Val Glu

355 360 365

Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys

370 375 380

Ala Ala Ser Gly Phe Thr Phe Thr Asn Tyr Gly Met His Trp Gly Arg

385 390 395 400

Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Val Ile Ser His Asp

405 410 415

Gly Asn Asn Lys Tyr Tyr Val Asp Ser Val Lys Gly Arg Phe Thr Ile

420 425 430

Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu

435 440 445

Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Glu Gly Ile Asp

450 455 460

Phe Trp Ser Gly Leu Asn Trp Phe Asp Pro Trp Gly Gln Gly Thr Leu

465 470 475 480

Val Thr Val Ser Ser Ala

485

<210> 52

<211> 510

<212> PRT

<213> Artificial

<220>

<223> CD5-sp-VL抗ANG-2-G4S-VH抗VEGF-A-(G4S)3-VL抗VEGF-A-G4S-VH抗ANG-2

<400> 52

Met Pro Met Gly Ser Leu Gln Pro Leu Ala Thr Leu Tyr Leu Leu Gly

1 5 10 15

Met Leu Val Ala Ser Cys Leu Gly Glu Ile Val Leu Thr Gln Ser Pro

20 25 30

Gly Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg

35 40 45

Ala Ser Gln Ser Ile Thr Gly Ser Tyr Leu Ala Trp Tyr Gln Gln Lys

50 55 60

Pro Gly Gln Ala Pro Arg Leu Leu Ile Cys Gly Ala Ser Ser Trp Ala

65 70 75 80

Thr Gly Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe

85 90 95

Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr

100 105 110

Cys Gln Gln Tyr Ser Ser Ser Pro Ile Thr Phe Gly Gln Gly Thr Arg

115 120 125

Leu Glu Ile Lys Arg Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu

130 135 140

Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys

145 150 155 160

Ala Ala Ser Gly Phe Thr Ile Ser Asp Tyr Trp Ile His Trp Val Arg

165 170 175

Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Gly Ile Thr Pro Ala

180 185 190

Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile

195 200 205

Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu

210 215 220

Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Phe Val Phe Phe

225 230 235 240

Leu Pro Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

245 250 255

Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

260 265 270

Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val

275 280 285

Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr

290 295 300

Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu

305 310 315 320

Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser

325 330 335

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln

340 345 350

Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Gly Asn Pro

355 360 365

Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly

370 375 380

Gly Ser Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro

385 390 395 400

Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr

405 410 415

Asn Tyr Gly Met His Trp Gly Arg Gln Ala Pro Gly Lys Gly Leu Glu

420 425 430

Trp Val Ala Val Ile Ser His Asp Gly Asn Asn Lys Tyr Tyr Val Asp

435 440 445

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr

450 455 460

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

465 470 475 480

Tyr Cys Ala Arg Glu Gly Ile Asp Phe Trp Ser Gly Leu Asn Trp Phe

485 490 495

Asp Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala

500 505 510

<210> 53

<211> 486

<212> PRT

<213> Artificial

<220>

<223> VL抗VEGF-A-G4S -VH抗ANG-2-(G4S)3-VL抗ANG-2-G4S -VH抗VEGF-A

<400> 53

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 Ser 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 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 Tyr Gly Asn Pro Phe

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly

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 Thr Asn

130 135 140

Tyr Gly Met His Trp Gly Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp

145 150 155 160

Val Ala Val Ile Ser His Asp Gly Asn Asn Lys Tyr Tyr Val 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 Glu Gly Ile Asp Phe Trp Ser Gly Leu Asn Trp Phe Asp

