CN112552412A - 一种包含TGF-β抑制剂、VEGF抑制剂和抗PDL1抗体的三功能融合蛋白 - Google Patents

一种包含TGF-β抑制剂、VEGF抑制剂和抗PDL1抗体的三功能融合蛋白 Download PDF

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CN112552412A
CN112552412A CN202011330365.2A CN202011330365A CN112552412A CN 112552412 A CN112552412 A CN 112552412A CN 202011330365 A CN202011330365 A CN 202011330365A CN 112552412 A CN112552412 A CN 112552412A
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屈向东
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Abstract

本发明提供一种包含TGF‑β抑制剂、VEGF抑制剂和PD‑L1抑制抗体的三功能融合蛋白及其用途。本发明的三功能融合蛋白对人VEGF蛋白和TGF‑β蛋白都具有非常高的亲和力，对人PD‑L1蛋白亲和力也很好，并且还能够阻断人PD‑1/PD‑L1蛋白的结合。

Description

一种包含TGF-β抑制剂、VEGF抑制剂和抗PDL1抗体的三功能融 合蛋白
相关申请的交叉引用
本申请要求于2019年12月5日申请的专利申请号为201911235055.X,发明名称为“一种含有TGF-β抑制剂的三功能融合蛋白及其应用”的优先权，其全部内容通过引用并入本文。
技术领域
本发明属于生物医药领域，更具体地，本发明涉及一种包含TGF-β抑制剂、VEGF抑制剂和抗PDL1抗体的三功能融合蛋白，以及该三功能融合蛋白的用途。
背景技术
在经典免疫监视理论中，免疫系统可以识别肿瘤抗原并将其消除。如果免疫系统能够完全消除肿瘤细胞，那么免疫清除可以稳定进行。如果肿瘤细胞通过突变逃避免疫系统的清除，免疫系统将会进行重新平衡。在这一过程中，肿瘤细胞的免疫原性逐渐降低。肿瘤细胞的增殖能力在免疫系统的压力下变弱，使得肿瘤细胞的侦测变得更为困难。
致癌基因的激活导致肿瘤细胞改变自身及肿瘤微环境，使得免疫系统和肿瘤细胞间的平衡被打破。当免疫系统和肿瘤细胞进入逃逸阶段，肿瘤细胞的恶性程度会提高，而肿瘤细胞丢失MHC分子使其避免被免疫细胞识别和消除。肿瘤微环境也可以通过释放免疫抑制因子抑制免疫系统，例如IL-10、TGF-β等。肿瘤细胞表面也会高表达免疫抑制蛋白(例如程序化死亡配体-1、PD-L1)，当效应T细胞与肿瘤细胞结合时，PD-L1与PD-1相互作用并诱导T细胞发生凋亡，这是肿瘤对免疫系统产生耐受的主要原因之一，肿瘤迅速生长，发生转移。如果人为激活宿主的免疫系统并将其重定向到肿瘤细胞，从理论上来讲肿瘤组织就能够被清除，而免疫治疗的理论已经在临床治疗中得到广泛证明。
免疫疗法可分为两类：特异性治疗和非特异性治疗。在类别中特定疗法，包括以下治疗策略：肿瘤疫苗通过注射肿瘤激活免疫细胞对患者的抗原。肿瘤疫苗包括：灭活的肿瘤细胞疫苗，肿瘤抗原疫苗，肿瘤DNA疫苗，树突状细胞(DC)疫苗和细菌疫苗。特异性ACT免疫疗法主要包括三种治疗方法：
a)肿瘤浸润淋巴细胞(TIL)：淋巴细胞从肿瘤组织中分离并在体外培养。TIL可以分泌具有特异性抗肿瘤的IL-2能力。
b)T细胞受体(TCR)治疗：T细胞识别肿瘤抗原通过其单链抗体片段(scFv)，并将单链抗体片段TCR通过病毒载体克隆到正常T细胞中。因此，正常T细胞变为特异性肿瘤杀伤T细胞。
c)CAR-T：T细胞通过基因修饰获得具有肿瘤特异性受体T细胞。与常规T细胞识别机制不同，CAR-T细胞识别肿瘤抗原不受到MHC分子的限制。因此，CAR-T细胞能够通过增加共刺激信号分子克服肿瘤的免疫逃逸机制，增强T细胞对肿瘤细胞的杀伤能能力。
在非特异性ACT免疫疗法中，有两种主要应用治疗方法：分别为淋巴因子激活的杀手(lymphokine activated killer,LAK)细胞治疗和细胞因子诱导的杀手(cytokineinduced killer,CIK)细胞治疗。
a)LAK细胞治疗：LAK细胞一方面利用IL-2刺激外周血淋巴细胞中的免疫细胞，包括NK细胞和T细胞等，另一方面通过过表达FAS配体，增强对靶细胞的识别能力，并通过释放穿孔素和颗粒酶细胞杀死肿瘤细胞。
b)CIK细胞治疗：CIK细胞来源于病人或健康人的外周血淋巴细胞(PBL)，在体外条件下受到抗CD3抗体、IFN-γ和IL-2刺激下扩增。CIK细胞主要通过FasL和穿孔素发挥抗肿瘤的作用。
免疫检查点是人体免疫系统中的保护性分子，在正常机体中防止由于T细胞过度活化引起的炎性损伤。肿瘤细胞能够利用这一特性，过度表达免疫检查点分子，抑制机体的免疫反应，逃避人体免疫系统的监视和杀伤，从而促进肿瘤细胞的生长。免疫检查点抑制剂治疗可以通过抑制肿瘤微环境中的免疫检查点活性，重新激活T细胞对肿瘤的免疫应答反应，实现抗肿瘤的效果。T细胞的完全活化受“双信号”系统调节：第一信号来自其自身TCR(T细胞受体)与抗原的MHC的特异性结合，即T细胞识别抗原；第二信号来自共刺激分子，该信号参与由抗原呈递细胞(APC)表达的共刺激分子与T细胞表面上的相应受体或配体(例如CD28)的相互作用。例如CD28-B7是正向共刺激信号，而负向共刺激分子主要是CTLA4-B7途径和PD-1/PD-L1途径。在肿瘤细胞侵入后，这种抑制途径被肿瘤细胞利于抑制T细胞活化，从而逃避免疫系统的清除作用。
PD-1(CD279)最早于1992年被报道，人PD-1编码基因PDCD1位于2q37.3，全长2097bp，由6个外显子组成，翻译产物为288个氨基酸组成的PD-1前体蛋白，剪切前20个氨基酸组成的信号肽后得到成熟蛋白质。PD-1包括胞外免疫球蛋白可变区IgV结构域，疏水跨膜结构域和胞内结构域，胞内尾部结构域N端ITIM基序包含2个磷酸化位点，C端则是一个ITSM基序。PD-1是膜蛋白，属于CD28免疫球蛋白超家族，主要表达在激活后的T细胞表面，此外还在胸腺的CD4-CD8-T细胞、活化的NK细胞和单核细胞有低丰度表达。PD-1有2个配体，分别是B7蛋白家族的PD-L1(CD274,B7-H1)和PD-L2(CD273,B7-DC)，PD-L1和PD-L2氨基酸序列有40％相同。两者区别主要在于表达模式不同，PD-L1组成性的低表达于APC、非造血细胞(如血管内皮细胞、胰岛细胞)和免疫豁免部位(如胎盘、睾丸和眼睛)，炎性细胞因子如I型和II型干扰素、TNF-α和VEGF等均可以诱导PD-L1的表达。PD-L2则只在被激活的巨噬细胞和树突细胞中有表达。PD-1与PD-L1在激活的T细胞结合后，PD-1的ITSM基序发生酪氨酸磷酸化，进而导致下游蛋白激酶Syk和PI3K的去磷酸化，抑制下游AKT、ERK等通路的活化，最终抑制T细胞活化所需基因及细胞因子的转录和翻译，发挥负向调控T细胞活性的作用。
在肿瘤细胞中，肿瘤细胞及肿瘤微环境通过上调PD-L1表达并与肿瘤特异的CD8+T细胞表面的PD-1结合，负调控T细胞活性，抑制免疫反应。肿瘤细胞可以通过以下4种途径上调PD-L1表达：1.编码PD-L1的基因扩增(9p24.1)；2.EGFR、MAPK、PI3K-Akt信号通路激活，HIF-1转录因子等可以从转录水平上调PD-L1表达；3.EB病毒的诱导(EB病毒阳性的胃癌和鼻咽癌表现为PD-L1高表达)；以及4.表观遗传学的调控。在肿瘤微环境中，干扰素-γ等炎症因子的刺激同样可以诱导PD-L1和PD-L2的表达。炎症因子可以诱导肿瘤微环境中其他细胞，包括巨噬细胞、树突状细胞和基质细胞表达PD-L1和PD-L2，而能够识别肿瘤抗原的肿瘤浸润性T细胞能够分泌干扰素-γ，进而诱导PD-L1表达上调，这一过程被称为“适应性免疫抵抗”，肿瘤细胞通过这一机制可以实现自我保护。有越来越多的证据表明肿瘤利用PD-1依赖的免疫抑制免疫逃避。在各种实体瘤和血液系统恶性肿瘤种均已经发现PD-L1和PD-L2的高表达。此外，PD-Ls的表达与肿瘤细胞的不良预后之间具有很强相关性，证明了包括食道癌、胃癌、肾癌、卵巢癌、膀胱癌、胰腺癌和黑色素瘤等。
目前FDA已经批准上市的PD-1治疗性单抗包括Nivolumab(Opdivo，2014年9月)、Pembrolizumab(Keytruda，2014年12月)和Cemiplimab(Libtayo，2018年9月)，并且已上市的PD-L1治疗性单抗包括Atezolizumab(Tecentriq，2014年9月)、Avelumab(Bavencio，2016年5月)和Duravulumab(Imfinzi，2017年5月)，已批准的适应症见下表1所示。
表1
Figure BDA0002795596830000041
此外还有诸如Pidilizumab、AMP-224、AMP-514和PDR001等的PD-1单抗、以及诸如BMS-936559和CK-301等的PD-L1单抗处于研发和临床试验中。
转化生长因子β(TGF-β)
转化生长因子β(TGF-β)是属于转化生长因子超家族的多功能细胞因子。TGF-β只在脊椎动物中存在，且包括三种亚型：TGF-β1、TGF-β2、TGF-β3，以及其他信号蛋白。TGF-β是脊椎动物心脏、眼睛、上颚等多个器官和组织正常发育所必需的成分，还具有人类和其他高等脊椎动物的长期生存所必需的调节适应性免疫系统和协调伤口愈合功能。
TGF-β三种亚型的一级结构相似度较高，同源性达到70-80％。TGF-β1有390个氨基酸，TGF-β2和TGF-β3各自则包含412个氨基酸，它们在N-端都具有从细胞分泌所必须的信号肽、一个LAP结构域和一个由112-114个氨基酸残基组成的C末端区域，这一肽段经蛋白水解作用从原区域释放后成为成熟的TGF-β分子，成熟的TGF-β蛋白二聚体化生成25KDa的活性蛋白。TGF-β都具有9个半胱氨酸残基，其中8个在分子内形成二硫键，从而形成TGF-β超家族的半胱氨酸结(cysteine knot)结构特征。第9个半胱氨酸与另一个TGF-β蛋白的对应半胱氨酸形成二硫键，从而形成具有功能的二聚体。
所有白细胞都能生成非活性形式TGF-β蛋白，此时TGF-β与LTBP(潜在TGF-β结合蛋白)和LAP(潜在相关肽)以形成复合物的方式分泌，而血清蛋白酶(如纤溶酶)可催化复合物中活性TGF-β的释放。活性TGF-β释放这一生理现象常发生在巨噬细胞表面：炎症刺激信号激活巨噬细胞，促进纤溶酶的激活，导致非活性TGF-β复合物通过其配体血小板反应蛋白1(TSP-1)与CD36结合，最终增强活性TGF-β的释放。巨噬细胞还可以吞噬浆细胞分泌的IgG结合型非活性TGF-β复合物的方式将活性TGF-β释放到细胞外液中。
TGF-β以细胞或组织特异的方式激活，包括蛋白酶、整合蛋白(integrin)、pH及活性氧等。TGF-β激活因子紊乱会导致TGF-β信号失调，引起多种并发症，包括炎症反应、自身免疫性疾病、组织纤维化、肿瘤和白内障。
活化的TGF-β释放后再与其他因子形成丝氨酸/苏氨酸激酶复合物，最终与TGF-β受体结合，激活下游信号通路。TGF-β受体由1型受体亚基和2型受体亚基组成。当TGF-β与受体结合后，2型受体激酶磷酸化并激活1型受体激酶，从而激活信号级联反应。TGF-β受体下游信号通路可分为经典通路(SMAD通路)和DAXX依赖凋亡通路(DAXX通路)。
1)经典通路：活化的TGF-β复合物与TGF-β受体的2型受体亚基结合，然后募集并磷酸化1型受体，后者募集并磷酸化受体调节的SMAD蛋白(R-SMAD)。R-SMAD再与coSMAD蛋白(共同SMAD，哺乳动物中为SMAD4)结合形成异源二聚体并进入细胞核，作为转录因子发挥调控基因表达的作用。SMAD途径受反馈抑制作用的调节，SMAD6和SMAD7可能会阻断I型受体亚基。也有大量研究发现经由SMAD-3的依赖TGF-β信号发挥免疫抑制功能，因此常常被认为与肿瘤发生有关。
2)TGF-β能够诱导人淋巴细胞和肝细胞凋亡或程序性细胞死亡，而在TGF-β缺失的小鼠中则会由于淋巴细胞过度增殖导致自身免疫失调。死亡相关蛋白6(DAXX)能够与死亡受体Fas结合导致凋亡，同时DAXX还能与2型TGF-β受体C末端结合，随后DAXX被同源结构域相互作用蛋白激酶2(HIPK2)磷酸化，激活凋亡信号诱导激酶1(ASK1)，ASK1随后激活Jun氨基-末端激酶(JNK)途径，最终导致凋亡。
TGF-β经典信号通路可以使不同下游底物和调节蛋白的激活，诱导免疫细胞的增殖、激活、分化和趋化性，因此在癌症、自身免疫疾病和传染性疾病领域中一直都是研究热点。
1)对T淋巴细胞的作用
TGF-β在T细胞调节和分化中起至关重要的作用。TGF-β1能够诱导CD4+T向诱导型Treg(iTreg)和分泌促炎性细胞因子的Th17细胞分化。TGF-β1可以促使活化的Th细胞上调表达Foxp3以及分化为Treg。在小鼠模型中，TGF-β1的这一诱导作用被证实与年龄有关。同时也有研究表明，在体外实验中中和TGF-β1可抑制Th细胞向Th17细胞的分化，换句话说，TGF-β1在Th17细胞生成中发挥了与它作为抗炎性细胞因子的这一传统观念相反的作用，但这也暗示了TGF-β1可以通过调节炎性细胞和抑炎细胞这两种细胞类型之间平衡，维持自身免疫稳态。
2)对B淋巴细胞的作用
TGF-β能够抑制B细胞增殖，目前确切机制尚不明确，但有数据表明TGF-β可以通过诱导转录因子Id3，诱导p21上调，同时还能抑制其他关键调控基因、例如c-myc和ATM来抑制B细胞增殖。CD40是激活先天免疫应答的关键表面分子，它可以通过诱导Smad7表达解除上文所述由TGF-β诱导的B细胞的生长抑制。TGF-β还能阻断B细胞活化，在人和小鼠中促进B细胞的IgA类转换，抑制抗体生成。
3)对巨噬细胞的作用
目前普遍认为TGF-β刺激静息状态的单核细胞，抑制活化的巨噬细胞。对于单核细胞而言，TGF-β具有化学吸引作用及促炎症反应。但也有报道指出TGF-β可通过抑制NF-κB信号通路下调单核细胞和巨噬细胞中促炎性细胞因子的产生。TGF-β的这种看似矛盾的作用暗示了TGF-β的作用高度依赖于免疫细胞当时所处的环境。
TGF-β表达上调起到免疫抑制功能，通常与许多恶性肿瘤发生相关。在许多自身免疫疾病中TGF-β也可能通过免疫抑制功能的失调参与。
1.肿瘤
在正常细胞中，TGF-β通过信号通路使其在G1期停止细胞周期和增殖，诱导分化或促进细胞凋亡。但在许多肿瘤细胞中，TGF-β信号通路的发生突变，而TGF-β失去对细胞的控制。肿瘤细胞增殖，伴随着周围的基质细胞(成纤维细胞)增殖，这两种细胞均增加其TGF-β的产生。这些TGF-β作用于周围的基质细胞、免疫细胞、内皮细胞和平滑肌细胞，抑制免疫反应，促进血管生成，从而使肿瘤具有更强侵入性。TGF-β还可以将能够通过炎症反应攻击肿瘤细胞的(免疫)效应T细胞转化为调节性(抑制)T细胞，从而抑制炎性反应。在正常组织中，不同细胞分别表达粘附分子和分泌细胞因子，组织完整性可以通过细胞和细胞之间的反馈作用得到维持，但在肿瘤组织中这些反馈机制受到破坏。当TGF-β信号无法控制肿瘤细胞中的NF-κB活性后，恶性肿瘤细胞能够在已经活化的免疫细胞存在情况下依然得以生存。
2.肥胖和糖尿病
TGF-β/SMAD3信号通路在调节葡萄糖和能量代谢稳态中非常重要，而这也可能在糖尿病性肾病中起作用。在自发肥胖小鼠中，TGF-β信号缺失通过炎症信号诱导了肥胖产生。
在IL-2存在下，由TGF-β诱导的iTreg通过Foxp3和IL-10介导的应答反应抑制了实验性自身免疫性脑脊髓炎(EAE)的发展，而EAE是一种多发性硬化症(MS)的动物模型。这表明TGF-β和iTreg可能在MS的调节和治疗中发挥作用。在临床上，多发性硬化的患者中的确观察到TGF-β水平降低。TGF-β在调节Th17细胞凋亡中的作用也许可以解释其在多发性硬化中的作用：TGF-β水平降低后，Th17细胞无法被凋亡，而过多分泌的TNF-α，通过TNFR1诱导少突胶质和神经元的脱髓因此，MS期间TGF-β水平降低也可能阻止神经元的髓鞘再生。
肿瘤微环境(TME)是肿瘤周围的环境，包括血管、免疫细胞、成纤维细胞、信号分子和细胞外基质(ECM)。肿瘤与周围的微环境密切相关并且不断相互作用，肿瘤可以通过释放细胞外信号，促进肿瘤血管生成，诱导免疫耐受影响微环境，而微环境中的免疫细胞也可以影响肿瘤细胞的生长和分化。
1)血管
据统计，80-90％的癌症是腺癌，或者上皮组织来源的癌症。这类肿瘤不会发生血管化，导致只有新生血管生成后肿瘤直径生长到大于2mm。血管新生作用在肿瘤微环境中上调，供给肿瘤细胞营养，其形成的脉管系统与正常组织多有不同，主要体现在通透性增强与滞留效应(EPR)和低氧环境两方面。
2)免疫细胞
骨髓来源抑制细胞(MDSC)是一类具有抑制T细胞反应潜力的异质细胞群体的统称。它们可以通过调节伤口的修复和炎症肿瘤中迅速扩增，这一现象在绝大多部分肿瘤部位可观察到的炎症反应都有关。肿瘤相关巨噬细胞(TAM)是MDSC中的研究热点之一。TAM能够响应与肿瘤相关的炎症反应被招募到肿瘤中，但与正常巨噬细胞不同，TAM没有细胞毒性作用，因此TAM是慢性炎症反应和癌症之间有密切联系的重要证据。在体外实验中，可以通过对巨噬细胞祖细胞给予不同的免疫调节细胞因子(如白介素4(IL-4)和白介素13(IL-13))来诱导TAM。TAM聚集在肿瘤的坏死区域，通过分泌IL-10使肿瘤细胞逃逸正常免疫细胞的监控，并通过分泌血管内皮生长因子(VEGF)和一氧化氮合酶(NOS)促进血管新生，通过分泌表皮生长因子(EGF)重构ECM促进肿瘤生长。TAM的NF-κB信号通路受到抑制，因此在肿瘤微环境中表现出慢性炎症反应。TAM的增加与预后不良有关。TAM代表了新型癌症治疗的潜在目标。
中性粒细胞是多形核免疫细胞，它是先天免疫系统的重要组成部分。中性粒细胞在肿瘤和某些癌症(例如肺腺癌)中富集，被认为与预后恶化有关。在某些实体瘤患者中，血液中的中性粒细胞数量(和髓样细胞前体)可以增加。在小鼠中进行的实验表明，与肿瘤相关的中性粒细胞促进肿瘤生长，但也有研究表明得出相反结论。
肿瘤浸润淋巴细胞(TIL)是穿透肿瘤的淋巴细胞。TIL与造血干细胞有共同的起源，但在发育上出现分化。肿瘤细胞可以通过分泌含有死亡配体FasL和TRAIL等的外泌体来诱导活化的T淋巴细胞凋亡，并通过相同的方法关闭NK细胞的正常细胞毒性反应。
3)成纤维细胞
腺癌相关的成纤维细胞(CAF)是来源不同的成纤维细胞群体，它们的功能被肿瘤细胞“盗版”(pirate)并且导致肿瘤发生。CAF细胞通常来源于周围基质的正常成纤维细胞，也可以来自周细胞、平滑肌细胞、纤维细胞，间充质干细胞，或通过EMT作用及EndMT作用转化生成。在体外实验中，CAF没有抑制肿瘤细胞生长的作用，反而可以通过分泌VEGF，成纤维细胞生长因子(FGFs)、血小板衍生生长因子(PDGF)以及其他促血管生成的信号来诱导血管新生，从而促进肿瘤生长。CAF还能分泌TGF-β促进EMT，而肿瘤细胞可以通过EMT发生转移、参与抑制细胞毒性T细胞和NKT细胞。CAF作为成纤维细胞还能够对ECM进行改造(remodeling)，使其包含更多的旁分泌生存信号，例如IGF-1和IGF-2，从而促进周围肿瘤细胞的生存。有研究证实CAF与逆向Warburg效应相关，CAF能够进行有氧糖酵解并将乳酸喂入肿瘤细胞。
血管内皮生长因子(VEGF)，是一类由细胞产生、能够刺激血管形成的信号蛋白，属于胱氨酸结生长因子家族中的血小板衍生生长因子(PDGF)家族，发现之初被称为血管通透性因子(VPF)。VEGF的最重要功能是参与血管生成(Vasculogenesis)和血管新生(Angiogenesis)。
VEGF家族包括VEGF-A、VEGF-B、VEGF-C、VEGF-D、PlGF、VEGF-E(Orf-VEGF)和svVEGF，而在哺乳动物中包含前5个基因。所有VEGF在固定位置都有8个保守的半胱氨酸残基，这与PDGF家族非常相似，例如M-CSF(CSF-1)，SCF(干细胞因子)和Flt3L(Flt3配体)。在8个半胱氨酸中，有6个残基形成3个分子内二硫键以产生3个环结构，剩余2个半胱氨酸残基则形成2个分子间二硫键，形成稳定的二聚体结构。与VEGF-A和VEGF-E中的loop-1和loop-3结合的结构对于VEGFR-2的结合和激活至关重要。
VEGF与细胞表面的VEGFR结合，并通过使受体形成二聚体、发生磷酸化作用激活刺激细胞反应。VEGFR是典型的酪氨酸激酶受体(TKR)，包含一个能与配体结合的胞外结构域、一个跨膜结构域和一个包括酪氨酸激酶结构域的胞内结构域。VEGFR有三种主要的亚型，分别为VEGFR-1(Flt-1)、2(KDR/Flk-1)和3。VEGFRs的细胞外结构域包含7个免疫球蛋白(Ig)类结构域，而胞内的酪氨酸激酶结构域与PDGFR类似，有一个长60至70个氨基酸的激酶插入(KI)序列，但不同之处在于VEGFRs的KI区或羧基末端不包含Y-x-x-M基序。