210 215 220

Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Gly Gly Gly

225 230 235 240

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Leu

245 250 255

Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr

260 265 270

Leu Ser Cys Arg Ala Ser Gln Ser Ile Thr Gly Ser Tyr Leu Ala Trp

275 280 285

Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Cys Gly Ala

290 295 300

Ser Ser Trp Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser

305 310 315 320

Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe

325 330 335

Ala Val Tyr Tyr Cys Gln Gln Tyr Ser Ser Ser Pro Ile Thr Phe Gly

340 345 350

Gln Gly Thr Arg Leu Glu Ile Lys Arg Gly Gly Gly Gly Ser Glu Val

355 360 365

Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu

370 375 380

Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Ser Asp Tyr Trp Ile

385 390 395 400

His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Gly

405 410 415

Ile Thr Pro Ala Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val Lys Gly

420 425 430

Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln

435 440 445

Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg

450 455 460

Phe Val Phe Phe Leu Pro Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

465 470 475 480

Leu Val Thr Val Ser Ser

485

<210> 54

<211> 510

<212> PRT

<213> Artificial

<220>

<223> CD5-sp-VL抗VEGF-A-G4S -VH抗ANG-2-(G4S)3-VL抗ANG-2-G4S -VH抗VEGF-A

<400> 54

Met Pro Met Gly Ser Leu Gln Pro Leu Ala Thr Leu Tyr Leu Leu Gly

1 5 10 15

Met Leu Val Ala Ser Cys Leu Gly Asp Ile Gln Met Thr Gln Ser Pro

20 25 30

Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg

35 40 45

Ala Ser Gln Asp Val Ser Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro

50 55 60

Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser

65 70 75 80

Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

85 90 95

Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

100 105 110

Gln Gln Gly Tyr Gly Asn Pro Phe Thr Phe Gly Gln Gly Thr Lys Val

115 120 125

Glu Ile Lys Arg Gly Gly Gly Gly Ser Gln Val Gln Leu Val Glu Ser

130 135 140

Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala

145 150 155 160

Ala Ser Gly Phe Thr Phe Thr Asn Tyr Gly Met His Trp Gly Arg Gln

165 170 175

Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Val Ile Ser His Asp Gly

180 185 190

Asn Asn Lys Tyr Tyr Val Asp Ser Val Lys Gly Arg Phe Thr Ile Ser

195 200 205

Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg

210 215 220

Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Glu Gly Ile Asp Phe

225 230 235 240

Trp Ser Gly Leu Asn Trp Phe Asp Pro Trp Gly Gln Gly Thr Leu Val

245 250 255

Thr Val Ser Ser Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

260 265 270

Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser

275 280 285

Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser

290 295 300

Ile Thr Gly Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala

305 310 315 320

Pro Arg Leu Leu Ile Cys Gly Ala Ser Ser Trp Ala Thr Gly Ile Pro

325 330 335

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

340 345 350

Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr

355 360 365

Ser Ser Ser Pro Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

370 375 380

Arg Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly

385 390 395 400

Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly

405 410 415

Phe Thr Ile Ser Asp Tyr Trp Ile His Trp Val Arg Gln Ala Pro Gly

420 425 430

Lys Gly Leu Glu Trp Val Ala Gly Ile Thr Pro Ala Gly Gly Tyr Thr

435 440 445

Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr

450 455 460

Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp

465 470 475 480

Thr Ala Val Tyr Tyr Cys Ala Arg Phe Val Phe Phe Leu Pro Tyr Ala

485 490 495

Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

500 505 510

<210> 55

<211> 27

<212> DNA

<213> Artificial

<220>

<223> VL抗VEGF-A（G6-31）-CDR3

<400> 55

cagcagggct atggcaatcc attcacc 27

<210> 56

<211> 324

<212> DNA

<213> Artificial

<220>

<223> VL抗VEGF-A（G6-31）

<400> 56

gatattcaga tgacacagtc cccatctagc ctgtctgcca gcgtgggcga cagggtgacc 60

atcacatgta gagcaagcca ggacgtgagc accgcagtgg catggtacca gcagaagcct 120

ggcaaggccc caaagctgct gatctactcc gcctctttcc tgtattctgg cgtgccaagc 180

aggtttagcg ggtccggatc tggaaccgac ttcaccctga caatctcctc tctgcagcct 240

gaggattttg ccacatacta ttgccagcag ggctatggca atccattcac ctttggccag 300

ggcacaaagg tggagatcaa gagg 324

<210> 57

<211> 1488

<212> DNA

<213> Artificial

<220>

<223> VH抗VEGF-A- (G4S)3-VL抗VEGF-A- G4S-VH抗ANG-2- (G4S)3-VL抗ANG-2

<400> 57

gaggtgcagc tggtggagag cggaggagga ctggtgcagc caggaggctc cctgcggctg 60

tcttgcgccg ccagcggctt taccatctcc gactactgga ttcactgggt gagacaggca 120

cctggcaagg gactggagtg ggtggcagga atcaccccag caggaggcta cacatactat 180

gccgacagcg tgaagggccg gttcaccatc tccgccgata cctctaagaa cacagcctat 240

ctgcagatga actccctgcg ggccgaggac acagccgtgt actattgcgc cagattcgtg 300

ttcttcctgc catacgccat ggattattgg ggccagggca ccctggtgac agtgagctcc 360

ggtggaggtg gctccggtgg cggtggcagc ggcggtggcg gctctgatat tcagatgaca 420

cagtccccat ctagcctgtc tgccagcgtg ggcgacaggg tgaccatcac atgtagagca 480

agccaggacg tgagcaccgc agtggcatgg taccagcaga agcctggcaa ggccccaaag 540

ctgctgatct actccgcctc tttcctgtat tctggcgtgc caagcaggtt tagcgggtcc 600

ggatctggaa ccgacttcac cctgacaatc tcctctctgc agcctgagga ttttgccaca 660

tactattgcc agcagggcta tggcaatcca ttcacctttg gccagggcac aaaggtggag 720

atcaagaggg gcggaggggg ctctcaggtg cagctggttg aatctggtgg cggagttgtg 780

cagcctggca gaagcctgag actgtcttgt gccgccagcg gcttcacctt caccaactat 840

ggaatgcact ggggcagaca ggcccctggc aaaggacttg aatgggtcgc cgtgatcagc 900

cacgacggca acaacaagta ctacgtggac agcgtgaagg gcagattcac catcagccgg 960

gacaacagca agaacaccct gtacctgcag atgaacagcc tgagagccga ggacaccgcc 1020

gtgtactatt gtgccagaga gggcatcgac ttttggagcg gcctgaattg gttcgaccct 1080

tggggacagg gcaccctggt tacagtttct tctgccggtg gaggtggctc cggtggcggt 1140