不同VEGF结合的受体及下游的生理反应不尽相同：VEGF-A结合并激活VEGFR-1和VEGFR-2，从而促进血管生成，血管通透性，细胞迁移和基因表达，VEGF-A及其受体系统的自分泌环存在于血管内皮细胞中，有助于内皮功能；VEGF-B和PIGF只能结合并激活VEGFR-1；VEGF-C和VEGF-D结合VEGFR-3和VEGFR-2，通过2种受体分别调节淋巴管生成及血管新生。
1)VEGFR-1主要表达在在血管内皮细胞和非内皮细胞中，包括造血干细胞、巨噬细胞和单核细胞。VEGFR-1可以结合VEGF-A和VEGF-B、VEGFR-1与VEGF-A亲和力(KD＝1-10pM)高于VEGFR-2与VEGF-A亲和力，但VEGFR-1胞内结构域络氨酸激酶活性比VEGFR-2低10倍，因此促进血管新生的信号主要来源VEGFR-2的信号通路。实验证实，VEGFR-1基因敲除小鼠由于内皮细胞过度生长和胚胎中血管的紊乱，在胚胎第8.5-9.0天即会发生死亡，而只表达VEGFR-1胞外和跨膜结构域的小鼠血管生成正常，进一步研究表明VEGFR-1的ECD而非胞内的激酶结构域可以通过捕获VEGF-A从而阻止了VEGFR-2的活化，在胚胎发育早期阶段起着抑制血管形成的作用。VEGFR-1酪氨酸残基的自磷酸化作用可以激活下游相对微弱的信号，参与内皮细胞、周细胞的生长和存活以及巨噬细胞的细胞迁移作用。MAPK途径的磷脂酶C(PLCγ)可以通过粘附在VEGFR-1的磷酸化Tyr1169上，调节内皮细胞的增殖，PI3K的p85亚基也被报道与活化和磷酸化后的VEGFR-1结合。
2)VEGFR-2表达于血管内皮、淋巴内皮细胞、巨核细胞和造血干细胞中，能结合VEGF-A、VEGF-C、VEGF-E，是VEGFR信号通路主要受体。在VEGFR-2基因敲除小鼠中，血管生成和造血发育存在缺陷，在8.5-9.0天胚胎致死，证明VEGFR-2在胚胎发育阶段的内皮细胞的生长和分化中起正向调节作用，与VEGFR-1作用相反。在VEGFR-2的自磷酸化酪氨酸残基中，磷酸化的Tyr1175导致PLCγ的结合，从而刺激参与DNA合成调控的MAPK途径，并参与与细胞存活有关的PI3K信号通路。Tyr951是VEGFR-2中的另一个磷酸化残基，可通过T细胞特异接头蛋白(TSA)与细胞质酪氨酸激酶Src相互作用，调节肌动蛋白应激纤维的形成和内皮细胞的迁移反应。
3)VEGFR-3主要在淋巴管内皮细胞中表达，在VEGF-C刺激后，PKC途径和Ras途径被激活，刺激淋巴管内皮细胞增殖、迁移和存活。但目前VEGFR-3中酪氨酸残基上的哪个自磷酸化位点直接参与这一生理过程尚不明确。在胚胎发育期间，VEGFR-3在血管网络和心血管系统的发育中也起着至关重要的作用。
VEGF-VEGFR是机体正常生理反应的一部分。除了参与胚胎发育过程中血管发育，当血液循环不足时(例如在低氧条件下)，可以通过新生血管恢复组织的氧气供应，例如运动后的肌肉产生新血管以及绕过阻塞血管的新血管(侧支循环)。细胞不受控制的生长和增殖会导致癌症，具有多种生物学特征，包括生长信号的自足、对生长信号的不敏感性、逃避凋亡、无限复制潜力、持续的血管生成、组织浸润和转移、异常的代谢途径、逃避免疫系统监控和基因组不稳定等。血管生长对于实体瘤的生长和转移至关重要。因此，对血管生成起到关键作用的VEGF-VEGFR系统被认为是研究肿瘤治疗的最重要目标之一。
贝伐珠单抗(Bevacizumab)是靶向VEGF-A的人源化单克隆抗体，可以选择性中和VEGF-A，但不能其他中和VEGF家族成员，目前已被批准用于治疗结直肠癌、乳腺癌、非小细胞肺癌、肾癌以及成胶质细胞瘤。贝伐珠单抗有一些可能危及生命的不良反应，包括高血压、蛋白尿、鼻出血、血栓形成和出血。此外，某些癌症通过数种机制实现对贝伐珠单抗的耐药性，例如增强替代性促血管生成信号通路，将骨髓来源的促血管生成细胞募集到肿瘤以及增加肿瘤中周细胞的数量。苹果酸舒尼替尼和甲苯磺酸索拉非尼可选择性靶向某些蛋白质受体，包括VEGFR，并抑制其激酶活性，因此被FDA批准用于胃基质瘤、晚期和高风险可切除肾细胞癌和某种罕见的胰腺癌治疗。除此之外，还有其他靶向VEGF-VEGFR系统的药物，包括诸如阿柏西普(Aflibercept，VEGF-Trap，已批准用于结肠癌)、雷莫芦单抗(Ramucirumab，抗VEGFR-2，已批准用于胃癌、结肠癌和转移性非小细胞肺癌)等大分子药物以及诸如Pazopanib、Axitinib和Cabozantinib等小分子药物。
发明内容
本发明的目的在于提供一种包含TGF-β抑制剂、VEGF抑制剂和抗PDL1抗体的三功能融合蛋白及其用途。
本发明采用了如下技术方案：
一种包含TGF-β抑制剂、VEGF抑制剂和抗PDL1抗体的三功能融合蛋白。
优选的，本发明的包含TGF-β抑制剂、VEGF抑制剂和抗PDL1抗体的三功能融合蛋白还具有这样的特征：所述三功能融合蛋白的氨基酸序列选自SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：6、SEQ ID NO：7、SEQ ID NO：8、SEQID NO：9、SEQ ID NO：10、SEQ ID NO：11和SEQ ID NO：12中的任意一个。
优选的，本发明的包含TGF-β抑制剂、VEGF抑制剂和抗PDL1抗体的三功能融合蛋白还具有这样的特征：所述TGF-β抑制剂为TGF-βRII胞外结构域或其N端处保持结合TGF-β活性的点突变或截短体形式，其中所述TGF-β抑制剂的氨基酸序列选自SEQ ID NO：13、SEQ IDNO：14、SEQ ID NO：15、SEQ ID NO：16、SEQ ID NO：17、SEQ ID NO：18和SEQ ID NO：19中的任意一个。
优选的，本发明的包含TGF-β抑制剂、VEGF抑制剂和抗PDL1抗体的三功能融合蛋白还具有这样的特征：所述VEGF抑制剂介于所述TGF-β抑制剂和所述抗PDL1抗体之间(如图1或图2所示)，其N端和C端均以共价键的方式与多肽连接，并且其与VEGF-A的亲和力KD值不高于100pM或者与VEGF-A、VEGF-B和PIGF结合的KD均不高于阿柏西普亲和力KD值的50倍，其中所述VEGF抑制剂的氨基酸序列选自SEQ ID NO：20、SEQ ID NO：32、SEQ ID NO：33和SEQID NO：34中的任意一个。
优选的，本发明的包含TGF-β抑制剂、VEGF抑制剂和抗PDL1抗体的三功能融合蛋白还具有这样的特征：所述抗PD-L1抗体为其单域抗体或scFv形式；和/或所述TGF-β抑制剂为TGF-βRI、TGF-βRII或其组合、或其单域抗体或scFv形式；和/或所述VEGF抑制剂为VEGF-Trap形式、或其单域抗体或scFv形式。
优选的，本发明的包含TGF-β抑制剂、VEGF抑制剂和抗PDL1抗体的三功能融合蛋白还具有这样的特征：所述抗PD-L1抗体的氨基酸序列为SEQ ID NO：21或SEQ ID NO：25，其中所述抗PD-L1抗体的CDR序列分别为SEQ ID NO：22、SEQ ID NO：23和SEQ ID NO：24；或SEQID NO：26、SEQ ID NO：27和SEQ ID NO：28中的任意一个。
本发明还提供上述任意一项所述的三功能融合蛋白在制备用于治疗癌症的药物中的用途。
本发明还提供上述任意一项所述的三功能融合蛋白在制备用于抑制肿瘤生长的药物中的用途。
本发明还提供上述任意一项所述的三功能融合蛋白在制备用于调节免疫功能的药物中的用途。
本发明还提供上述任意一项所述的三功能融合蛋白在制备用于抗感染的药物中的用途。
本发明还提供上述任意一项所述的三功能融合蛋白在制备用于抑制血管增长的药物中的用途。
本发明的有益效果
本发明的三功能融合蛋白对人VEGF蛋白具有非常高的亲和力，对人PD-L1蛋白结合亲和力很好，并且能够阻断人PD-1/PD-L1蛋白的结合，阻断活性好于PD-L1对照抗体。
附图说明
附图是用来提供对本发明的进一步理解，并且构成说明书的一部分，与下面的具体实施方式一起用于解释本发明，但并不构成对本发明的限制。在附图中：
图1是本发明的三功能融合蛋白的一种结构的示意图；
图2是本发明的三功能融合蛋白的另一种结构的示意图；
图3示出了ELISA检测QP363蛋白结合人PD-L1的亲和力曲线；
图4示出了ELISA检测QP363蛋白阻断人PD-1/PD-L1结合的活性曲线；
图5示出了QP361、QP363、QP364等分子抑制TGFβ-SMAD3荧光素酶报告基因实验；
图6示出了FACS检测三功能融合蛋白CHO48结合高表达PDL1的人肺癌细胞HCC827活性；
图7示出了ELISA检测CHO48等分子与人PD-L1重组蛋白结合；
图8示出了ELISA检测CHO48等分子阻断人PD-L1与人PD-1结合；
图9示出了混合淋巴细胞反应5天后细胞上清检测IFNγ的表达；
图10示出了CHO48抑制TGFβ-SMAD3荧光素酶报告基因实验；并且
图11和12示出了在转基因鼠模型中评价CHO48对肿瘤生长的抑制作用的结果。
具体实施方式
以下对本发明的具体实施方式进行详细说明。应当理解的是，此处所描述的具体实施方式仅用于说明和解释本发明，并不用于限制本发明。
在本文中所披露的范围的端点和任何值都不限于该精确的范围或值，这些范围或值应当理解为包含接近这些范围或值的值。对于数值范围来说，各个范围的端点值之间、各个范围的端点值和单独的点值之间，以及单独的点值之间可以彼此组合而得到一个或多个新的数值范围，这些数值范围应被视为在本文中具体公开。
本实施方式中未注明具体条件的实验方法，通常是按照常规条件，或按照原料或商品制造厂商所建议的条件。如分子克隆，实验室手册，冷泉港实验室，当代分子生物学方法，细胞生物学等等。未注明具体来源的试剂，为市场购买的常规试剂。
实施例1：TGF-β抑制剂、VEGF抑制剂和抗PDL1抗体三功能的融合蛋白的克隆和表达
采用TGFβRII胞外结构域(SEQ ID NO：13的全长、N端突变(SEQ ID NO：14、SEQ IDNO：15、SEQ ID NO：16)及N端截短形式如SEQ ID NO：17、SEQ ID NO：18、SEQ ID NO：19))作为融合蛋白中TGF-β抑制剂。研究发现，TGFβRII胞外结构域N端突变或N端截短形式的结构和功能较为稳定，本发明TGFβRII胞外结构域全长、N端突变及N端截短形式的序列如下表2所示：
表2TGFβRII胞外结构域序列
Figure BDA0002795596830000151
三功能融合蛋白中VEGF抑制剂采用VEGF-Trap(SEQ ID NO：20、SEQ ID NO：32、SEQID NO：33、SEQ ID NO：34)，序列如下所示：
SEQ ID NO：20 QD375 pQD-VEGF-Trap-FC
SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO：32
SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEK
SEQ ID NO：33
GRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKPFVAFGSGMESLVEATVGERVRIPAKYLGYPPPEIKWYKNGIPLESNHTIKAGHVLTIMEVSERDTGNYTVILTNPISKEKQSHVVSLVVYVPPGPG
SEQ ID NO：34
GRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHENLSVAFGSGMESLVEATVGERVRIPAKYLGYPPPEIKWYKNGIPLESNHTIKAGHVLTIMEVSERDTGNYTVILTNPISKEKQSHVVSLVVYVPPGPG
三功能融合蛋白中PD-L1单域抗体序列如下所示(SEQ ID NO：21、SEQ ID NO：25，其中CDR序列分别为SEQ ID NO：22、SEQ ID NO：23、SEQ ID NO：24；SEQ ID NO：26、SEQ IDNO：27、SEQ ID NO：28)：
SEQ ID NO：21 QD322 anti-PD-L1 VHH
EVQLLESGGGLVQPGGSLRLSCAASGFTYGTYAMSWFRQAPGKGREGVACIDIYGRASYTDPVKGRFTISQDNSKNTLYLQMNSLKAEDTAVYYCAARDFGYCTASWVHEGFSRYWGQGTLVTVSS
CDR序列分别如下：
SEQ ID NO：22 QD322-CDR1
GFTYGTYAMS
SEQ ID NO：23 QD322-CDR2
CIDIYGRASYTDPVKG
SEQ ID NO：24 QD322-CDR3
AARDFGYCTASWVHEGFSRY
SEQ ID NO：25 QD509 anti-PD-L1 VHH
EVQLLESGGGLVQPGGSLRLSCAASGFTLDDYAIGWFRQAPGKGREGVSCISKSGETTFFVESVKDRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATGSWCTVGSMSRQFYRQFFHSWGQGTLVTVSS
CDR序列分别如下：
SEQ ID NO：26 QD509-CDR1
GFTLDDYAIG
SEQ ID NO：27 QD509-CDR2
KSGETTFFVESVKD
SEQ ID NO：28 QD509-CDR3
ATGSWCTVGSMSRQFYRQFFHS
三功能融合蛋白克隆设计及蛋白序列如下表3所示：
表3
Figure BDA0002795596830000171
克隆编号及序列：
SEQ ID NO:1QD361 pQD-TGFβRII-VEGF-Trap-antiPDL1(QD322)
IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSGGGGSSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTYGTYAMSWFRQAPGKGREGVACIDIYGRASYTDPVKGRFTISQDNSKNTLYLQMNSLKAEDTAVYYCAARDFGYCTASWVHEGFSRYWGQGTLVTVSS
SEQ ID NO:2QD362 pQD-TGFβRII(M1)-VEGF-Trap-antiPDL1(QD322)
IPPHVQKSVNSTMIVTDNSTGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSGGGGSSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTYGTYAMSWFRQAPGKGREGVACIDIYGRASYTDPVKGRFTISQDNSKNTLYLQMNSLKAEDTAVYYCAARDFGYCTASWVHEGFSRYWGQGTLVTVSS
SEQ ID NO:3QD363 pQD-antiPDL1(QD322)-VEGF-Trap-TGFβRII
EVQLLESGGGLVQPGGSLRLSCAASGFTYGTYAMSWFRQAPGKGREGVACIDIYGRASYTDPVKGRFTISQDNSKNTLYLQMNSLKAEDTAVYYCAARDFGYCTASWVHEGFSRYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD
SEQ ID NO:4 QD364 pQD-antiPDL1(QD322)-VEGF-Trap-TGFβRII(M1)
EVQLLESGGGLVQPGGSLRLSCAASGFTYGTYAMSWFRQAPGKGREGVACIDIYGRASYTDPVKGRFTISQDNSKNTLYLQMNSLKAEDTAVYYCAARDFGYCTASWVHEGFSRYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSIPPHVQKSVNSTMIVTDNSTGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD
SEQ ID NO:5 QD388 pQD-antiPDL1(QD322)-VEGF-Trap-TGFβRII(M2)
EVQLLESGGGLVQPGGSLRLSCAASGFTYGTYAMSWFRQAPGKGREGVACIDIYGRASYTDPVKGRFTISQDNSKNTLYLQMNSLKAEDTAVYYCAARDFGYCTASWVHEGFSRYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSIPPHVQKSVNSTMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD
SEQ ID NO:6 QD389 pQD-antiPDL1(QD322)-VEGF-Trap-TGFβRII(M3)
EVQLLESGGGLVQPGGSLRLSCAASGFTYGTYAMSWFRQAPGKGREGVACIDIYGRASYTDPVKGRFTISQDNSKNTLYLQMNSLKAEDTAVYYCAARDFGYCTASWVHEGFSRYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNSTGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD
SEQ ID NO:7 QD525 pQD-TGFβRII-VEGF-Trap-antiPDL1(QD509)
IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSGGGGSSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTYGTYAMSWFRQAPGKGREGVACIDIYGRASYTDPVKGRFTISQDNSKNTLYLQMNSLKAEDTAVYYCAARDFGYCTASWVHEGFSRYWGQGTLVTVSS
SEQ ID NO:8 QD526 pQD-TGFβRII(M1)-VEGF-Trap-antiPDL1(QD509)
IPPHVQKSVNSTMIVTDNSTGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSGGGGSSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTLDDYAIGWFRQAPGKGREGVSCISKSGETTFFVESVKDRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATGSWCTVGSMSRQFYRQFFHSWGQGTLVTVSSSEQ ID NO:9QD527 pQD-antiPDL1(QD509)-VEGF-Trap-TGFβRII
EVQLLESGGGLVQPGGSLRLSCAASGFTLDDYAIGWFRQAPGKGREGVSCISKSGETTFFVESVKDRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATGSWCTVGSMSRQFYRQFFHSWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD
SEQ ID NO:10 QD528 pQD-antiPDL1(QD509)-VEGF-Trap-TGFβRII(M1)
EVQLLESGGGLVQPGGSLRLSCAASGFTLDDYAIGWFRQAPGKGREGVSCISKSGETTFFVESVKDRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATGSWCTVGSMSRQFYRQFFHSWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSIPPHVQKSVNSTMIVTDNSTGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD
SEQ ID NO:11 QD529 pQD-antiPDL1(QD509)-VEGF-Trap-TGFβRII(M2)