ggcagcggcg gtggcggctc tgagatcgtg ctgacacaga gccctggcac actgtcactg 1200

tctccaggcg aaagagccac actgagctgt agagccagcc agagcatcac cggctcttac 1260

ctggcttggt atcagcagaa gcctggacag gcccctagac tgctgatttg tggcgcctct 1320

tcttgggcca ccggcattcc cgatagattt tctggcagcg gctccggcac cgacttcacc 1380

ctgacaatca gcagactgga acccgaggac ttcgccgtgt actactgcca gcagtacagc 1440

agcagcccca tcacctttgg ccagggcaca agactggaaa tcaagcgg 1488

<210> 58

<211> 1560

<212> DNA

<213> Artificial

<220>

<223> CD5-sp-VH抗VEGF-A- (G4S)3-VL抗VEGF-A- G4S-VH抗ANG-2-

(G4S)3-VL抗ANG-2

<400> 58

atgccaatgg ggtccctaca acccctggca acactatatc tgctaggtat gctcgtggcc 60

tcctgtttag gagaggtgca gctggtggag agcggaggag gactggtgca gccaggaggc 120

tccctgcggc tgtcttgcgc cgccagcggc tttaccatct ccgactactg gattcactgg 180

gtgagacagg cacctggcaa gggactggag tgggtggcag gaatcacccc agcaggaggc 240

tacacatact atgccgacag cgtgaagggc cggttcacca tctccgccga tacctctaag 300

aacacagcct atctgcagat gaactccctg cgggccgagg acacagccgt gtactattgc 360

gccagattcg tgttcttcct gccatacgcc atggattatt ggggccaggg caccctggtg 420

acagtgagct ccggtggagg tggctccggt ggcggtggca gcggcggtgg cggctctgat 480

attcagatga cacagtcccc atctagcctg tctgccagcg tgggcgacag ggtgaccatc 540

acatgtagag caagccagga cgtgagcacc gcagtggcat ggtaccagca gaagcctggc 600

aaggccccaa agctgctgat ctactccgcc tctttcctgt attctggcgt gccaagcagg 660

tttagcgggt ccggatctgg aaccgacttc accctgacaa tctcctctct gcagcctgag 720

gattttgcca catactattg ccagcagggc tatggcaatc cattcacctt tggccagggc 780

acaaaggtgg agatcaagag gggcggaggg ggctctcagg tgcagctggt tgaatctggt 840

ggcggagttg tgcagcctgg cagaagcctg agactgtctt gtgccgccag cggcttcacc 900

ttcaccaact atggaatgca ctggggcaga caggcccctg gcaaaggact tgaatgggtc 960

gccgtgatca gccacgacgg caacaacaag tactacgtgg acagcgtgaa gggcagattc 1020

accatcagcc gggacaacag caagaacacc ctgtacctgc agatgaacag cctgagagcc 1080

gaggacaccg ccgtgtacta ttgtgccaga gagggcatcg acttttggag cggcctgaat 1140

tggttcgacc cttggggaca gggcaccctg gttacagttt cttctgccgg tggaggtggc 1200

tccggtggcg gtggcagcgg cggtggcggc tctgagatcg tgctgacaca gagccctggc 1260

acactgtcac tgtctccagg cgaaagagcc acactgagct gtagagccag ccagagcatc 1320

accggctctt acctggcttg gtatcagcag aagcctggac aggcccctag actgctgatt 1380

tgtggcgcct cttcttgggc caccggcatt cccgatagat tttctggcag cggctccggc 1440

accgacttca ccctgacaat cagcagactg gaacccgagg acttcgccgt gtactactgc 1500

cagcagtaca gcagcagccc catcaccttt ggccagggca caagactgga aatcaagcgg 1560

<210> 59

<211> 1458

<212> DNA

<213> Artificial

<220>

<223> VL抗ANG-2-G4S-VH抗VEGF-A-(G4S)3-VL抗VEGF-A-G4S-VH抗ANG-2

<400> 59

gagatcgtgc tgacacagag ccctggcaca ctgtcactgt ctccaggcga aagagccaca 60

ctgagctgta gagccagcca gagcatcacc ggctcttacc tggcttggta tcagcagaag 120

cctggacagg cccctagact gctgatttgt ggcgcctctt cttgggccac cggcattccc 180

gatagatttt ctggcagcgg ctccggcacc gacttcaccc tgacaatcag cagactggaa 240

cccgaggact tcgccgtgta ctactgccag cagtacagca gcagccccat cacctttggc 300

cagggcacaa gactggaaat caagcggggc ggagggggct ctgaggtgca gctggtggag 360

agcggaggag gactggtgca gccaggaggc tccctgcggc tgtcttgcgc cgccagcggc 420

tttaccatct ccgactactg gattcactgg gtgagacagg cacctggcaa gggactggag 480

tgggtggcag gaatcacccc agcaggaggc tacacatact atgccgacag cgtgaagggc 540

cggttcacca tctccgccga tacctctaag aacacagcct atctgcagat gaactccctg 600

cgggccgagg acacagccgt gtactattgc gccagattcg tgttcttcct gccatacgcc 660

atggattatt ggggccaggg caccctggtg acagtgagct ccggtggagg tggctccggt 720

ggcggtggca gcggcggtgg cggctctgat attcagatga cacagtcccc atctagcctg 780

tctgccagcg tgggcgacag ggtgaccatc acatgtagag caagccagga cgtgagcacc 840

gcagtggcat ggtaccagca gaagcctggc aaggccccaa agctgctgat ctactccgcc 900

tctttcctgt attctggcgt gccaagcagg tttagcgggt ccggatctgg aaccgacttc 960

accctgacaa tctcctctct gcagcctgag gattttgcca catactattg ccagcagggc 1020

tatggcaatc cattcacctt tggccagggc acaaaggtgg agatcaagag gggcggaggg 1080

ggctctcagg tgcagctggt tgaatctggt ggcggagttg tgcagcctgg cagaagcctg 1140

agactgtctt gtgccgccag cggcttcacc ttcaccaact atggaatgca ctggggcaga 1200

caggcccctg gcaaaggact tgaatgggtc gccgtgatca gccacgacgg caacaacaag 1260

tactacgtgg acagcgtgaa gggcagattc accatcagcc gggacaacag caagaacacc 1320

ctgtacctgc agatgaacag cctgagagcc gaggacaccg ccgtgtacta ttgtgccaga 1380

gagggcatcg acttttggag cggcctgaat tggttcgacc cttggggaca gggcaccctg 1440

gttacagttt cttctgcc 1458

<210> 60

<211> 1530

<212> DNA

<213> Artificial

<220>

<223> CD5-sp-VL抗ANG-2-G4S-VH抗VEGF-A-(G4S)3-VL抗VEGF-A-G4S-VH抗ANG-2

<400> 60

atgccaatgg ggtccctaca acccctggca acactatatc tgctaggtat gctcgtggcc 60

tcctgtttag gagagatcgt gctgacacag agccctggca cactgtcact gtctccaggc 120

gaaagagcca cactgagctg tagagccagc cagagcatca ccggctctta cctggcttgg 180

tatcagcaga agcctggaca ggcccctaga ctgctgattt gtggcgcctc ttcttgggcc 240

accggcattc ccgatagatt ttctggcagc ggctccggca ccgacttcac cctgacaatc 300

agcagactgg aacccgagga cttcgccgtg tactactgcc agcagtacag cagcagcccc 360

atcacctttg gccagggcac aagactggaa atcaagcggg gcggaggggg ctctgaggtg 420

cagctggtgg agagcggagg aggactggtg cagccaggag gctccctgcg gctgtcttgc 480

gccgccagcg gctttaccat ctccgactac tggattcact gggtgagaca ggcacctggc 540

aagggactgg agtgggtggc aggaatcacc ccagcaggag gctacacata ctatgccgac 600

agcgtgaagg gccggttcac catctccgcc gatacctcta agaacacagc ctatctgcag 660

atgaactccc tgcgggccga ggacacagcc gtgtactatt gcgccagatt cgtgttcttc 720

ctgccatacg ccatggatta ttggggccag ggcaccctgg tgacagtgag ctccggtgga 780

ggtggctccg gtggcggtgg cagcggcggt ggcggctctg atattcagat gacacagtcc 840

ccatctagcc tgtctgccag cgtgggcgac agggtgacca tcacatgtag agcaagccag 900

gacgtgagca ccgcagtggc atggtaccag cagaagcctg gcaaggcccc aaagctgctg 960

atctactccg cctctttcct gtattctggc gtgccaagca ggtttagcgg gtccggatct 1020

ggaaccgact tcaccctgac aatctcctct ctgcagcctg aggattttgc cacatactat 1080

tgccagcagg gctatggcaa tccattcacc tttggccagg gcacaaaggt ggagatcaag 1140

aggggcggag ggggctctca ggtgcagctg gttgaatctg gtggcggagt tgtgcagcct 1200

ggcagaagcc tgagactgtc ttgtgccgcc agcggcttca ccttcaccaa ctatggaatg 1260

cactggggca gacaggcccc tggcaaagga cttgaatggg tcgccgtgat cagccacgac 1320