EVQLLESGGGLVQPGGSLRLSCAASGFTLDDYAIGWFRQAPGKGREGVSCISKSGETTFFVESVKDRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATGSWCTVGSMSRQFYRQFFHSWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSIPPHVQKSVNSTMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD
SEQ ID NO:12 QD530 pQD-antiPDL1(QD509)-VEGF-Trap-TGFβRII(M3)
EVQLLESGGGLVQPGGSLRLSCAASGFTLDDYAIGWFRQAPGKGREGVSCISKSGETTFFVESVKDRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATGSWCTVGSMSRQFYRQFFHSWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNSTGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD
PD-L1阳性抗体序列如下：
SEQ ID NO：29QD1180阿特珠单抗重链序列(atezolizumab)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO：30QD1181阿特珠单抗轻链序列(atezolizumab)
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
克隆构建方法：引物设计：利用在线软件DNAWorks(v3.2.4)(http://helixweb.nih.gov/dnaworks/)设计多条引物合成含重组所需基因片段：5’-30bp信号肽+VH/VK+30bp CH1/CL-3’。
片段拼接：按照TaKaRa公司Primer STAR GXL DNA聚合酶操作说明书，用上面设计的多条引物，PCR扩增得到重组所需基因片段。第一步PCR：PCR反应体系50μL包括10μLPrimerSTAR GXL Buffer(5×)；4μL dNTP Mixture(2.5mmol·L-1)；引物如上所示各1μL；1μL PrimeSTAR GXL DNA聚合酶。PCR反应条件为98℃2min，98℃20s，55℃15s，68℃30s，30个循环；68℃5min。第二步PCR：以第一步PCR产物为模板，首尾两条引物进行PCR扩增，条件同第一步。PCR搭建扩增目的片段。
表达载体pQD构建及酶切：利用一些特殊的限制性内切酶，如BsmBI，识别序列与酶切位点不同的特性设计构建表达载体pQD(带信号肽)，BsmBI酶切载体，切胶回收备用。
重组构建表达载体：重组目的基因片段与BsmBI酶切回收表达载体pQD(带信号肽片段)按3：1比例分别加入DH5α感受态细胞中，0℃冰浴30min，42℃热击90s，加入5倍体积LB培养基，37℃孵育45min，涂布LB-Amp平板，37℃培养过夜，挑取单克隆送测序得到各目的克隆。
蛋白表达：根据克隆设计蛋白表达组合如下表4：
表4
Figure BDA0002795596830000231
293E细胞培养密度维持在0.2-3×106/ml之间，转染前一天待转染细胞离心换液，调整细胞密度为0.5-0.8×106/ml。转染当天，293E细胞密度为1-1.5×106/ml。准备质粒和转染试剂PEI，需转染质粒量为300ug/300ml细胞，使用PEI和质粒的质量比为2:1。将质粒和PEI进行混匀，静置15min，不宜超过20min。将质粒和PEI混合物缓慢加入293E的细胞中，放入8％CO2，120rpm，37℃的摇床中培养。转染第五到六天，水平离心机4700rpm离心20min收集细胞上清纯化。
实施例2：融合蛋白的纯化鉴定
蛋白亲和层析法：细胞培养液经过高速离心后取上清，利用GE的Protein A层析柱进行亲和层析。层析使用平衡缓冲液为1×PBS(pH 7.4)，细胞上清上样结合后利用PBS洗涤至紫外线回到基线，然后再利用洗脱缓冲液0.1M甘氨酸(pH 3.0)洗脱目的蛋白，利用Tris调节pH至中性保存；
蛋白体积排阻层析SEC：将离子交换所得产物超滤浓缩后进行体积排阻层析，如利用GE的Superdex200凝胶进行分离，以去除可能的聚体及其它成分，获得高纯度的目的产物。所得蛋白纯度分析可以通过SDS-PAGE及SEC-HPLC检测进行分析。蛋白浓度通过紫外分光光度法测定。
Protein A纯化结果如下表5所示。
表5：三功能融合蛋白瞬转表达蛋白产量
Figure BDA0002795596830000241
结果表面，本发明三功能融合蛋白均表现出较高的蛋白表达水平。
三功能融合蛋白体外生化或生物功能活性
实施例3：Biacore检测三功能融合蛋白与人TGFβ1体外结合亲和力和动力学实验
测试样品：QP361、QP364、QP388、QP389
通过Biacore S200测定待测分子与人TGFβ1的亲和力，实验过程如下：
A.主要材料
1.仪器：Biacore S200；
B.实验步骤：用Protein A芯片亲和捕获一定量的待测三功能融合蛋白，然后于芯片表面流经人TGFβ1(QPP12.1，SinoBio，10804-HNAC)蛋白，利用Biacore实时检测反应信号，从而获得结合和解离曲线。
C.实验结果表6所示。
表6：Biacore检测三功能融合蛋白与人TGFβ1蛋白结合亲和力结果
Figure BDA0002795596830000251
结果表明，本发明三功能融合蛋白QP361、QP364、QP388、QP389均对人TGFβ1具有极高的亲和力，KD值均小于等于0.106pM。
实施例4：Biacore检测三功能融合蛋白与人VEGF体外结合亲和力和动力学实验
测试样品：QP361、QP364、阿柏西普(Aflibercept)(蛋白编号QP375)
通过Biacore S200测定待测分子与人VEGF的亲和力，实验过程如下：
A.主要材料
1.仪器：Biacore S200；
B.实验步骤：用Protein A芯片亲和捕获一定量的待测三功能融合蛋白，然后于芯片表面流经人VEGF165(QPP13.1，SinoBio，11066-HNAH)蛋白，利用Biacore实时检测反应信号，从而获得结合和解离曲线。
C.实验结果如表7所示。
表7：Biacore检测三功能融合蛋白与人VEGF蛋白结合亲和力
Figure BDA0002795596830000252
结果表明，本发明三功能融合蛋白QP361、QP364均对人VEGF蛋白具有极高的亲和力，KD值≤5.32pM。与阿柏西普和人VEGF蛋白结合亲和力相当。
实施例5：ELISA检测QP361等分子对人PD-L1蛋白结合
实验样品：QP361、QP363、QP36693670(对照M7824序列引用专利US9676863B2)，阿特珠单抗
实验步骤：包被兔His抗体(金斯瑞，A00174)2μg/ml 50μl/孔，4℃过夜。PBS洗3次。封闭：3％BSA 250μl/孔，室温孵育1h。孵育人PD-L1重组蛋白(SinoBio，10084-H08H)1μg/ml，50μl/孔，25℃1h，PBS洗3遍。孵育抗体阿特珠单抗，QP361等蛋白，稀释8个梯度。25℃1h，PBS洗6遍。孵育二抗HRP-抗人FC，1:5000倍稀释,25℃1h，PBST洗6遍。TMB显色，H2SO4终止反应。酶标仪设置450nm读数。
结果如图3所示，ELISA检测QP361等分子与人PD-L1重组蛋白结合。
实施例6：ELISA检测QP361等分子阻断人PD-L1和PD-1蛋白结合
实验样品：QP361、QP363、QP36693670(对照M7824序列引用专利US9676863B2)
包被蛋白QP1138(PD1-FC)2μg/ml 50μl/孔，4℃过夜。PBS洗3次。封闭：3％BSA 250μl/孔，室温孵育1h。分别配制2μg/ml PDL1-小鼠FC和不同浓度抗体，等体积混匀，室温孵育1h。PBST洗3次，PBS洗3次。孵育二抗：HRP-小鼠IgG(1：5000)50μl/孔，PBST洗6次，PBS洗3次。显色：TMB 100μl/孔，显色10min。2M H2SO4 50μl/孔终止。
结果如图4所示，QP361等分子能阻断人PD-L1与人PD-1结合。
实施例7：SMAD3荧光素酶报告基因抑制实验
实验样品：QP361、QP363、QP364、人IgG
实验材料：HepG2细胞(人肝癌细胞株)，购于中国医学科学院基础医学研究所细胞资源中心。pGM-SEB luc质粒，购自吉满生物，货号GM-0211086；JetPRIME Transfection，购自Polyplus，货号114-15；TGFβ1，购自Sino Biological，货号10804-HNAC；ONE-GloTMLuciferase Assay System，购自Promega，货号E6120；人IgG，购自Sigma，货号I4506；其他抗体来自内部制备。
实验方法：HepG2细胞使用含有10％FBS的DMEM高糖培养基于37℃5％CO2培养箱中培养，实验第一天接种2×106于10cm培养皿中培养过夜；第二天每个培养皿转染15μg pGM-SEB luc质粒，继续于37℃5％CO2培养箱中培养24小时；第三天用胰酶消化转染后的HepG2细胞，800rpm离心5分钟弃上清，用不完全培养基(含0.5％FBS的DMEM高糖培养基)重悬细胞并计数，将细胞按5×104个，50μl每孔接种于96孔板中，37℃5％CO2培养箱中培养6小时；将25μl每孔不完全培养基配制的终浓度为1ng/ml的TGFβ1和25μl每孔梯度稀释后的各个待测抗体加入96孔板中，与细胞悬液混合均匀，37℃5％CO2培养箱中继续培养18小时；将ONE-GloTM Luciferase Assay System试剂按100μl每孔加入待测的96孔板中，室温避光孵育10分钟，于酶标仪中检测发光值。
实验结果如图5所示，在TGFβ-SMAD3荧光素酶报告基因实验中，QP361、QP363、QP364能抑制其信号通路，表现出很好的TGFβ抑制活性。
三功能融合蛋白中的PD-L1纳米抗体为QP509，构建生产了蛋白CHO48(antiPDL1(QD509)-VEGF-Trap-TGFβRII)，蛋白序列如SEQ ID NO：9所示。
SEQ ID NO:9QD527 pQD-antiPDL1(QD509)-VEGF-Trap-TGFβRII
EVQLLESGGGLVQPGGSLRLSCAASGFTLDDYAIGWFRQAPGKGREGVSCISKSGETTFFVESVKDRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATGSWCTVGSMSRQFYRQFFHSWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD
实施例8：FACS检测三功能融合蛋白结合高表达PDL1的人肺癌细胞HCC827活性
实验样品：CHO48
实验步骤：HCC827细胞计数，1E5/孔。PBS洗1遍，3％FBS 200ul/孔，冰上封闭1h。孵育CHO48蛋白，66.7nM起5倍稀释，共稀释7个梯度，最后一孔100倍稀释，100ul/孔，冰上1h。PBS洗2遍。孵育PE-抗人FC抗体，按照1:200稀释比例，50ul/孔，冰上孵育1h，PBS洗3遍。120ul/孔PBS重悬细胞，上机检测。使用graphpad prism软件分析平均荧光值。
结果如图6所示，三功能融合蛋白CHO48结合高表达PDL1的人肺癌细胞HCC827。
实施例9：ELISA检测CHO48等分子对人PD-L1蛋白结合
实验样品：CHO48、QP509(CHO48中PD-L1纳米抗体FC融合蛋白)，QP36693670(对照M7824序列引用专利US9676863B2)
实验步骤：包被兔His抗体(金斯瑞，A00174)2μg/ml 50μl/孔，4℃过夜。PBS洗3次。封闭：3％BSA 250μl/孔，室温孵育1h。孵育人PD-L1重组蛋白(SinoBio，10084-H08H)1μg/ml，50μl/孔，25℃1h，PBS洗3遍。孵育抗体阿特珠单抗，CHO48等抗体，稀释8个梯度。25℃1h，PBS洗6遍。孵育二抗HRP-抗人FC，1:5000倍稀释,25℃1h，PBST洗6遍。TMB显色，H2SO4终止反应。酶标仪设置450nm读数。
结果如图7所示，ELISA检测CHO48等分子与人PD-L1重组蛋白结合。
实施例10：ELISA检测CHO48等分子阻断人PD-L1和PD-1蛋白结合
实验样品：CHO48、QP509(CHO48中PD-L1纳米抗体FC融合蛋白)，QP36693670(对照M7824序列引用专利US9676863B2)
包被蛋白QP1138(PD1-FC)2μg/ml 50μl/孔，4℃过夜。PBS洗3次。封闭：3％BSA 250μl/孔，室温孵育1h。分别配制2μg/ml PDL1-小鼠FC和不同浓度抗体，等体积混匀，室温孵育1h。PBST洗3次，PBS洗3次。孵育二抗：HRP-小鼠IgG(1：5000)50μl/孔，PBST洗6次，PBS洗3次。显色：TMB 100μl/孔，显色10min。2M H2SO4 50μl/孔终止。
结果如图8所示，CHO48等分子能阻断人PD-L1与人PD-1结合。
实施例11：Biacore检测三功能融合蛋白与人PD-L1体外结合亲和力和动力学实验
测试样品：CHO48、QP509(CHO48中PD-L1纳米抗体FC融合蛋白)、阿特珠单抗
通过Biacore S200测定待测分子与人PD-L1的亲和力，实验过程如下：
A.仪器：Biacore S200；
B.实验步骤：用Protein A芯片亲和捕获一定量的待测三功能融合蛋白，然后于芯片表面流经人PD-L1(QPP09，SinoBio，10084-H08H)蛋白，利用Biacore实时检测反应信号，从而获得结合和解离曲线。
C.实验结果如下表8所示。
表8：三功能融合蛋白CHO48与人PD-L1结合亲和力
Figure BDA0002795596830000291
结果表明，本发明三功能融合蛋白CHO48对人PD-L1蛋白具有极高的亲和力，KD值＝28.7pM。PD-L1单抗QP509(VHH-FC)与PD-L1蛋白结合的KD值分别29.9pM。由此可知三功能融合蛋白CHO48中PD-L1抗体仍能保持很高亲和力。对照抗体阿特珠单抗，与PD-L1蛋白结合KD值为1.39nM。
实施例12：Biacore检测三功能融合蛋白与人TGFβ1体外结合亲和力和动力学实验
测试样品：CHO48、QP36693670(对照M7824序列引用专利US9676863B2)
通过Biacore S200测定待测分子与人TGFβ1的亲和力，实验过程如下：
A.主要材料
1.仪器：Biacore S200；
B.实验步骤：用Protein A芯片亲和捕获一定量的待测三功能融合蛋白，然后于芯片表面流经人TGFβ1(QPP12.1，SinoBio，10804-HNAC)蛋白，利用Biacore实时检测反应信号，从而获得结合和解离曲线。
C.实验结果如下表9所示。
表9：三功能融合蛋白CHO48与人TGFβ1结合亲和力结果
Figure BDA0002795596830000301
结果表明，本发明三功能融合蛋白CHO48对人TGFβ1具有极高的亲和力，KD值0.445pM。与对照分子M7824(QP36693670)相当。
实施例13：Biacore检测三功能融合蛋白与人VEGF体外结合亲和力和动力学实验
测试样品：CHO48、阿柏西普(aflibercept)(蛋白编号QP375)
通过Biacore S200测定待测分子与人VEGF的亲和力，实验过程如下：
A.主要材料
1.仪器：Biacore S200；
B.实验步骤：用Protein A芯片亲和捕获一定量的待测三功能融合蛋白，然后于芯片表面流经人VEGF165(QPP13.1，SinoBio，11066-HNAH)蛋白，利用Biacore实时检测反应信号，从而获得结合和解离曲线。
C.实验结果如表10所示。
表10：三功能融合蛋白CHO48与人VEGF蛋白结合亲和力
Figure BDA0002795596830000302
结果表明，本发明三功能融合蛋白CHO48对人VEGF蛋白具有极高的亲和力，KD值2.67pM。与阿柏西普对人VEGF蛋白亲和力相当。
实施例14：在混合淋巴细胞反应中能刺激T细胞增殖，增强IFN-γ的产生
实验样品：CHO48，人IgG
混合淋巴细胞反应是指将人T细胞和同种异体树突细胞以10:1的比例于96孔圆底培养板中混合，淋巴细胞接受同种异型抗原的刺激而发生活化、增殖，产生种类众多的细胞因子，PD-L1抗体通过阻断PD-1/PD-L1结合的免疫抑制信号，刺激T细胞增殖释放细胞因子如IFN-γ等。
实验步骤：分离PBMC中的单核细胞，加入rhGM-CSF(1000U/ml)和rhIL4(500U/ml)，诱导为iDC(诱导性树突状细胞)；分离PBMC中的CD4+T细胞。将DC细胞与T细胞按适宜比例混合，加入不同浓度(2.7nM、0.9nM)的CHO48及人IgG(SIGMA，I4506-10MG)，混合培养5天，检测培养上清中IFNγ的表达。结果如图9所示，CHO48在混合淋巴细胞反应(MLR)中能刺激T细胞增殖，增强IFN-γ的产生。
实施例15：CHO48抑制TGFβ-SMAD3荧光素酶报告基因实验
实验样品：CHO48，人IgG
实验材料：HepG2细胞(人肝癌细胞株)，购于中国医学科学院基础医学研究所细胞资源中心。pGM-SEB luc质粒，购自吉满生物，货号GM-0211086；JetPRIME Transfection，购自Polyplus，货号114-15；TGFβ1，购自Sino Biological，货号10804-HNAC；ONE-GloTMLuciferase Assay System，购自Promega，货号E6120；人IgG，购自Sigma，货号I4506；其他抗体来自内部制备。
实验方法：HepG2细胞使用含有10％FBS的DMEM高糖培养基于37℃5％CO2培养箱中培养，实验第一天接种2×106于10cm培养皿中培养过夜；第二天每个培养皿转染15μg pGM-SEB luc质粒，继续于37℃5％CO2培养箱中培养24小时；第三天用胰酶消化转染后的HepG2细胞，800rpm离心5分钟弃上清，用不完全培养基(含0.5％FBS的DMEM高糖培养基)重悬细胞并计数，将细胞按5×104个，50μl每孔接种于96孔板中，37℃5％CO2培养箱中培养6小时；将25μl每孔不完全培养基配制的终浓度为1ng/ml的TGFβ1和25μl每孔梯度稀释后的各个待测抗体加入96孔板中，与细胞悬液混合均匀，37℃5％CO2培养箱中继续培养18小时；将ONE-GloTM Luciferase Assay System试剂按100μl每孔加入待测的96孔板中，室温避光孵育10分钟，于酶标仪中检测发光值。
实验结果如图10所示，在TGFβ-SMAD3荧光素酶报告基因实验中，CHO48能抑制其信号通路，表现出很好的TGFβ抑制活性。
实施例16：在C57BL/6鼠中评价三功能分子CHO48对肿瘤生长的抑制作用
实验样品：CHO48
实验步骤：收集对数生长期的MC38-hPDL1(tg)-mPDL1(KO)细胞，PBS重悬至适合浓度接种。每只实验小鼠于右侧背部皮下接种5×105MC38-hPDL1细胞，定期观察肿瘤生长情况，待肿瘤生长至平均体积约60mm3时根据肿瘤大小和小鼠体重随机分组给药。分组给药当天定义为第0天。给药方案如表11所示：
表11
Figure BDA0002795596830000321
给药体积为10ul/g体重。
每周观察3次小鼠体重和肿瘤体积，用游标卡尺测量肿瘤长短径，并按公式肿瘤体积(mm3)＝0.5×(a×b2)计算、记录肿瘤生长情况，绘制肿瘤生长曲线。实验结果如图11-12和下表12所示：
表12
Figure BDA0002795596830000322
根据实验终点时D20天肿瘤体积和肿瘤重量统计分析，与对照组相比，CHO48实验组的肿瘤体积(TGItv％＝48.66％)没有统计学意义差异(p＝0.078)，肿瘤重量(TGItw％＝57.48％)有显著性差异(P<0.05)，CHO48在该模型中表现出一定的抗肿瘤活性。
以上详细描述了本发明的优选实施方式，但是，本发明并不限于上述实施方式中的具体细节，在本发明的技术构思范围内，可以对本发明的技术方案进行多种简单变型，这些简单变型均属于本发明的保护范围。
另外需要说明的是，在上述具体实施方式中所描述的各个具体技术特征，在不矛盾的情况下，可以通过任何合适的方式进行组合，为了避免不必要的重复，本发明对各种可能的组合方式不再另行说明。