ggcaacaaca agtactacgt ggacagcgtg aagggcagat tcaccatcag ccgggacaac 1380

agcaagaaca ccctgtacct gcagatgaac agcctgagag ccgaggacac cgccgtgtac 1440

tattgtgcca gagagggcat cgacttttgg agcggcctga attggttcga cccttgggga 1500

cagggcaccc tggttacagt ttcttctgcc 1530

<210> 61

<211> 1458

<212> DNA

<213> Artificial

<220>

<223> VL抗VEGF-A-G4S -VH抗ANG-2-(G4S)3-VL抗ANG-2-G4S -VH抗VEGF-A

<400> 61

gatattcaga tgacacagtc cccatctagc ctgtctgcca gcgtgggcga cagggtgacc 60

atcacatgta gagcaagcca ggacgtgagc accgcagtgg catggtacca gcagaagcct 120

ggcaaggccc caaagctgct gatctactcc gcctctttcc tgtattctgg cgtgccaagc 180

aggtttagcg ggtccggatc tggaaccgac ttcaccctga caatctcctc tctgcagcct 240

gaggattttg ccacatacta ttgccagcag ggctatggca atccattcac ctttggccag 300

ggcacaaagg tggagatcaa gaggggcgga gggggctctc aggtgcagct ggttgaatct 360

ggtggcggag ttgtgcagcc tggcagaagc ctgagactgt cttgtgccgc cagcggcttc 420

accttcacca actatggaat gcactggggc agacaggccc ctggcaaagg acttgaatgg 480

gtcgccgtga tcagccacga cggcaacaac aagtactacg tggacagcgt gaagggcaga 540

ttcaccatca gccgggacaa cagcaagaac accctgtacc tgcagatgaa cagcctgaga 600

gccgaggaca ccgccgtgta ctattgtgcc agagagggca tcgacttttg gagcggcctg 660

aattggttcg acccttgggg acagggcacc ctggttacag tttcttctgc cggtggaggt 720

ggctccggtg gcggtggcag cggcggtggc ggctctgaga tcgtgctgac acagagccct 780

ggcacactgt cactgtctcc aggcgaaaga gccacactga gctgtagagc cagccagagc 840

atcaccggct cttacctggc ttggtatcag cagaagcctg gacaggcccc tagactgctg 900

atttgtggcg cctcttcttg ggccaccggc attcccgata gattttctgg cagcggctcc 960

ggcaccgact tcaccctgac aatcagcaga ctggaacccg aggacttcgc cgtgtactac 1020

tgccagcagt acagcagcag ccccatcacc tttggccagg gcacaagact ggaaatcaag 1080

cggggcggag ggggctctga ggtgcagctg gtggagagcg gaggaggact ggtgcagcca 1140

ggaggctccc tgcggctgtc ttgcgccgcc agcggcttta ccatctccga ctactggatt 1200

cactgggtga gacaggcacc tggcaaggga ctggagtggg tggcaggaat caccccagca 1260

ggaggctaca catactatgc cgacagcgtg aagggccggt tcaccatctc cgccgatacc 1320

tctaagaaca cagcctatct gcagatgaac tccctgcggg ccgaggacac agccgtgtac 1380

tattgcgcca gattcgtgtt cttcctgcca tacgccatgg attattgggg ccagggcacc 1440

ctggtgacag tgagctcc 1458

<210> 62

<211> 1530

<212> DNA

<213> Artificial

<220>

<223> CD5-sp-VL抗VEGF-A-G4S -VH抗ANG-2-(G4S)3-VL抗ANG-2-G4S -VH抗VEGF-A

<400> 62

atgccaatgg ggtccctaca acccctggca acactatatc tgctaggtat gctcgtggcc 60

tcctgtttag gagatattca gatgacacag tccccatcta gcctgtctgc cagcgtgggc 120

gacagggtga ccatcacatg tagagcaagc caggacgtga gcaccgcagt ggcatggtac 180

cagcagaagc ctggcaaggc cccaaagctg ctgatctact ccgcctcttt cctgtattct 240

ggcgtgccaa gcaggtttag cgggtccgga tctggaaccg acttcaccct gacaatctcc 300

tctctgcagc ctgaggattt tgccacatac tattgccagc agggctatgg caatccattc 360

acctttggcc agggcacaaa ggtggagatc aagaggggcg gagggggctc tcaggtgcag 420

ctggttgaat ctggtggcgg agttgtgcag cctggcagaa gcctgagact gtcttgtgcc 480

gccagcggct tcaccttcac caactatgga atgcactggg gcagacaggc ccctggcaaa 540

ggacttgaat gggtcgccgt gatcagccac gacggcaaca acaagtacta cgtggacagc 600