此外，本发明的各种不同的实施方式之间也可以进行任意组合，只要其不违背本发明的思想，其同样应当视为本发明所公开的内容。
序列表
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530 535 540
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
545 550 555 560
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
565 570 575
Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
580 585 590
Gly Gly Gly Gly Ser Ile Pro Pro His Val Gln Lys Ser Val Asn Asn
595 600 605
Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu
610 615 620
Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser
625 630 635 640
Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu
645 650 655
Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu
660 665 670
Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu
675 680 685
Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly
690 695 700
Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn
705 710 715 720
Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp
725 730
<210> 4
<211> 734
<212> PRT
<213> 人工序列()
<400> 4
Glu Val Gln Leu Leu 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 Tyr Gly Thr Tyr
20 25 30
Ala Met Ser Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Gly Val
35 40 45
Ala Cys Ile Asp Ile Tyr Gly Arg Ala Ser Tyr Thr Asp Pro Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Gln Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Ala Arg Asp Phe Gly Tyr Cys Thr Ala Ser Trp Val His Glu Gly Phe
100 105 110
Ser Arg Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly
115 120 125
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
130 135 140
Gly Ser Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile
145 150 155 160
Pro Glu Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys
165 170 175
Arg Val Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu
180 185 190
Asp Thr Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys
195 200 205
Gly Phe Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr
210 215 220
Cys Glu Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr
225 230 235 240
His Arg Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His
245 250 255
Gly Ile Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala
260 265 270
Arg Thr Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser
275 280 285
Ser Lys His Gln His Lys Lys Leu Val Asn Arg Asp Leu Lys Thr Gln
290 295 300
Ser Gly Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly
305 310 315 320
Val Thr Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly
325 330 335
Leu Met Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys Asp
340 345 350
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
355 360 365
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
370 375 380
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
385 390 395 400
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
405 410 415
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
420 425 430
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
435 440 445
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
450 455 460
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
465 470 475 480
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
485 490 495
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
500 505 510
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
515 520 525
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
530 535 540
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
545 550 555 560
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
565 570 575
Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
580 585 590
Gly Gly Gly Gly Ser Ile Pro Pro His Val Gln Lys Ser Val Asn Ser
595 600 605
Thr Met Ile Val Thr Asp Asn Ser Thr Gly Ala Val Lys Phe Pro Gln
610 615 620
Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys
625 630 635 640
Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln
645 650 655
Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu
660 665 670
Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu
675 680 685
Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro
690 695 700
Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp
705 710 715 720
Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp
725 730
<210> 5
<211> 733
<212> PRT
<213> 人工序列()
<400> 5
Glu Val Gln Leu Leu 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 Tyr Gly Thr Tyr
20 25 30
Ala Met Ser Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Gly Val
35 40 45
Ala Cys Ile Asp Ile Tyr Gly Arg Ala Ser Tyr Thr Asp Pro Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Gln Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Ala Arg Asp Phe Gly Tyr Cys Thr Ala Ser Trp Val His Glu Gly Phe
100 105 110
Ser Arg Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly
115 120 125
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
130 135 140
Gly Ser Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile
145 150 155 160
Pro Glu Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys
165 170 175
Arg Val Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu
180 185 190
Asp Thr Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys
195 200 205
Gly Phe Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr
210 215 220
Cys Glu Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr
225 230 235 240
His Arg Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His
245 250 255
Gly Ile Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala
260 265 270
Arg Thr Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser
275 280 285
Ser Lys His Gln His Lys Lys Leu Val Asn Arg Asp Leu Lys Thr Gln
290 295 300
Ser Gly Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly
305 310 315 320
Val Thr Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly
325 330 335
Leu Met Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys Asp
340 345 350
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
355 360 365
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
370 375 380
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
385 390 395 400
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
405 410 415
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
420 425 430
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
435 440 445
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
450 455 460
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
465 470 475 480
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
485 490 495
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
500 505 510
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
515 520 525
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
530 535 540
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
545 550 555 560
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
565 570 575
Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
580 585 590
Gly Gly Gly Gly Ser Ile Pro Pro His Val Gln Lys Ser Val Asn Ser
595 600 605
Thr Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu
610 615 620
Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser
625 630 635 640
Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu
645 650 655
Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu
660 665 670
Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu
675 680 685
Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly
690 695 700
Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn
705 710 715 720
Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp
725 730
<210> 6
<211> 734
<212> PRT
<213> 人工序列()
<400> 6
Glu Val Gln Leu Leu 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 Tyr Gly Thr Tyr
20 25 30
Ala Met Ser Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Gly Val
35 40 45
Ala Cys Ile Asp Ile Tyr Gly Arg Ala Ser Tyr Thr Asp Pro Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Gln Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Ala Arg Asp Phe Gly Tyr Cys Thr Ala Ser Trp Val His Glu Gly Phe
100 105 110
Ser Arg Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly
115 120 125
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
130 135 140
Gly Ser Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile
145 150 155 160
Pro Glu Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys
165 170 175
Arg Val Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu
180 185 190
Asp Thr Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys
195 200 205
Gly Phe Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr
210 215 220
Cys Glu Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr
225 230 235 240
His Arg Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His
245 250 255
Gly Ile Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala
260 265 270
Arg Thr Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser
275 280 285
Ser Lys His Gln His Lys Lys Leu Val Asn Arg Asp Leu Lys Thr Gln
290 295 300
Ser Gly Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly
305 310 315 320
Val Thr Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly
325 330 335
Leu Met Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys Asp
340 345 350
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
355 360 365
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
370 375 380
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
385 390 395 400
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
405 410 415
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
420 425 430
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
435 440 445
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
450 455 460
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
465 470 475 480
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
485 490 495
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
500 505 510
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
515 520 525
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
530 535 540
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
545 550 555 560
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
565 570 575
Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
580 585 590
Gly Gly Gly Gly Ser Ile Pro Pro His Val Gln Lys Ser Val Asn Asn
595 600 605
Asp Met Ile Val Thr Asp Asn Ser Thr Gly Ala Val Lys Phe Pro Gln
610 615 620
Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys
625 630 635 640
Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln
645 650 655
Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu
660 665 670
Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu
675 680 685
Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro
690 695 700
Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp
705 710 715 720
Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp
725 730
<210> 7
<211> 733
<212> PRT
<213> 人工序列()
<400> 7
Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr
1 5 10 15
Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp
20 25 30
Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys
35 40 45
Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val
50 55 60
Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp
65 70 75 80
Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro
85 90 95
Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met
100 105 110
Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu
115 120 125
Glu Tyr Asn Thr Ser Asn Pro Asp Gly Gly Gly Gly Ser Gly Gly Gly
130 135 140
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Asp Thr Gly
145 150 155 160
Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu Ile Ile His Met
165 170 175
Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val Thr Ser Pro Asn
180 185 190
Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr Leu Ile Pro Asp
195 200 205
Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser Asn
210 215 220
Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr Val Asn
225 230 235 240
Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg Gln Thr Asn Thr
245 250 255
Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val
260 265 270
Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val
275 280 285
Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys
290 295 300
Lys Leu Val Asn Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys
305 310 315 320
Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln
325 330 335
Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn
340 345 350
Ser Thr Phe Val Arg Val His Glu Lys Asp Lys Thr His Thr Cys Pro
355 360 365
Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
370 375 380
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
385 390 395 400
Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
405 410 415
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
420 425 430
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
435 440 445
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
450 455 460
Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
465 470 475 480
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
485 490 495
Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
500 505 510
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
515 520 525
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
530 535 540
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
545 550 555 560
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
565 570 575
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly Gly Ser
580 585 590
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu
595 600 605
Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser
610 615 620
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Tyr Gly Thr Tyr Ala
625 630 635 640
Met Ser Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Gly Val Ala
645 650 655
Cys Ile Asp Ile Tyr Gly Arg Ala Ser Tyr Thr Asp Pro Val Lys Gly
660 665 670
Arg Phe Thr Ile Ser Gln Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln
675 680 685
Met Asn Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala
690 695 700
Arg Asp Phe Gly Tyr Cys Thr Ala Ser Trp Val His Glu Gly Phe Ser
705 710 715 720
Arg Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
725 730
<210> 8
<211> 737
<212> PRT
<213> 人工序列()
<400> 8
Ile Pro Pro His Val Gln Lys Ser Val Asn Ser Thr Met Ile Val Thr
1 5 10 15
Asp Asn Ser Thr Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys
20 25 30
Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn
35 40 45
Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala
50 55 60
Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His
65 70 75 80
Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser
85 90 95
Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe
100 105 110
Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser
115 120 125
Glu Glu Tyr Asn Thr Ser Asn Pro Asp Gly Gly Gly Gly Ser Gly Gly
130 135 140
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Asp Thr
145 150 155 160
Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu Ile Ile His
165 170 175
Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val Thr Ser Pro
180 185 190
Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr Leu Ile Pro
195 200 205
Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser
210 215 220
Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr Val
225 230 235 240
Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg Gln Thr Asn
245 250 255
Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser
260 265 270
Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn
275 280 285
Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His
290 295 300
Lys Lys Leu Val Asn Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met
305 310 315 320
Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp
325 330 335
Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys
340 345 350
Asn Ser Thr Phe Val Arg Val His Glu Lys Asp Lys Thr His Thr Cys
355 360 365
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
370 375 380
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
385 390 395 400
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
405 410 415
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
420 425 430
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
435 440 445
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
450 455 460
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
465 470 475 480
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
485 490 495
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
500 505 510
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
515 520 525
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
530 535 540
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
545 550 555 560
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
565 570 575
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly Gly
580 585 590
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
595 600 605
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
610 615 620
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Asp Tyr
625 630 635 640
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Gly Val
645 650 655
Ser Cys Ile Ser Lys Ser Gly Glu Thr Thr Phe Phe Val Glu Ser Val
660 665 670
Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
675 680 685
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
690 695 700
Ala Thr Gly Ser Trp Cys Thr Val Gly Ser Met Ser Arg Gln Phe Tyr
705 710 715 720
Arg Gln Phe Phe His Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser
725 730 735
Ser
<210> 9
<211> 736
<212> PRT
<213> 人工序列()
<400> 9
Glu Val Gln Leu Leu 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 Leu Asp Asp Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Lys Ser Gly Glu Thr Thr Phe Phe Val Glu Ser Val
50 55 60
Lys Asp 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 Thr Gly Ser Trp Cys Thr Val Gly Ser Met Ser Arg Gln Phe Tyr
100 105 110
Arg Gln Phe Phe His Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
130 135 140
Gly Gly Gly Gly Ser Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr
145 150 155 160