gtgaagggca gattcaccat cagccgggac aacagcaaga acaccctgta cctgcagatg 660

aacagcctga gagccgagga caccgccgtg tactattgtg ccagagaggg catcgacttt 720

tggagcggcc tgaattggtt cgacccttgg ggacagggca ccctggttac agtttcttct 780

gccggtggag gtggctccgg tggcggtggc agcggcggtg gcggctctga gatcgtgctg 840

acacagagcc ctggcacact gtcactgtct ccaggcgaaa gagccacact gagctgtaga 900

gccagccaga gcatcaccgg ctcttacctg gcttggtatc agcagaagcc tggacaggcc 960

cctagactgc tgatttgtgg cgcctcttct tgggccaccg gcattcccga tagattttct 1020

ggcagcggct ccggcaccga cttcaccctg acaatcagca gactggaacc cgaggacttc 1080

gccgtgtact actgccagca gtacagcagc agccccatca cctttggcca gggcacaaga 1140

ctggaaatca agcggggcgg agggggctct gaggtgcagc tggtggagag cggaggagga 1200

ctggtgcagc caggaggctc cctgcggctg tcttgcgccg ccagcggctt taccatctcc 1260

gactactgga ttcactgggt gagacaggca cctggcaagg gactggagtg ggtggcagga 1320

atcaccccag caggaggcta cacatactat gccgacagcg tgaagggccg gttcaccatc 1380

tccgccgata cctctaagaa cacagcctat ctgcagatga actccctgcg ggccgaggac 1440

acagccgtgt actattgcgc cagattcgtg ttcttcctgc catacgccat ggattattgg 1500

ggccagggca ccctggtgac agtgagctcc 1530

Claims

1.一种双特异性抗体，其包括抗VEGF-A和ANG-2的结合结构域，所述双特异性抗体重链可变区(VH)和轻链可变区(VL)从N-末端到C-末端按照如下顺序排列：

1)VH_抗VEGF-A-VL_抗VEGF-A-VH_抗ANG-2-VL_抗ANG-2；

2)VL_抗ANG-2-VH_抗VEGF-A-VL_抗VEGF-A-VH_抗ANG-2；或

3)VL_抗VEGF-A-VH_抗ANG-2-VL_抗ANG-2-VH_抗VEGF-A；

其中，所述VH_抗VEGF-A包括SEQ ID NO:1所示的CDR1，SEQ ID NO:2所示的CDR2；SEQ IDNO:3所示的CDR3；

所述VL_抗ANG-2包括SEQ ID NO:10所示的CDR1，SEQ ID NO:11所示的CDR2；SEQ ID NO:12所示的CDR3。

2.如权利要求1所述的双特异性抗体，其中

所述VH_抗VEGF-A包括SEQ ID NO:13所示的序列或与SEQ ID NO:13序列同一性为70％、80％、90％、95％、或99％的序列；

3.如权利要求1所述的双特异性抗体，其中所述抗体包括SEQ ID NO:17、18、19、49、51或53所示的序列或与SEQ ID NO:17、18、19、49、51或53序列同一性为70％、80％、90％、95％、或99％的序列。

4.如权利要求1所述的双特异性抗体，所述双特异性抗体的N-末端包含信号肽序列或标签序列，优选所述信号肽序列为如SEQ ID NO:23所示的CD5-sp信号肽。

5.如权利要求4所述的双特异性抗体，其中所述抗体包括SEQ ID NO:20、21、22、50、52或54所示的序列或与SEQ ID NO:20、21、22、50、52或54序列同一性为70％、80％、90％、95％、或99％的序列。

6.如权利要求1所述的双特异性抗体，其中所述重链可变区(VH)和轻链可变区(VL)之间由(G₄S)n链接，其中n＝1至4间任意整数。

7.如权利要求1所述的双特异性抗体，其中所述双特异性抗体的结构为：

VH_抗VEGF-A-(G₄S)₃-VL_抗VEGF-A-G₄S-VH_抗ANG-2-(G₄S)₃-VL_抗ANG-2；

VL_抗ANG-2-G₄S-VH_抗VEGF-A-(G₄S)₃-VL_抗VEGF-A-G₄S-VH_抗ANG-2；

VL_抗VEGF-A-G₄S-VH_抗ANG-2-(G₄S)₃-VL_抗ANG-2-G₄S-VH_抗VEGF-A；

CD5-sp-VH_抗VEGF-A-(G₄S)₃-VL_抗VEGF-A-G₄S-VH_抗ANG-2-(G₄S)_3-VL_抗ANG-2；

CD5-sp-VL_抗ANG-2-G₄S-VH_抗VEGF-A-(G₄S)₃-VL_抗VEGF-A-G₄S-VH_抗ANG-2；或

CD5-sp-VL_抗VEGF-A-G₄S-VH_抗ANG-2-(G₄S)₃-VL_抗ANG-2-G₄S-VH_抗VEGF-A。

8.一种核酸序列，其编码如权利要求1-7所述的双特异性抗体。

9.一种载体，其包括编码如权利要求1-7所述的双特异性抗体的核酸序列，所述载体优选为AAV病毒载体。

10.如权利要求9所述的AAV病毒载体，进一步包括：5’ITR和3’ITR、启动子、polyA序列。

11.一种AAV病毒颗粒，包括如权利要求9或10所述的AAV病毒载体和衣壳蛋白。

12.如权利要求11所述的AAV病毒颗粒，其中所述衣壳蛋白的血清型为AAV1、AAV2、AAV4、AAV5、AAV6、AAV7、AAV8、或AAV9。

13.一种药物组合物，包括如权利要求1-7所述的双特异性抗体、如权利要求8所述的核酸序列、如权利要求9或10所述的载体、如权利要求11或12所述的AAV病毒颗粒中的至少一种，以及药学上可接受的载体。

14.如权利要求1-7所述的双特异性抗体、如权利要求8所述的核酸序列、如权利要求9或10所述的载体、如权利要求11或12所述的AAV病毒颗粒或如权利要求13所述的药物组合物在制备用于治疗或预防癌症、眼内新生血管综合征、类风湿性关节炎、银屑病、增殖性视网膜病变、年龄相关性黄斑变性或糖尿病性黄斑水肿的药物中的用途。

15.如权利要求14所述的用途，所述年龄相关性黄斑变性为湿性年龄相关性黄斑变性。

16.如权利要求14或15所述的用途，其中所述药物通过玻璃体腔或视网膜下腔注射施用。