Ser Glu Ile Pro Glu Ile Ile His Met Thr Glu Gly Arg Glu Leu Val
165 170 175
Ile Pro Cys Arg Val Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys
180 185 190
Phe Pro Leu Asp Thr Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp
195 200 205
Ser Arg Lys Gly Phe Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly
210 215 220
Leu Leu Thr Cys Glu Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn
225 230 235 240
Tyr Leu Thr His Arg Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser
245 250 255
Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn
260 265 270
Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu
275 280 285
Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu Val Asn Arg Asp Leu
290 295 300
Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr
305 310 315 320
Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala
325 330 335
Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe Val Arg Val His
340 345 350
Glu Lys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
355 360 365
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
370 375 380
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
385 390 395 400
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
405 410 415
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
420 425 430
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
435 440 445
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
450 455 460
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
465 470 475 480
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
485 490 495
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
500 505 510
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
515 520 525
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
530 535 540
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
545 550 555 560
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
565 570 575
Leu Ser Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
580 585 590
Gly Gly Ser Gly Gly Gly Gly Ser Ile Pro Pro His Val Gln Lys Ser
595 600 605
Val Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe
610 615 620
Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn
625 630 635 640
Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys
645 650 655
Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile
660 665 670
Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe
675 680 685
Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys
690 695 700
Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys
705 710 715 720
Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp
725 730 735
<210> 10
<211> 737
<212> PRT
<213> 人工序列()
<400> 10
Glu Val Gln Leu Leu 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 Leu Asp Asp Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Lys Ser Gly Glu Thr Thr Phe Phe Val Glu Ser Val
50 55 60
Lys Asp 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 Thr Gly Ser Trp Cys Thr Val Gly Ser Met Ser Arg Gln Phe Tyr
100 105 110
Arg Gln Phe Phe His Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
130 135 140
Gly Gly Gly Gly Ser Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr
145 150 155 160
Ser Glu Ile Pro Glu Ile Ile His Met Thr Glu Gly Arg Glu Leu Val
165 170 175
Ile Pro Cys Arg Val Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys
180 185 190
Phe Pro Leu Asp Thr Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp
195 200 205
Ser Arg Lys Gly Phe Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly
210 215 220
Leu Leu Thr Cys Glu Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn
225 230 235 240
Tyr Leu Thr His Arg Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser
245 250 255
Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn
260 265 270
Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu
275 280 285
Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu Val Asn Arg Asp Leu
290 295 300
Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr
305 310 315 320
Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala
325 330 335
Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe Val Arg Val His
340 345 350
Glu Lys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
355 360 365
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
370 375 380
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
385 390 395 400
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
405 410 415
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
420 425 430
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
435 440 445
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
450 455 460
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
465 470 475 480
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
485 490 495
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
500 505 510
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
515 520 525
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
530 535 540
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
545 550 555 560
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
565 570 575
Leu Ser Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
580 585 590
Gly Gly Ser Gly Gly Gly Gly Ser Ile Pro Pro His Val Gln Lys Ser
595 600 605
Val Asn Ser Thr Met Ile Val Thr Asp Asn Ser Thr Gly Ala Val Lys
610 615 620
Phe Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp
625 630 635 640
Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu
645 650 655
Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn
660 665 670
Ile Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp
675 680 685
Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys
690 695 700
Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu
705 710 715 720
Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro
725 730 735
Asp
<210> 11
<211> 736
<212> PRT
<213> 人工序列()
<400> 11
Glu Val Gln Leu Leu 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 Leu Asp Asp Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Lys Ser Gly Glu Thr Thr Phe Phe Val Glu Ser Val
50 55 60
Lys Asp 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 Thr Gly Ser Trp Cys Thr Val Gly Ser Met Ser Arg Gln Phe Tyr
100 105 110
Arg Gln Phe Phe His Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
130 135 140
Gly Gly Gly Gly Ser Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr
145 150 155 160
Ser Glu Ile Pro Glu Ile Ile His Met Thr Glu Gly Arg Glu Leu Val
165 170 175
Ile Pro Cys Arg Val Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys
180 185 190
Phe Pro Leu Asp Thr Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp
195 200 205
Ser Arg Lys Gly Phe Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly
210 215 220
Leu Leu Thr Cys Glu Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn
225 230 235 240
Tyr Leu Thr His Arg Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser
245 250 255
Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn
260 265 270
Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu
275 280 285
Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu Val Asn Arg Asp Leu
290 295 300
Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr
305 310 315 320
Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala
325 330 335
Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe Val Arg Val His
340 345 350
Glu Lys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
355 360 365
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
370 375 380
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
385 390 395 400
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
405 410 415
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
420 425 430
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
435 440 445
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
450 455 460
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
465 470 475 480
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
485 490 495
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
500 505 510
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
515 520 525
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
530 535 540
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
545 550 555 560
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
565 570 575
Leu Ser Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
580 585 590
Gly Gly Ser Gly Gly Gly Gly Ser Ile Pro Pro His Val Gln Lys Ser
595 600 605
Val Asn Ser Thr Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe
610 615 620
Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn
625 630 635 640
Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys
645 650 655
Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile
660 665 670
Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe
675 680 685
Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys
690 695 700
Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys
705 710 715 720
Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp
725 730 735
<210> 12
<211> 737
<212> PRT
<213> 人工序列()
<400> 12
Glu Val Gln Leu Leu 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 Leu Asp Asp Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Lys Ser Gly Glu Thr Thr Phe Phe Val Glu Ser Val
50 55 60
Lys Asp 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 Thr Gly Ser Trp Cys Thr Val Gly Ser Met Ser Arg Gln Phe Tyr
100 105 110
Arg Gln Phe Phe His Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
130 135 140
Gly Gly Gly Gly Ser Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr
145 150 155 160
Ser Glu Ile Pro Glu Ile Ile His Met Thr Glu Gly Arg Glu Leu Val
165 170 175
Ile Pro Cys Arg Val Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys
180 185 190
Phe Pro Leu Asp Thr Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp
195 200 205
Ser Arg Lys Gly Phe Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly
210 215 220
Leu Leu Thr Cys Glu Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn
225 230 235 240
Tyr Leu Thr His Arg Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser
245 250 255
Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn
260 265 270
Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu
275 280 285
Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu Val Asn Arg Asp Leu
290 295 300
Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr
305 310 315 320
Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala
325 330 335
Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe Val Arg Val His
340 345 350
Glu Lys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
355 360 365
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
370 375 380
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
385 390 395 400
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
405 410 415
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
420 425 430
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
435 440 445
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
450 455 460
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
465 470 475 480
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
485 490 495
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
500 505 510
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
515 520 525
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
530 535 540
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
545 550 555 560
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
565 570 575
Leu Ser Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
580 585 590
Gly Gly Ser Gly Gly Gly Gly Ser Ile Pro Pro His Val Gln Lys Ser
595 600 605
Val Asn Asn Asp Met Ile Val Thr Asp Asn Ser Thr Gly Ala Val Lys
610 615 620
Phe Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp
625 630 635 640
Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu
645 650 655
Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn
660 665 670
Ile Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp
675 680 685
Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys
690 695 700
Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu
705 710 715 720
Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro
725 730 735
Asp
<210> 13
<211> 136
<212> PRT
<213> 人工序列()
<400> 13
Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr
1 5 10 15
Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp
20 25 30
Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys
35 40 45
Ser Thr Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val
50 55 60
Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp
65 70 75 80
Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro
85 90 95
Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met
100 105 110
Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu
115 120 125
Glu Tyr Asn Thr Ser Asn Pro Asp
130 135
<210> 14
<211> 138
<212> PRT
<213> 人工序列()
<400> 14
Ile Pro Pro His Val Gln Lys Ser Val Asn Ser Thr Met Ile Val Thr
1 5 10 15
Asp Asn Ser Thr Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys
20 25 30
Asp Val Arg Phe Ser Thr Cys Asp Asp Asn Gln Lys Ser Cys Met Ser
35 40 45
Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val
50 55 60
Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys
65 70 75 80
His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala
85 90 95
Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe
100 105 110
Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe
115 120 125
Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp
130 135
<210> 15
<211> 136
<212> PRT
<213> 人工序列()
<400> 15
Ile Pro Pro His Val Gln Lys Ser Val Asn Ser Thr Met Ile Val Thr
1 5 10 15
Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp
20 25 30
Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys
35 40 45
Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val
50 55 60
Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp
65 70 75 80
Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro
85 90 95
Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met
100 105 110
Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu
115 120 125
Glu Tyr Asn Thr Ser Asn Pro Asp
130 135
<210> 16
<211> 137
<212> PRT
<213> 人工序列()
<400> 16
Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr
1 5 10 15
Asp Asn Ser Thr Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys
20 25 30
Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn
35 40 45
Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala
50 55 60
Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His
65 70 75 80
Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser
85 90 95
Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe
100 105 110
Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser
115 120 125
Glu Glu Tyr Asn Thr Ser Asn Pro Asp
130 135
<210> 17
<211> 117
<212> PRT
<213> 人工序列()
<400> 17
Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe
1 5 10 15
Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr
20 25 30
Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys
35 40 45
Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu
50 55 60
Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile
65 70 75 80
Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys
85 90 95
Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn
100 105 110
Thr Ser Asn Pro Asp
115
<210> 18
<211> 115
<212> PRT
<213> 人工序列()
<400> 18
Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr
1 5 10 15
Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile
20 25 30
Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp
35 40 45
Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr
50 55 60
His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys
65 70 75 80
Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser
85 90 95
Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser
100 105 110
Asn Pro Asp
115
<210> 19
<211> 122
<212> PRT
<213> 人工序列()
<400> 19
Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe
1 5 10 15
Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser
20 25 30
Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val
35 40 45
Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys
50 55 60
His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Phe Asp Ala Ala
65 70 75 80
Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe
85 90 95
Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe
100 105 110
Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp
115 120
<210> 20
<211> 432
<212> PRT
<213> 人工序列()
<400> 20
Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu
1 5 10 15
Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val
20 25 30
Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr
35 40 45
Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe
50 55 60
Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu
65 70 75 80
Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg
85 90 95
Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile
100 105 110
Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr
115 120 125
Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys
130 135 140
His Gln His Lys Lys Leu Val Asn Arg Asp Leu Lys Thr Gln Ser Gly
145 150 155 160
Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr
165 170 175
Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met
180 185 190
Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys Asp Lys Thr
195 200 205
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
210 215 220
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
225 230 235 240
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
245 250 255
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
260 265 270
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
275 280 285
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
290 295 300
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
305 310 315 320
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
325 330 335
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys
340 345 350
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
355 360 365
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
370 375 380
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
385 390 395 400
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
405 410 415
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
420 425 430
<210> 21
<211> 126
<212> PRT
<213> 人工序列()
<400> 21
Glu Val Gln Leu Leu 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 Tyr Gly Thr Tyr
20 25 30
Ala Met Ser Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Gly Val
35 40 45
Ala Cys Ile Asp Ile Tyr Gly Arg Ala Ser Tyr Thr Asp Pro Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Gln Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Ala Arg Asp Phe Gly Tyr Cys Thr Ala Ser Trp Val His Glu Gly Phe
100 105 110
Ser Arg Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 22
<211> 10
<212> PRT
<213> 人工序列()
<400> 22
Gly Phe Thr Tyr Gly Thr Tyr Ala Met Ser
1 5 10
<210> 23
<211> 16
<212> PRT
<213> 人工序列()
<400> 23
Cys Ile Asp Ile Tyr Gly Arg Ala Ser Tyr Thr Asp Pro Val Lys Gly
1 5 10 15
<210> 24
<211> 20
<212> PRT
<213> 人工序列()
<400> 24
Ala Ala Arg Asp Phe Gly Tyr Cys Thr Ala Ser Trp Val His Glu Gly
1 5 10 15
Phe Ser Arg Tyr
20
<210> 25
<211> 129
<212> PRT
<213> 人工序列()
<400> 25
Glu Val Gln Leu Leu 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 Leu Asp Asp Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Lys Ser Gly Glu Thr Thr Phe Phe Val Glu Ser Val
50 55 60
Lys Asp 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 Thr Gly Ser Trp Cys Thr Val Gly Ser Met Ser Arg Gln Phe Tyr
100 105 110
Arg Gln Phe Phe His Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser
<210> 26
<211> 10
<212> PRT
<213> 人工序列()
<400> 26
Gly Phe Thr Leu Asp Asp Tyr Ala Ile Gly
1 5 10
<210> 27
<211> 14
<212> PRT
<213> 人工序列()
<400> 27
Lys Ser Gly Glu Thr Thr Phe Phe Val Glu Ser Val Lys Asp
1 5 10
<210> 28
<211> 22
<212> PRT
<213> 人工序列()
<400> 28
Ala Thr Gly Ser Trp Cys Thr Val Gly Ser Met Ser Arg Gln Phe Tyr
1 5 10 15
Arg Gln Phe Phe His Ser
20
<210> 29
<211> 448
<212> PRT
<213> 人工序列()
<400> 29
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 Phe Ser Asp Ser
20 25 30
Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Trp Ile Ser Pro Tyr Gly Gly Ser 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 Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
130 135 140
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
145 150 155 160
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
210 215 220
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
225 230 235 240
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
260 265 270
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
275 280 285
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val
290 295 300
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
305 310 315 320
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
325 330 335
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
340 345 350
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
355 360 365
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
370 375 380
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
385 390 395 400
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
405 410 415
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
420 425 430
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 30
<211> 214
<212> PRT
<213> 人工序列()
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Claims (11)

1.一种包含TGF-β抑制剂、VEGF抑制剂和抗PDL1抗体的三功能融合蛋白。
2.根据权利要求1所述的三功能融合蛋白，其特征在于，
所述三功能融合蛋白的氨基酸序列选自SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3、SEQID NO：4、SEQ ID NO：5、SEQ ID NO：6、SEQ ID NO：7、SEQ ID NO：8、SEQ ID NO：9、SEQ IDNO：10、SEQ ID NO：11和SEQ ID NO：12中的任意一个。
3.根据权利要求1所述的三功能融合蛋白，其特征在于，
所述TGF-β抑制剂为TGF-βRII胞外结构域或其N端处保持结合TGF-β活性的点突变或截短体形式，其中所述TGF-β抑制剂的氨基酸序列选自SEQ ID NO：13、SEQ ID NO：14、SEQ IDNO：15、SEQ ID NO：16、SEQ ID NO：17、SEQ ID NO：18和SEQ ID NO：19中的任意一个。
4.根据权利要求1所述的三功能融合蛋白，其特征在于，
所述VEGF-Trap(抑制剂)形式，在其N端和C端均以共价键的方式与多肽连接，并且其与VEGF-A的亲和力KD值不高于100pM或者与VEGF-A、VEGF-B和PIGF结合的KD均不高于阿柏西普亲和力KD值的50倍，其中所述VEGF抑制剂的氨基酸序列选自SEQ ID NO：20、SEQ ID NO：32、SEQ ID NO：33和SEQ ID NO：34中的任意一个。
5.根据权利要求1所述的三功能融合蛋白，其特征在于，
所述抗PD-L1抗体为其单域抗体或scFv形式；和/或所述TGF-β抑制剂为TGF-βRI、TGF-βRII或其组合、或其单域抗体或scFv形式；和/或所述VEGF抑制剂为VEGF-Trap形式、或其单域抗体或scFv形式。
6.根据权利要求1所述的三功能融合蛋白，其特征在于，
所述抗PD-L1抗体的氨基酸序列为SEQ ID NO：21或SEQ ID NO：25，其中所述抗PD-L1抗体的CDR序列分别为SEQ ID NO：22、SEQ ID NO：23和SEQ ID NO：24；或SEQ ID NO：26、SEQID NO：27和SEQ ID NO：28中的任意一个。
7.根据权利要求1至6中任一项所述的三功能融合蛋白在制备用于治疗癌症的药物中的用途。
8.根据权利要求1至6中任一项所述的三功能融合蛋白在制备用于抑制肿瘤生长的药物中的用途。
9.根据权利要求1至6中任一项所述的三功能融合蛋白在制备用于调节免疫功能的药物中的用途。
10.根据权利要求1至6中任一项所述的三功能融合蛋白在制备用于抗感染的药物中的用途。
11.根据权利要求1至6中任一项所述的三功能融合蛋白在制备用于抑制血管增长的药物中的用途。
CN202011330365.2A 2019-12-05 2020-11-24 一种包含TGF-β抑制剂、VEGF抑制剂和抗PDL1抗体的三功能融合蛋白 Active CN112552412B (zh)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114480252A (zh) * 2022-01-24 2022-05-13 四川大学华西医院 功能增强型内皮细胞的驯化方法及驯化得到的细胞制剂
WO2022105832A1 (zh) * 2019-12-05 2022-05-27 启愈生物技术(上海)有限公司 抗pd-l1纳米抗体及三功能融合蛋白
WO2022174781A1 (zh) * 2021-02-22 2022-08-25 浙江道尔生物科技有限公司 多结构域融合蛋白及其应用
WO2022228445A1 (en) * 2021-04-26 2022-11-03 I-Mab Biopharma Co., Ltd. Single domain pd-l1 antibodies
WO2023030433A1 (zh) * 2021-09-03 2023-03-09 开拓药业(广东)有限公司 抗alk-1/抗vegf双特异性抗体及其应用
WO2024041477A1 (zh) * 2022-08-22 2024-02-29 浙江道尔生物科技有限公司 多结构域融合蛋白的用途

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115925895A (zh) * 2021-04-13 2023-04-07 石河子大学 抗bvdv e0的纳米抗体及其制备方法及应用
CN113234168B (zh) * 2021-06-04 2022-09-13 浙江大学 一种融合蛋白、基因及应用
CN115028726B (zh) * 2022-03-31 2024-01-09 浙江特瑞思药业股份有限公司 一种抗pd-1纳米抗体及其应用
CN117164719A (zh) * 2022-05-28 2023-12-05 启愈生物技术(上海)有限公司 靶向SIRPα和PD-L1的双特异性抗体或其抗原结合片段及应用
WO2023241480A1 (zh) * 2022-06-13 2023-12-21 三优生物医药(上海)有限公司 抗pd-l1、vegf和egfr三特异性抗体及其应用
CN117736323A (zh) * 2022-09-21 2024-03-22 三优生物医药(上海)有限公司 抗pd-l1抗体及其用途
CN115925947B (zh) * 2022-09-27 2023-08-22 上海百英生物科技股份有限公司 一种亲和力成熟方法及抗人pd-l1单域抗体的亲和力成熟

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104066447A (zh) * 2011-11-23 2014-09-24 拜奥文斯瑞有限公司 重组蛋白及其治疗用途
US20180051085A1 (en) * 2016-06-17 2018-02-22 Immunomedics, Inc. Novel anti-pd-1 checkpoint inhibitor antibodies that block binding of pd-l1 to pd-1
WO2018205985A1 (zh) * 2017-05-12 2018-11-15 江苏恒瑞医药股份有限公司 含有TGF-β受体的融合蛋白及其医药用途
CN108864290A (zh) * 2017-05-08 2018-11-23 上海津曼特生物科技有限公司 双特异性重组蛋白及其应用
US20180358128A1 (en) * 2017-06-13 2018-12-13 Alexander Bagaev Systems and methods for identifying cancer treatments from normalized biomarker scores

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2952717T3 (es) * 2014-10-14 2023-11-03 Novartis Ag Moléculas de anticuerpos contra PD-L1 y usos de las mismas
CN114272371A (zh) * 2015-07-29 2022-04-05 诺华股份有限公司 包含抗pd-1抗体分子的联合疗法
CN105524173B (zh) * 2016-01-28 2022-09-23 东南大学 一种针对人源抗体Fc片段的纳米抗体及其应用
CN109096396B (zh) * 2017-06-20 2022-01-04 华兰生物工程股份有限公司 一种抗pd-l1人源化纳米抗体及其应用
CN113164777A (zh) * 2018-09-27 2021-07-23 马伦戈治疗公司 Csf1r/ccr2多特异性抗体
CN112646031B (zh) * 2019-10-10 2021-12-07 天境生物科技(上海)有限公司 抗4-1bb纳米抗体及其用途
CN112574309B (zh) * 2019-12-05 2023-06-16 启愈生物技术(上海)有限公司 一种抗pd-l1纳米抗体及其用途
CN111909272B (zh) * 2020-08-12 2022-09-23 华东理工大学 抗pd-l1纳米抗体及其应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104066447A (zh) * 2011-11-23 2014-09-24 拜奥文斯瑞有限公司 重组蛋白及其治疗用途
US20180051085A1 (en) * 2016-06-17 2018-02-22 Immunomedics, Inc. Novel anti-pd-1 checkpoint inhibitor antibodies that block binding of pd-l1 to pd-1
CN108864290A (zh) * 2017-05-08 2018-11-23 上海津曼特生物科技有限公司 双特异性重组蛋白及其应用
WO2018205985A1 (zh) * 2017-05-12 2018-11-15 江苏恒瑞医药股份有限公司 含有TGF-β受体的融合蛋白及其医药用途
US20180358128A1 (en) * 2017-06-13 2018-12-13 Alexander Bagaev Systems and methods for identifying cancer treatments from normalized biomarker scores

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ITALIA GRENGA, M.D. ET AL.: "Anti-PD-L1/TGFβR2 (M7824) fusion protein induces immunogenic modulation of human urothelial carcinoma cell lines, rendering them more susceptible to immune-mediated recognition and lysis", 《UROLOGIC ONCOLOGY: SEMINARS AND ORIGINAL INVESTIGATIONS》 *
KARIN M. KNUDSON ET AL.: "M7824, a novel bifunctional anti-PD-L1/TGFβ Trap fusion protein, promotes anti-tumor efficacy as monotherapy and in combination with vaccine", 《ONCOIMMUNOLOGY》 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022105832A1 (zh) * 2019-12-05 2022-05-27 启愈生物技术(上海)有限公司 抗pd-l1纳米抗体及三功能融合蛋白
WO2022174781A1 (zh) * 2021-02-22 2022-08-25 浙江道尔生物科技有限公司 多结构域融合蛋白及其应用
WO2022174451A1 (zh) * 2021-02-22 2022-08-25 浙江道尔生物科技有限公司 一种具有抗癌活性的多结构域融合蛋白
CN116761814A (zh) * 2021-02-22 2023-09-15 浙江道尔生物科技有限公司 多结构域融合蛋白及其应用
WO2022228445A1 (en) * 2021-04-26 2022-11-03 I-Mab Biopharma Co., Ltd. Single domain pd-l1 antibodies
WO2023030433A1 (zh) * 2021-09-03 2023-03-09 开拓药业(广东)有限公司 抗alk-1/抗vegf双特异性抗体及其应用
CN114480252A (zh) * 2022-01-24 2022-05-13 四川大学华西医院 功能增强型内皮细胞的驯化方法及驯化得到的细胞制剂
CN114480252B (zh) * 2022-01-24 2023-04-25 四川大学华西医院 功能增强型内皮细胞的驯化方法及驯化得到的细胞制剂
WO2024041477A1 (zh) * 2022-08-22 2024-02-29 浙江道尔生物科技有限公司 多结构域融合蛋白的用途

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